Posts Tagged ‘Clopidogrel’

Platelet Endothelial Aggregation Receptor-1 (PEAR1) Gene to be most strongly associated with Dual Antiplatelet Therapy Response: Genetic Determinants of Variable Response to Aspirin (alone and in combination with Clopidogrel)

Reporter: Aviva Lev-Ari, PhD, RN

4 Genetic Variation in PEAR1 is Associated with Platelet Aggregation and Cardiovascular Outcomes

Joshua P. Lewis1Kathleen Ryan1Jeffrey R. O’Connell1Richard B. Horenstein1,Coleen M. Damcott1Quince Gibson1Toni I. Pollin1Braxton D. Mitchell1Amber L. Beitelshees1Ruth Pakzy1Keith Tanner1Afshin Parsa1Udaya S. Tantry2Kevin P. Bliden2Wendy S. Post3Nauder Faraday3William Herzog4Yan Gong5Carl J. Pepine6Julie A. Johnson5Paul A. Gurbel2 and Alan R. Shuldiner7*

Author Affiliations

1University of Maryland School of Medicine, Baltimore, MD

2Sinai Hospital of Baltimore, Baltimore, MD

3Johns Hopkins University School of Medicine, Baltimore, MD

4Sinai Hospital of Baltimore & Johns Hopkins University School of Medicine, Baltimore, MD

5University of Florida College of Pharmacy, Gainesville, FL

6University of Florida College of Medicine, Gainesville, FL

7University of Maryland School of Medicine & Veterans Administration Medical Center, Baltimore, MD

* University of Maryland School of Medicine & Veterans Administration Medical Center, Baltimore, MD


Background-Aspirin or dual antiplatelet therapy (DAPT) with aspirin and clopidogrel is standard therapy for patients at increased risk for cardiovascular events. However, the genetic determinants of variable response to aspirin (alone and in combination with clopidogrel) are not known.

Methods and Results-We measured ex-vivo platelet aggregation before and after DAPT in individuals (n=565) from the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study and conducted a genome-wide association study (GWAS) of drug response. Significant findings were extended by examining genotype and cardiovascular outcomes in two independent aspirin-treated cohorts: 227 percutaneous coronary intervention (PCI) patients, and 1,000 patients of the International VErapamil SR/trandolapril Study (INVEST) GENEtic Substudy (INVEST-GENES). GWAS revealed a strong association between single nucleotide polymorphisms on chromosome 1q23 and post-DAPT platelet aggregation. Further genotyping revealed rs12041331 in the platelet endothelial aggregation receptor-1 (PEAR1) gene to be most strongly associated with DAPT response (P=7.66×10-9). In Caucasian and African American patients undergoing PCI, A-allele carriers of rs12041331 were more likely to experience a cardiovascular event or death compared to GG homozygotes (hazard ratio = 2.62, 95%CI 0.96-7.10, P=0.059 and hazard ratio = 3.97, 95%CI 1.10-14.31, P=0.035 respectively). In aspirin-treated INVEST-GENES patients, rs12041331 A-allele carriers had significantly increased risk of myocardial infarction compared to GG homozygotes (OR=2.03, 95%CI 1.01-4.09, P=0.048).

Conclusions – Common genetic variation in PEAR1 may be a determinant of platelet response and cardiovascular events in patients on aspirin, alone and in combination with clopidogrel.

Clinical Trial Registration; Identifiers:NCT00799396 and NCT00370045


Circulation: Cardiovascular Genetics.2013; 6: 184-192 Published online before print February 7, 2013,doi: 10.1161/​CIRCGENETICS.111.964627





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Anticoagulation Genotype guided Dosing

Author and Curator: Larry H. Bernstein, MD, FCAP 


A common complication of patients on Warfarin (coumadin) is massive hematoma associated with a fall.  Warfarin is an inhibitor of the liver production of prothrombin.  Inadequate Warfarin dosing results in a risk of thromboembolism to the lungs or the brain, depending on where the clot is initiated.  However, there is a pharmacogenomic problem in that there are individuals who have a genetic polymorphism (CYP2C9 and VKORC1) that affects the coagulation effect of the coumadin.  This introduces the question of whether such individuals should be identified and dosed according to pharmacogenomic testing.  The usual measurement of the effect of the drug is the International Normalized Ratio (INR) from the Prothrombin Time (PT).  The existence of chronic liver disease and the use of coumadin are perhaps almost the exclusive value for the PT.
A similar question arises for the genotyping of clopidogrel for antiplatelet guidance. Does it reduce risk in stenting?

A Pharmacogenetic versus a Clinical Algorithm for Warfarin Dosing

Stephen E. Kimmel, M.D., Benjamin French, Ph.D., Scott E. Kasner, M.D., Julie A. Johnson, Pharm.D., and others

Center for Therapeutic Effectiveness Research,  Philadelphia, PA 19104-6021, or at

A complete list of investigators and committees in the Clarification of Optimal Anticoagulation through Genetics (COAG) trial is provided in the Supplementary Appendix, available at

November 19, 2013 | NEJM

Comparison of  genotype-guided dosing algorithm with the clinically guided dosing algorithm for Warfarin dosing




The clinical utility of genotype-guided (pharmacogenetically based) dosing of warfarin has been tested only in small clinical trials or observational studies, with equivocal results.
We randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy. The primary outcome was the percentage of time that the international normalized ratio (INR) was in the therapeutic range from day 4 or 5 through day 28 of therapy.


At 4 weeks, the mean percentage of time in the therapeutic range was 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference, [genotype-guided group minus clinically guided group], −0.2; 95% confidence interval, −3.4 to 3.1; P=0.91). There also was no significant between-group difference among patients with a predicted dose difference between the two algorithms of 1 mg per day or more. There was, however, a significant interaction between dosing strategy and race (P=0.003). Among black patients, the mean percentage of time in the therapeutic range was less in the genotype-guided group than in the clinically guided group. The rates of the combined outcome of any INR of 4 or more, major bleeding, or thromboembolism did not differ significantly according to dosing strategy.


Genotype-guided dosing of warfarin did not improve anticoagulation control during the first 4 weeks of therapy. (Funded by the National Heart, Lung, and Blood Institute and others; COAG number, NCT00839657.)
Figure 1 Distribution of Time in the Therapeutic Range.
nejmoa1310669_f1 Distribution of Time in the Therapeutic Range.
Figure 2 Range of INRs during the 4-Week Study.
nejmoa1310669_f2 Range of INRs during the 4-Week Study.


The need for clinical trials before widespread adoption of genotype-guided drug dosing and selection remains widely debated.1-4 Warfarin therapy has served as a model for the potential for pharmacogenetics to improve patient care.1 Observational studies have identified two genes, CYP2C9 and VKORC1, that are associated with variation in warfarin maintenance doses. However, the clinical utility of starting warfarin at the maintenance dose predicted by genotype-guided algorithms has been tested only in small trials, none of which were definitive.5-8 In contrast, observational studies have suggested potential benefits from genotype-guided dosing.9,10 In addition, previous clinical trials could not determine the usefulness of current dosing algorithms among black patients, for whom genotype-guided algorithms perform less well than for other populations.11-13
On the basis of available data, the Food and Drug Administration (FDA) has updated the label for warfarin twice, suggesting that variants in CYP2C9 and VKORC1 may be taken into consideration when choosing the initial warfarin dose. However, the Centers for Medicare and Medicaid Services did not find sufficient evidence to cover the cost of genotyping for warfarin dosing.14 Our study, called the Clarification of Optimal Anticoagulation through Genetics (COAG) trial, was designed to test the effect of genotype-guided dosing on anticoagulation control.


Study Design and Oversight

The COAG trial was a multicenter, double-blind, randomized, controlled trial that compared a genotype-guided warfarin-dosing strategy with a clinically based dosing strategy during the first 5 days of therapy among patients initiating warfarin treatment.15-17 The study was designed by the authors and approved by the institutional review board at the University of Pennsylvania and at each participating clinical center. The data were collected, analyzed, and interpreted by the authors. A steering committee provided oversight of the trial (for details, see the Supplementary Appendix, available with the full text of this article at An independent data and safety monitoring board monitored the trial and made recommendations to the National Heart, Lung, and Blood Institute. The first two authors wrote the first draft of the manuscript, which was edited and approved by all the authors. The National Heart, Lung, and Blood Institute supported this study. Bristol-Myers Squibb donated Coumadin (warfarin). GenMark Diagnostics and AutoGenomics loaned genotyping platforms to the clinical centers. None of the companies supporting the trial had any role in the design of the protocol or in the collection, analysis, or interpretation of the data. The authors vouch for the data and the analyses, and for the fidelity of this report to the trial protocol, which is available at

Study Patients and Randomization

From September 2009 through April 2013, we enrolled both inpatients and outpatients at 18 clinical centers in the United States. All the patients were adults initiating warfarin therapy with a target international normalized ratio (INR) of 2 to 3. Detailed inclusion and exclusion criteria are provided in the Supplementary Appendix. All patients provided written informed consent.
Patients were randomly assigned, in a 1:1 ratio, to the use of a dosing algorithm that included both clinical variables and genotype data or to a clinically guided dosing strategy. Randomization was stratified according to clinical center and self-reported race (black vs. nonblack).
Genotyping for CYP2C9 and VKORC1 at each clinical center was performed with the use of one of two FDA-approved platforms, the GenMark Dx eSensor XT-8 or the AutoGenomics INFINITI Analyzer. Per protocol, genotyping was performed in all patients immediately after blood-sample collection to maintain blinding to the treatment assignment. Genotyping was repeated at the central laboratory with the use of either pyrosequencing or real-time polymerase-chain-reaction assay to measure the accuracy at clinical centers.

Study Intervention and Follow-up

The study intervention period was the first 5 days of warfarin therapy. During this period, the prespecified algorithms were used to determine the warfarin dose. For each dosing strategy, a dose-initiation algorithm was used during the first 3 days of therapy, and a dose-revision algorithm was used on day 4, 5, or both. The algorithms for the genotype-guided dosing strategy12,18 included clinical variables and genotype data for CYP2C9*2, CYP2C9*3, and VKORC1. The algorithms for the clinically based dosing strategy included clinical variables only. The dosing algorithms are provided in the Supplementary Appendix. If genotype information was not available for a patient in the genotype-guided dosing group before the administration of warfarin on any given day in the first 5 days, the clinical algorithm was used on that day.
During the first 4 weeks of therapy, patients and clinicians were unaware of the actual dose of warfarin that was administered, because the pills were encapsulated to prevent identification of the dose (see the Supplementary Appendix). After the 5-day initiation period, we adjusted the dose during the first 4 weeks using standardized dose-adjustment techniques,5,10 starting with the doses predicted by the algorithms and making the same relative adjustments on the basis of the INR in the two study groups. Clinicians were informed of the relative dose change (e.g., a 10% dose increase) at each INR measurement but not the actual dose of warfarin. Clinicians could contact the medical monitor (who was aware of the study-group assignments) to request an override of these relative dose changes without being informed of the actual dose. All patients were to be followed for a total of 6 months.

Study Outcomes

The primary outcome was the percentage of time in the therapeutic range (INR, 2 to 3) from the completion of the intervention period (day 4 or 5) through day 28 of therapy. We calculated the percentage of time in the therapeutic range using a standard linear interpolation method between successive INR values,19 as detailed in the Supplementary Appendix. Each clinical center measured INRs with the use of instruments certified by the Clinical Laboratory Improvement Amendments and following strict quality assurance.
Secondary outcomes included a composite outcome of any INR of 4 or more, major bleeding, or thromboembolism in the first 4 weeks (principal secondary outcome); the time to the first therapeutic INR; the time to the determination of a maintenance dose (which was defined as the time to the first of two consecutive INR measurements, measured at least 1 week apart, that were in the therapeutic range without a dose change); and the time to an adverse event (death from any cause, major bleeding, thromboembolism, or any clinically relevant nonmajor bleeding event20,21) in the first 4 weeks. Two physicians who were unaware of the study-group assignments adjudicated major bleeding and thromboembolic serious adverse events. The definitions of major bleeding,22 clinically relevant nonmajor bleeding, and thromboembolism are provided in the Supplementary Appendix.

Statistical Analysis

We analyzed the primary outcome in the modified intention-to-treat population, which included all patients who underwent randomization with the exception of patients for whom INR data were not available (Fig. S1 in the Supplementary Appendix). Safety outcomes were analyzed in the entire cohort, regardless of whether patients received the study drug. We used regression models to analyze the primary and secondary outcomes, using linear regression for the percentage of time in the therapeutic range and Cox regression for time-to-event outcomes. The protocol specified that we conduct coprimary analyses in which we evaluated the primary outcome in all patients and in a primary subgroup, which comprised patients who had an absolute difference of 1.0 mg or more in the predicted initial daily dose between the genotype-guided dosing algorithm and the clinical dosing algorithm. We used an alpha allocation approach, which formally allows for the evaluation of the treatment benefit in an enriched subgroup as a coprimary end point. In this approach, the overall type I error rate of 0.05 for the primary outcome was split between the analyses performed among all patients and among those in the primary subgroup.17 All models were adjusted for the stratification variables (center and race). Additional subgroups, which were prespecified, were race (black vs. nonblack), sex, and the total number of allelic variants (1 variant vs. 0 or >1 variant in either CYP2C9 or VKORC1 5). All statistical tests were two-sided. All analyses were performed with the use of the R statistical package, version 3.0.1 (R Development Core Team).
We specified a minimum detectable difference of 5.5% in the mean percentage of time in the therapeutic range between the genotype-guided group and the clinically guided group in the entire study population.16 We assumed a standard deviation for the percentage of time in therapeutic range of 25% and a potential dropout rate of 10%. On the basis of recruitment rates,15 the initial sample size of 1238 patients was revised to 1022 patients on September 16, 2012 (with the approval of the data and safety monitoring board). The revised sample size provided a power of at least 80% to detect a between-group difference of 5.5% at a type I error rate of 0.04 among all patients and a 9.0% difference at a type I error rate of 0.01 among patients in the coprimary analysis.


Patients, Genotyping, and Follow-up

A total of 1015 patients were enrolled and randomly assigned to either the genotype-guided dosing algorithm or the clinically guided dosing algorithm (Fig.S1 in the Supplementary Appendix). There were no significant between-group differences at baseline (Table 1Table 1Characteristics of the Patients at Baseline.). The characteristics of the patients according to self-reported race are provided in Table S1 in the Supplementary Appendix. A total of 60 participants (30 in each group) withdrew before completing the intervention period and did not have an available percentage of time in the therapeutic range, resulting in an analytic sample size of 955. A median of six INRs were measured during the first 4 weeks in each of the two study groups. Dispensed doses during the intervention period are summarized in Table S2 in the Supplementary Appendix.
Genotype data were available in the genotype-guided group for 45% of the patients before the first warfarin dose, for 94% before the second warfarin dose, and for 99% before the application of the dose-revision algorithm on day 4 or 5. The mean (±SD) difference between the dose calculated for patients without genotype data on day 1, as compared with the dose they would have received if genotype data had been available, was −0.1±0.4 mg per day during the first 3 days. The central laboratory confirmed 99.8% of all genotyping results from the clinical centers. All genotype distributions were in Hardy–Weinberg equilibrium (P>0.20 for all comparisons).

Primary Outcome

At 4 weeks, there was no significant between-group difference in the mean percentage of time in the therapeutic range: 45.2% in the genotype-guided group and 45.4% in the clinically guided group (adjusted mean difference [genotype-guided group minus clinically guided group], −0.2%; 95% confidence interval, −3.4 to 3.1; P=0.91) (Table 2Table 2Percentage of Time in the Therapeutic INR Range through Week 4 of Therapy, According to Subgroup. and Figure 1Figure 1Distribution of Time in the Therapeutic Range.). There was also no significant between-group difference in the percentage of time in the therapeutic range among patients in the coprimary analysis (Table 2). When the 4-week trial was divided into two 2-week intervals, there was also no significant difference between the groups in either interval (Table 2).
However, there was a significant interaction between race and dosing strategy (P=0.003) (Table 2). Among black patients, the mean percentage of time in the therapeutic range was less in the genotype-guided group than in the clinically guided group (35.2% vs. 43.5%; adjusted mean difference, −8.3%; P=0.01). Among nonblack patients, the mean percentage of time in the therapeutic range was slightly higher in the genotype-guided group than in the clinically guided group (48.8% vs. 46.1%; adjusted mean difference, 2.8%; P=0.15). There were no significant differences in the percentage of time in the therapeutic range according to sex or the total number of genetic variants (Table 2).

Anticoagulation Control and Dose Prediction

There were no significant between-group differences in the mean percentage of time above the therapeutic range (INR, >3) or below the therapeutic range (INR, <2) (Figure 2Figure 2Range of INRs during the 4-Week Study., and Table S3 in the Supplementary Appendix). However, black patients in the genotype-guided group were more likely to have INRs above the therapeutic range than were those in the clinically guided group (Fig. S2 and Table S3 in the Supplementary Appendix).
There was no overall between-group difference in the time to the first INR in the therapeutic range (Table S4 in the Supplementary Appendix). However, black patients in the genotype-guided group took longer on average to reach the first therapeutic INR than did those in the clinically guided group (Table S4 and Fig. S3 in the Supplementary Appendix). The time to the determination of the maintenance dose did not differ significantly between the two groups overall or according to the primary subgroup, race, or total number of genetic variants (Table S5 in the Supplementary Appendix).
The performance characteristics of the dosing algorithms with respect to the maintenance dose that was determined are shown in Table S6 (which includes the accuracy of a hypothetical, empirical dosing strategy of 5 mg per day) and in Fig. S4, both in the Supplementary Appendix. The genotype-guided algorithms performed better at predicting the maintenance dose among nonblack patients than among black patients. Dose overrides during the first 4 weeks were rare, occurring in only 3.9% of doses in the genotype-guided group and 3.6% of those in the clinically guided group; rates of overrides did not differ according to race.

Adverse Events

At 4 weeks, there were no significant between-group differences in the principal secondary outcome (the time to any INR of ≥4, major bleeding, or thromboembolism) or any other adverse events (Table 3Table 3Adverse Events through Day 28 of Warfarin Therapy., and Table S7 in the Supplementary Appendix). Safety data for the entire duration of follow-up (i.e., past the primary outcome duration) are provided in Table S8 in the Supplementary Appendix.


In our study, we found no benefit of genotype-guided dosing of warfarin with respect to the primary outcome of the percentage of time in the therapeutic INR range, either overall or among patients with a predicted dose difference between the genotype-guided algorithm and the clinically guided algorithm of at least 1 mg per day. Our findings exclude a meaningful effect of genotype-guided dosing on the percentage of time in the therapeutic range during the first month of warfarin treatment. However, there was a significant difference in the effects of the algorithms in the prespecified subgroup of black patients, as compared with nonblack patients. Although the interaction between race and dosing strategy with respect to the primary outcome could be due to chance, the analysis was prespecified and was consistent with our a priori hypothesis that there would be race-based differences.
The dosing algorithms that we used in the trial have been validated and account for race (specifically black vs. nonblack).11-13,18 The genotype-guided algorithm performed as well as anticipated on the basis of previous studies,5,8,10-12,18,23 with an R2 of 0.48 and a mean absolute error of 1.3 mg per day for the dose-initiation algorithm and an R2 of 0.69 and a mean absolute error of 1.0 mg per day for the dose-revision algorithm. Despite this accuracy in predicting maintenance doses, there was no benefit of genotype-guided dosing with respect to anticoagulation control.
Observational studies have shown an association between the use of genetic algorithms and improved outcomes, but because of limitations in the study design, they were unable to assess whether the observed associations were causal.1,9,10 Previous clinical trials have produced equivocal results,5-8 but these trials were limited by a small size and lack of blinding to the warfarin dose. The two trials that suggested possible benefit also were limited by large numbers of dropouts6 and a comparison with nonalgorithm-based dosing.8 Previous studies also enrolled either no black patients6-8 or a minimal number of black patients5 (a total of 3) (Anderson J: personal communication).
The average percentage of time in the therapeutic range of 45% in our study is similar to that in other trials, taking into account the range of INRs used for the calculation and the timing and duration of therapy (Tables S9A and S9B in the Supplementary Appendix).5,10,24,25 Unlike previous trials that used only a baseline genotype-guided algorithm, our study used both a dose-initiation and a dose-revision algorithm. A recent study comparing a similar initiation algorithm with a combined initiation and revision algorithm showed no effect on the percentage of time in the therapeutic range with the addition of the revision algorithm.10
There are several questions that our study was not designed to answer. First, the trial did not compare genotype-based dosing with usual care or a fixed initial dose (e.g., 5 mg per day). However, such a comparison could not have discerned whether differences in outcomes were due to the marginal benefit of genetic information or to the use of the clinical information that is included in all genotype-guided dosing algorithms. Second, our study does not address the question of whether a longer duration of genotype-guided dosing would have improved INR control,26 an issue that is being addressed in another trial.27 Third, the dosing algorithms that we used included the three single-nucleotide polymorphisms among the two genes that are most likely to influence warfarin dosing. Although other genes may contribute to warfarin dosing, it is unlikely that they have a substantial effect, particularly in white populations.28 Fourth, although there were no significant between-group differences in the rates of bleeding or thromboembolic events during the primary follow-up period of 4 weeks, the trial was not powered for these outcomes. Fifth, the first dose of warfarin was not informed by genotyping in 55% of the patients; whether this influenced the results is unknown. However, the effect of missing genetics data on day 1 on the dose administered during the first 3 days of therapy was trivial.
In conclusion, our findings do not support the hypothesis that initiating warfarin therapy at a genotype-guided maintenance dose for the first 5 days, as compared with initiating warfarin at a clinically predicted maintenance dose, improves anticoagulation control during the first 4 weeks of therapy. Our results emphasize the importance of performing randomized trials for pharmacogenetics, particularly for complex regimens such as warfarin.


1 Ginsburg GS, Voora D. The long and winding road to warfarin pharmacogenetic testing. J Am Coll Cardiol 2010;55:2813-2815
2 Burke W, Laberge AM, Press N. Debating clinical utility. Public Health Genomics 2010;13:215-223
3 Ashley EA, Hershberger RE, Caleshu C, et al. Genetics and cardiovascular disease: a policy statement from the American Heart Association. Circulation 2012;126:142-157
4 Woodcock J, Lesko LJ. Pharmacogenetics — tailoring treatment for the outliers. N Engl J Med 2009;360:811-813
5 Anderson JL, Horne BD, Stevens SM, et al. Randomized trial of genotype-guided versus standard warfarin dosing in patients initiating oral anticoagulation. Circulation 2007;116:2563-2570
6 Caraco Y, Blotnick S, Muszkat M. CYP2C9 genotype-guided warfarin prescribing enhances the efficacy and safety of anticoagulation: a prospective randomized controlled study. Clin Pharmacol Ther 2008;83:460-470
A complete list of investigators and committees in the Clarification of Optimal Anticoagulation through Genetics (COAG) trial is provided in the Supplementary Appendix, available at


Do Pharmacogenetics Have a Role in the Dosing of Vitamin K Antagonists?

Bruce Furie, M.D.
Nov 19, 2013



Vitamin K plays a single role in human biology — as a cofactor for the synthesis of γ-carboxyglutamic acid. This amino acid is a component of at least 14 proteins, including 4 blood-coagulation proteins (factor IX, factor VII, factor X, and prothrombin) and 2 regulatory proteins (protein C and protein S), and it is critical for the physiologic function of these proteins. Humans do not synthesize vitamin K. Rather, we ingest it in our diet. The vitamin K quinone is reduced to the semiquinone, and this reduced vitamin K is a cofactor that is required for the conversion of specific glutamic-acid residues on the vitamin K–dependent proteins to γ-carboxyglutamic acid by the vitamin K–dependent carboxylase. Vitamin K epoxide, a product of this reaction, is converted back to the vitamin K quinone by the vitamin K epoxide reductase, otherwise known as VKOR. This vitamin K cycle can be broken, and a state of vitamin K deficiency at the carboxylase level effected, by the inhibition of VKOR by vitamin K antagonists, including warfarin.
Warfarin and its analogues have been used as oral anticoagulant agents for more than 50 years. By targeting VKOR, the post-translational modification of the vitamin K–dependent blood-coagulation proteins is impaired. A reduced functional level of factor IX, factor VII, factor X, and prothrombin leads to delayed blood coagulation. This inhibition is monitored in the clinical laboratory with the use of the prothrombin time and is corrected for the varied potencies of tissue factor used in the assay by means of a calibration factor, yielding the international normalized ratio (INR). The intensity of therapy with vitamin K antagonists varies according to the indication for anticoagulation, and the INR is adjusted by varying the dose of the vitamin K antagonist.
The goal of therapy is to keep the INR in the therapeutic range, since patients with an INR that is subtherapeutic are at increased risk for thrombosis and patients with an INR that is supratherapeutic are at increased risk for bleeding. Keeping the INR within the therapeutic range can be challenging. Warfarin binds to albumin, and only about 3% is free and pharmacologically active. A number of medications can displace warfarin, leading to its increased activity and subsequent increased rate of degradation. Diet, specifically the intake of foods containing vitamin K, can offset the effect of the daily dose of the vitamin K antagonist. Age, weight, and sex are other factors that influence the dose.
In addition, the catabolic rate of the vitamin K antagonists appears to have a genetic basis. Genetic polymorphisms in the cytochrome P-450 enzyme CYP2C9 include two variants, C144R in CYP2C9*2 and I359L in CYP2C9*3. These variants have substantially reduced activity, as compared with CYP2C9*1, and are associated with reduced clearance and thus a decrease in the warfarin-dose requirement.1 Similarly, mutations in VKOR, the target of the vitamin K antagonists, lead to various degrees of warfarin resistance. Polymorphisms in VKORC1, the gene encoding this protein, lead to variability in the sensitivity to vitamin K antagonists.2
Might genotyping of CYP2C9 and VKORC1 in patients initiating anticoagulant therapy with vitamin K antagonists lead to more precise dosing and, by extrapolation, reduce the risk of thrombotic and bleeding complications? Numerous anecdotal, observational, and small clinical trials have been published on the use of this information, with many authorities promoting this approach.
The results of three large, randomized clinical trials that test this hypothesis have now been published in the Journal.3-5 Although they vary in organization and structure (duration of study, vitamin K antagonist used, double-blind vs. single-blind design, racial characteristics of the study group, and method for dosing in the control group), they are also similar (large, multicenter, randomized studies; primary end point of the time in the therapeutic range; genotyping of CYP2C9 and VKORC1; and the use of the INR target as a biomarker for the risk of bleeding and thrombosis). Importantly, these trials all examine the initiation of therapy with vitamin K antagonists and use as a primary end point the percentage of time that a patient is within the therapeutic range during the initial phase of treatment. The more important end point, the rate of bleeding and thrombotic complications, was beyond the power design of these trials.
Despite design differences, the conclusions of the three studies are similar. In an initial period of 4 weeks of anticoagulation with warfarin, the randomized, double-blind study by Kimmel et al.3 showed results in the study group that included pharmacogenetic information to supplement clinically guided dosing that were nearly identical to the results in the group that used clinically guided dosing alone (percentage of time in the therapeutic INR range, 45.2% vs. 45.4%). In the 12 weeks after the initiation of anticoagulation with acenocoumarol and phenprocoumon, Verhoef et al.4 used a new point-of-care device and found that a genotype-guided algorithm that included clinical variables yielded results that were similar to those achieved with an algorithm based on clinical variables (61.6% vs. 60.2% at 12 weeks). Pirmohamed et al.5 compared genotype-guided dosing of warfarin, also using a point-of-care device, with standard dosing methods used in clinical practice. The results at 12 weeks were 67.4% and 60.3%, which are significantly different albeit similar, indicating a modest improvement.
What can we conclude from these trials? First, we must recall that these trials address the process of the initiation of anticoagulant therapy — during the very first week — and not an approach to intermediate or long-term anticoagulation. Second, it would appear that, despite the variation in trial design, these trials indicate that this pharmacogenetic testing has either no usefulness in the initial dosing of vitamin K antagonists or, at best, marginal usefulness, given the cost and effort required to perform this testing.
Improved safety with the use of vitamin K antagonists is nonetheless an important goal, and it remains so, despite the introduction of new oral anticoagulants. Perhaps we should concentrate on improvements in the infrastructure for INR testing, including better communication among the laboratory, the physician, and the patient (e.g., through social media); in the use of formal algorithms for dosing, without concern for genotype; in patient adherence to therapy and possibly more responsibility for dosing being assumed by the patient; and in increased diligence by medical and paramedical personnel in testing, monitoring, and dosing on the basis of the INR, given the high percentage of medical mismanagement associated with these anticoagulant agents.


1 Aithal GP, Day CP, Kesteven PJ, Daly AK. Association of polymorphisms in the cytochrome P450 CYP2C9 with warfarin dose requirement and risk of bleeding complications. Lancet 1999;353:717-719
2 D’Andrea G, D’Ambrosio RL, Di Perna P, et al. A polymorphism in the VKORC1 gene is associated with an interindividual variability in the dose-anticoagulant effect of warfarin. Blood 2005;105:645-649
3 Kimmel SE, French B, Kasner SE, et al. A pharmacogenetic versus a clinical algorithm for warfarin dosing. N Engl J Med 2013. DOI: 10.1056/NEJMoa1310669.
4 Verhoef TI, Ragia G, de Boer A, et al. A randomized trial of genotype-guided dosing of acenocoumarol and phenprocoumon. N Engl J Med 2013. DOI: 10.1056/NEJMoa1311388.
5 Pirmohamed M, Burnside G, Eriksson N, et al. A randomized trial of genotype-guided dosing of warfarin. N Engl J Med 2013. DOI: 10.1056/NEJMoa1311386.

Rapid Warfarin Reversal With 4-Factor Prothrombin Complex Concentrate

Samuel Z. Goldhaber, MD    Nov 07, 2013     Clotblog at

Hello. This is Dr. Sam Goldhaber from the Clotblog at, speaking to you from Brigham and Women’s Hospital and Harvard Medical School on the important topic of 4-factor prothrombin complex concentrate (4F-PCC), which is the optimal approach to urgent warfarin reversal.
The US Food and Drug Administration only recently approved 4F-PCC to reverse excessive bleeding from warfarin. We have had 3-factor PCC around for a long time but it doesn’t have much factor XII in it. Our European colleagues have had 4F-PCC for the past few years.
I was very pleased recently to have had the opportunity to use 4F-PCC to reverse an intracranial hemorrhage in a patient who had an international normalized ratio (INR) of 2.8 and a spontaneous head bleed. She was receiving warfarin for anticoagulation, and within about 15 minutes the 4F-PCC, along with 10 mg of intravenous vitamin K, returned her INR to normal. Of greater importance, the head bleeding stopped and she regained virtually all of her neurologic function. It seemed miraculous to me and it happened so quickly that it was very gratifying.
Almost simultaneously with my clinical experience, we published an observational study in Circulation [1] with about 300 patients who bled on warfarin. Approximately 80% of these patients were on warfarin because of permanent atrial fibrillation, and they had an average age in the 70s. Initially patients received fresh frozen plasma (before 4F-PCC became available in Canada) and their outcomes with fresh frozen plasma were tracked very carefully. When 4F-PCC came along and practice switched to that therapy, those outcomes were tracked as well.

Clopidogrel Genotyping for Antiplatelet Guidance in MI Stenting: Maybe Reduced Ischemic Risk

Steve Stiles    Nov 06, 2013

SAN FRANCISCO, CA — A novel study of genotype-guided antiplatelet therapy in patients who received a stent for acute MI saw a sharp drop in ischemic events over one year among those who tested positive for a clopidogrel loss-of-function (LOF) gene pattern and had their originally prescribed antiplatelet therapy altered based on the assay results.
In the prospective GIANT trial with 1445 patients, reported here at TCT 2013 , it was discretionary whether clinicians raised the clopidogrel dosage or switched thienopyridine agents based on the assay results, which they had in hand within 48 hours after stenting. Such changes were made in 86% of the 316 who tested positive for the LOF genotype, a group known to be at increased ischemic risk on standard clopidogrel-containing antiplatelet therapy after stenting.
Among those 272 patients with assay-guided antiplatelet changes, the one-year composite risk of death, MI, or stent thrombosis closely matched that of patients lacking the high-risk genotype, according to co–principal investigator Dr Bernard R Chevalier (L’Institut CardioVasculaire Paris-Sud, Massy, France), who presented the study.
Dr Bernard R Chevalier
Of note, the composite end point was about five times higher for the remaining 14% of LOF-genotype patients whose antiplatelet therapy wasn’t changed based the assay.
“These are really the first clinical-trial data in the genotype space compared with the phenotype space, and I think it’s long overdue,” according to Dr Daniel I Simon (University Hospitals Case Medical Center, Cleveland, OH), speaking from the panel charged with critiquing GIANT after its formal presentation. As did many throughout TCT 2013, Simon was weighing two different approaches to sharpening thienopyridine selection for dual-agent antiplatelet therapy after coronary interventions, specifically those focusing on genotyping for the CYP2C19 clopidogrel loss-of-function variant vs platelet-function assays like VerifyNow (Accumetrics).
Such platelet-function testing with coronary stenting has its supporters and detractors but hasn’t found a consistent role in managing patients undergoing PCI, even for acute coronary syndromes, as heartwire has long reported.
One-Year Rates (%) of Primary End Point (Death, MI, or Stent Thrombosis) by Clopidogrel LOF Genotype Status
End point Normal
LOF, treatment is adjusted LOF, treatment is not adjusted
                              n=1118           n=272                     n=55
Primary              3.04                 3.3*                        15.6
 *p=0.83 vs normal; p<0.0001 for noninferiority; p=0.04 vs LOF-treatment-is-not-adjusted
“I think these are amazing results,” Dr Cindy L Grines (Detroit Medical Center Cardiovascular Institute, MI) said at a press briefing on GIANT, referring to both the high event rate in LOF-genotype patients whose treatment wasn’t changed and similarly lower event rates in the other two groups. “Both of those [findings] are a little bit unexpected, I’d guess?” She asked Chevalier why clinicians did not modify antiplatelet therapy in 14% of patients positive for the LOF genotype.
In GIANT, said Chevalier in his presentation, investigators were “strongly recommended” to give such patients prasugrel (Effient, Lilly/Daiichi-Sanyo) or, if they had contraindications to prasugrel, to double their clopidogrel dosage.
But prasugrel, a more potent antiplatelet than clopidogrel, had already been chosen for initial antiplatelet therapy in more than half of patients in the study. Perhaps clinicians believed such patients would benefit from it regardless of their ultimate genotype status. Indeed, some patients later found not to have the clopidogrel LOF genotype were switched from prasugrel to clopidogrel, perhaps satisfied by the assay that the latter drug would be adequate after all.
Chevalier speculated that clinicians also may not have boosted antiplatelet therapy in some LOF-genotype patients if it was considered too risky, such as for those with bleeding risk factors. The high event rate in patients with the LOF genotype whose antiplatelet therapy wasn’t adjusted, therefore, may be more related to how sick the patient was, rather than any cues from genotyping. He said his group is currently looking for the answer in further analyses.
Prevalence of Thienopyridine Use and Dosages, Before and After Genotyping, by Assay Outcome
Thienopyridine and dosage by timing
n=1118                                     treatment is  adjusted,   (%)                 n=272 (%)
                                                                           Normal,     LOF          p
Clopidogrel 75 mg/d (preassay)           35.6           34.7      NS
Clopidogrel 75 mg/d (postassay)        44.5             0       <0.001
Clopidogrel 150 mg/d (preassay)        10               9.1         NS
Clopidogrel 150 mg/d (postassay)    8.9             16.8   <0.05
Prasugrel 10 mg/d (preassay)            53.3           55.5       NS
Prasugrel 10 mg/d (postassay)         46.1            83.1    <0.001
GIANT was funded by Biotronik. Chevalier discloses consulting for or receiving research grants or speaker fees from Abbott Vascular, Asahi, Astra Zeneca, AVI, Boston Scientific, Biotronik, Colibri, Cook, Cordis, Daiichi Sankyo, Eli-Lilly, Iroko, Medtronic, and Terumo and being general director of and owning equity interest in the European Cardiovascular Research Center.

 Bivalirudin Started during Emergency Transport for Primary PCI

Philippe Gabriel Steg, M.D., Arnoud van ‘t Hof, M.D., Ph.D., Christian W. Hamm, M.D., Peter Clemmensen, M.D., Ph.D., Frédéric Lapostolle, M.D., Ph.D., Pierre Coste, M.D., Jurrien Ten Berg, M.D., Ph.D., Pierre Van Grunsven, M.D., Gerrit Jan Eggink, M.D., Lutz Nibbe, M.D., Uwe Zeymer, M.D., Marco Campo dell’ Orto, M.D., Holger Nef, M.D., Jacob Steinmetz, M.D., Ph.D., Louis Soulat, M.D., Kurt Huber, M.D., Efthymios N. Deliargyris, M.D., Debra Bernstein, Ph.D., Diana Schuette, Ph.D., Jayne Prats, Ph.D., Tim Clayton, M.Sc., Stuart Pocock, Ph.D., Martial Hamon, M.D., and Patrick Goldstein, M.D. for the EUROMAX Investigators

N Engl J Med 2013; 369:2207-2217December 5, 2013DOI: 10.1056/NEJMoa1311096



Bivalirudin, as compared with heparin and glycoprotein IIb/IIIa inhibitors, has been shown to reduce rates of bleeding and death in patients undergoing primary percutaneous coronary intervention (PCI). Whether these benefits persist in contemporary practice characterized by prehospital initiation of treatment, optional use of glycoprotein IIb/IIIa inhibitors and novel P2Y12 inhibitors, and radial-artery PCI access use is unknown.



We randomly assigned 2218 patients with ST-segment elevation myocardial infarction (STEMI) who were being transported for primary PCI to receive either bivalirudin or unfractionated or low-molecular-weight heparin with optional glycoprotein IIb/IIIa inhibitors (control group). The primary outcome at 30 days was a composite of death or major bleeding not associated with coronary-artery bypass grafting (CABG), and the principal secondary outcome was a composite of death, reinfarction, or non-CABG major bleeding.


Bivalirudin, as compared with the control intervention, reduced the risk of the primary outcome (5.1% vs. 8.5%; relative risk, 0.60; 95% confidence interval [CI], 0.43 to 0.82; P=0.001) and the principal secondary outcome (6.6% vs. 9.2%; relative risk, 0.72; 95% CI, 0.54 to 0.96; P=0.02). Bivalirudin also reduced the risk of major bleeding (2.6% vs. 6.0%; relative risk, 0.43; 95% CI, 0.28 to 0.66; P<0.001). The risk of acute stent thrombosis was higher with bivalirudin (1.1% vs. 0.2%; relative risk, 6.11; 95% CI, 1.37 to 27.24; P=0.007). There was no significant difference in rates of death (2.9% vs. 3.1%) or reinfarction (1.7% vs. 0.9%). Results were consistent across subgroups of patients.


Bivalirudin, started during transport for primary PCI, improved 30-day clinical outcomes with a reduction in major bleeding but with an increase in acute stent thrombosis. (Funded by the Medicines Company; EUROMAX number, NCT01087723.)

  Source Information

The authors’ affiliations are listed in the Appendix.
Dr. Steg at Cardiologie, Département Hospitalo-Universitaire FIRE, Hôpital Bichat, Assistance Publique–Hôpitaux de Paris,  Paris, France, or at
A complete list of the European Ambulance Acute Coronary Syndrome Angiography (EUROMAX) investigators is provided in the Supplementary Appendix, available at


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Platelets in Translational Research – Part 2

Subtitle: Discovery of Potential Anti-platelet Targets

Reviewer and Curator: Larry H. Bernstein, MD, FCAP 


This presentation is the the second of a series on Platelets in Translational Medicine: Part I:  Platelet structure, interactions between platelets and endothelium, and intracellular transcription

Part II: Discovery of Potential Anti-platelet Targets

Endothelium-dependent vasodilator effects of platelet activating factor on rat resistance vessels

1Katsuo Kamata, Tatsuya Mori, *Koki Shigenobu & Yutaka Kasuya Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo and *Department of Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba, Jp Br. J. Pharmacol. (1989), 98, 1360-1364 To elucidate the mechanisms of the powerful and long-lasting hypotension produced by platelet activating factor (PAF), its effects on perfusion pressure in the perfused mesenteric arterial bed of the rat were examined. 2 Infusion of PAF (10-11 to 3 x 10-10M; EC50 = 4.0 x 10′ m; 95%CL = 1.6 x 10-11 — 9.4 x 10-11 M) and acetylcholine (ACh) (10′ to 10-6m; EC50 = 3.0 ± 0.1 x 10-9m) produced marked concentration-dependent vasodilatations which were significantly inhibited by treatment with detergents (0.1% Triton X-100 for 30 s or 0.3% CHAPS for 90 s). 3 Pretreatment with CV-6209, a PAF antagonist, inhibited PAF- but not ACh-induced vasodila­tation. 4 Treatment with indomethacin (10-6m) had no effect on PAF- or ACh-induced vasodilatation. 5


These results demonstrate that extremely low concentrations of PAF produce vasodilatation of resistance vessels through the release of endothelium-derived relaxing factor (EDRF). This may account for the strong hypotension produced by PAF in vivo. Platelet activating factor (PAF, acetyl glyceryl ether phosphorylcholine) has been shown to produce strong and long-lasting hypotension in various animal species, e.g. normotensive and spontaneously hypertensive rats, rabbits, guinea-pigs, and dogs (Tanaka et al., 1983). This action of PAF is thought to be endothelium-dependent (Kamitani et al., 1984; Kasuya et al., 1984a,b; Shigenobu et al., 1985; 1987). In a previous study (Shigenobu et al., 1987), we found that relatively low concentrations of PAF (10-9-10-7m) produced endothelium-dependent relaxation of the rat aorta in the presence of bovine serum albumin. This vasodilator action of PAF at low concentrations might be the cause of its hypo­tensive action in vivo. While the aorta will offer a resistance to flow, it is obvious that the contribution of vessels of smaller diameter to peripheral vascular resistance is much greater. In this regard, the mesen­teric circulation of the rat receives approximately one-fifth of the cardiac output (Nichols et al., 1985) and, thus, regulation of this bed may make a signifi­cant contribution towards systemic blood pressure and circulating blood volume.  Therefore, we examined the effect of PAF on the resistance vessels of the rat mesenteric vascular bed and found that extremely low concentrations (10 -11 to 3 x 10-16 m) can produce endothelium-dependent vasodilatation. Figure 1 Effects of PAF on the perfusion pressure of the methoxamine (10-3N)-constricted mesenteric vascu­lar bed. (a) Upper panel: relaxation induced by PAF (3 x 10-10 M). Lower panel: effects of the PAF-antagonist, CV-6209 (3 x 10-914), on the relaxation induced by PAF (3 x 10“N). (b) Concentration-response curve for the relaxation produced by PAF (10-11 to 3 x 10-10N) in the methoxamine (10-51)-constricted mesenteric vascular bed. Each point is the mean and vertical bars represent the s.e.mean from 5 experiments. Figure 2 Effects of detergents on acetylcholine (ACh)-induced relaxation of the methoxamine (10-5M)-con­stricted mesenteric vascular bed. Concentration-response curves are shown for ACh-induced vasodilatation before (0) and after treatment with 0.3% CHAPS (❑) or 0.1% Triton X-100 (0). Each point is the mean and vertical bars represent the s.e.mean from 5 experiments. Infusions of extremely low concentrations of PAF (10-11 to 3.1 x 10-1° m) produced a marked and long-lasting vasodilatation which was significantly suppressed by treatment with detergents ar bed. Concentration-response curves are shown for ACh-induced vasodilatation before (0) and after treatment with 0.3% CHAPS (❑) or 0.1% Triton X-100 (0). Each point is the mean and vertical bars represent the s.e.mean from 5 experiments. Since Furchgott & Zawadzki (1980) demonstrated the obligatory role of endothelium in vascular relax­ation by ACh, many studies have suggested that endothelium-derived relaxing factor (EDRF) is re­leased from endothelial cells in response to a large number of agonists (Furchgott, 1984). In the present study with perfused resistance vessels, ACh produced vasodilatation in a concentration-dependent manner and the vasorelaxant responses were significantly suppressed by perfusion with detergents such as CHAPS or Triton X-100.  These data strongly suggest the pos­sible involvement of the endothelium in the relax­ation induced by PAF. CV-6209, a PAF antagonist, inhibited PAF-induced but not ACh-induced vasodilatation in a concentration-dependent manner. Specific antago­nism by CV-6209 has already been obtained with respect to PAF-induced hypotension or platelet aggregation (Terashita et al., 1987). An accumulating body of evidence suggests that hypotension resulting from endotoxin challenge is due to the endogenous release of PAF from endothelial cells (Camussi et al., 1983), leukocytes (Demopoules et al., 1979), macro­phages (Mencia-Huerta & Benveniste, 1979; Camussi et al., 1983) and platelets (Chingard et al., 1979). Indeed, PAF antagonists can reverse estab­lished endotoxin-induced hypotension (Terashita et al., 1985; Handley et al., 1985a,b). From the above data and the results of the present study, one pos­sible explanation for endotoxin-induced hypotension may be that the release of PAF occurs, which then binds to its receptors located on the endothelial cells, stimulating production of EDRF. In conclusion, we demonstrated that extremely low concentrations of PAF produce long-lasting vasodilatation in a resistance vessel of the mesenteric vasculature. Moreover, we showed that this PAF-induced vasodilatation is mediated by a vasodilator substance released from endothelial cells (EDRF) which is not a prostaglandin. Since the PAF-induced endothelium-dependent relaxation observed in the present study was elicited at low concentrations and was long-lasting, it may be the main mechanism by which PAF induces hypotension in vivo.

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

Andreas C Eriksson*1, Lena Jonasson2, Tomas L Lindahl3, Bo Hedbäck2 and Per A Whiss1 1Divisions of Drug Research/Pharmacology and 2Cardiology, Department of Medical and Health Sciences, Linköping University, Linköpin, Sw, and 3Department of Clinical Chemistry, University Hospital, Linköping, Sw Journal of Translational Medicine 2009, 7:42     http:/


Background: Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.

Methods: With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.

Results: The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5′-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone. Conclusion: The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB2 for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment. The benefits of ASA have been clearly demonstrated by the Anti-platelet Trialists’ Collaboration. They found that ASA therapy reduces the risk by 25% of myocardial infarction, stroke or vascular death in “high-risk” patients. When using the same outcomes as the Anti-platelet Trialists’ Collaboration on a comparable set of “high-risk” patients, the CAPRIE-study showed a slight benefit of clopidogrel over ASA. Furthermore, the combination of clopidogrel and ASA has been shown to be more effective than ASA alone for preventing vascu­lar events in patients with unstable angina and myo­cardial infarction as well as in patients undergoing percutaneous coronary intervention (PCI). Despite the obvious benefits from anti-platelet therapy in coro­nary disease, low response to clopidogrel has been described by several investigators. A lot of attention has also been drawn towards low response to ASA, often called “ASA resistance”. The concept of ASA resistance is complicated for several reasons. First of all, different stud­ies have defined ASA resistance in different ways. In its broadest sense, ASA resistance can be defined either as the inability of ASA to inhibit platelets in one or more platelet function tests (laboratory resistance) or as the inability of ASA to prevent recurrent thrombosis (i.e. treatment fail­ure, here denoted clinical resistance). The lack of a general definition of ASA resistance results in difficulties when trying to measure the prevalence of this phenome­non. Estimates of laboratory resistance range from approximately 5 to 60% depending on the assay used, the patients studied and the way of defining ASA resistance. Likewise, lack of a standardized definition of low response to clopidogrel makes it difficult to estimate the prevalence of this phenomenon as well. The principles of existing platelet assays, as well as their advantages and disadvantages, have been described elsewhere. In short, assays potentially useful for monitoring treatment effects include those commonly used in research such as platelet aggregometry and flow cytometry as well as immunoassays for measuring metabolites of thromboxane A2 (TXA2). Also, the PFA-100TM, MultiplateTM and the VerifyNowTM are examples of instruments commercially developed for evaluation of anti-platelet therapy. How­ever, no studies have investigated the usefulness of alter­ing treatment based on laboratory findings of ASA resistance. Regarding clopidogrel, there are recent studies showing that adjustment of clopidogrel loading doses according to vasodilator-stimulated phosphoprotein phosphorylation index measured utilising flow cytometry decrease major adverse cardiovascular events in patients with clopidogrel resistance. Static adhesion is an aspect of platelet function that has not been investigated in earlier studies of the effects of platelet inhibiting drugs. Consequently, static platelet adhesion is not measured by any of the current candidate assays for clinical evaluation of platelet function. The static platelet adhesion assay offers an opportunity for simultaneous measurements of the combined effects of several different platelet activators on platelet function. In this study, platelet adhesion to albumin, collagen and fibrinogen was investigated in the presence of soluble platelet activators including adenosine 5′-diphosphate (ADP), adrenaline, lysophosphatidic acid (LPA) and ris-tocetin. Collagen, fibrinogen, ADP and adrenaline are physiological agents that are well-known for their interac­tions with platelets. Ristocetin is a compound derived from bacteria that facilitates the interaction between von Willebrand factor (vWf) and glycoprotein (GP)-Ib-IX-V on platelets, which otherwise occurs only at flow condi­tions. The static nature of the assay therefore prompted us to include ristocetin in order to get a rough estimate on GPIb-IX-V dependent events. LPA is a phospholipid that is produced and released by activated platelets and that also can be generated through mild oxi­dation of LDL. It was included in the present study since it is present in atherosclerotic vessels and suggested to be important for platelet activation after plaque rup­ture. Finally, albumin was included as a surface since the platelet activating effect of LPA can be detected when measuring adhesion to such a surface. Thus, by the use of different platelet activators, several measures of platelet adhesion were obtained simultaneously This means that the possibilities to screen for conditions potentially important for detecting effects of platelet-inhibiting drugs far exceeds the screening abilities of other platelet function tests. Consequently, the static platelet adhesion assay is very well suited for development into a clinically useful device for monitoring platelet inhibiting treatment. Also, it has earlier been proposed that investi­gating the combined effects of two activators on platelet activity might be necessary in order to detect effects of ASA and other antiplatelet agents [26]. This is a criterion that can easily be met by the static platelet adhesion assay. Through the screening procedure we found different con­ditions where the static adhesion was influenced by the drug given.

The inclusion of patients and controls. Patients and controls were included consecutively. Blood samples from controls were drawn at two different occasions separated by 2–5.5 months. All patients entering the study received ASA combined with clopidogrel and blood sampling was performed 1.5–6.5 months after initiating the treatment. This was followed by a randomised cross-over enabling all patients to receive monotherapy with both ASA and clopidogrel. The patients received monotherapy for at least 3 weeks and for a maximum of 4.5 months before performing blood sampling. A total of 33 patients and 30 controls entered the study. In the end, 29 patients and 29 controls completed the study. Blood was drawn from patients at three different occa­sions (Figure 1). The first sample was drawn after all patients had received combined treatment with ASA (75 mg/day) and clopidogrel (75 mg/day) for 1.5–6.5 months after the index event. The study then used a randomised cross-over design meaning that half of the patients received ASA as monotherapy while half received only clopidogrel (75 mg/day for both monotherapies). The monotherapy was then switched for every patient so that all patients in total received all three therapies. Samples for evaluation of the monotherapies were drawn after therapy for at least 3 weeks and at the most for 4.5 months. Most of the differences in treatment length can be ascribed to the fact that the national recommendations for treatment in this patient group were changed during the course of the study. The allocation to monotherapy was blinded for the laboratory personnel. In general, the use of three different treatments for intra-individual com­parisons in a cross-over design is different from previous studies on ASA and clopidogrel, which have mainly been concerned with only two treatment alternatives.

Intra-individual variation in healthy controls

Measurements of platelet adhesion and serum TXB2-levels were performed on healthy controls on two separate occa­sions (2–5.5 months interval) in order to investigate the presence of intraindividual variation in platelet reactivity and clotting-induced TXB2-production. The standardised Z-scores from the simplified factors were used for analysis by Repeated Measures ANOVA of the data from the healthy controls. We found significantly decreased plate­let adhesion at the second compared to the first visit for ADP-induced adhesion (Factor 1, p = 0.012) and for adhe­sion to fibrinogen (Factor 5, p = 0.012). This intra-indi-vidual variability over time makes it difficult to draw any conclusions regarding effects of anti-platelet treatment. We therefore further analysed the individual variables constituting Factors 1 and 5 with Repeated Measures ANOVA in order to distinguish the variables that varied significantly over time. Variables being significantly dif­ferent between visit 1 and visit 2 were then excluded and a new Repeated Measures ANOVA was performed on the new factors. After this modification, none of the factors corresponding to adhesion showed variation over time and these factors were then used for analysis on patients. Serum levels of TXB2, which constituted a separate factor, varied significantly in healthy controls at two separate occasions (Figure 2). flow chart of patients and controls_Image_1 Effect of platelet inhibiting treatment on serum TXB2-levels (Factor 13). Serum TXB2-levels (Factor 13) for patients (n = 29) and healthy controls (n = 29) are presented as mean + SEM. ASA alone or in combination with clopidogrel was signif­icantly different from clopidogrel alone and compared to the mean of the controls (p < 0.001). Also, the difference between controls at visit 1 and visit 2 was significant. ***p < 0.001, ns = not significant. When investigating possible effects of platelet-inhibiting treatment with Repeated Measures ANOVA, significant effects were seen for four of the factors corresponding to platelet adhesion. The factors that were not able to detect significant treatment effects were adrenaline-induced adhesion (Factor 3), ristocetin-induced adhesion (Factor 4) and adhesion to fibrinogen (Factor 5). Regarding adhe­sion factors detecting treatment effects, ADP-induced adhesion (Factor 1, Figure 3A inset) was significantly decreased by clopidogrel alone or by clopidogrel plus ASA compared with ASA alone. Surprisingly, platelet adhesion induced by ADP was lower for the monotherapy with clopidogrel compared to dual therapy. ADP-induced adhesion to albumin is shown as a representative example of the variables of Factor 1 (Figure 3A). Ristocetin-induced adhesion to albumin (Factor 6, Figure 3B inset) was signif­icantly decreased by clopidogrel alone compared with ASA alone. This difference was also seen for ristocetin combined with LPA, which is shown as an example of a variable belonging to Factor 6 (Figure 3B). In Factor 7 (Figure 3C inset), corresponding to LPA-induced adhe­sion to albumin, we found clopidogrel to decrease adhe­sion compared with ASA and compared with ASA plus clopidogrel. These differences were reflected by the com­bined activation through LPA and adrenaline, which was a variable included in Factor 7 (Figure 3C). Finally, adhe­sion to collagen (Factor 8, Figure 3D) was significantly decreased by dual therapy compared with ASA alone or clopidogrel alone. As can be seen from the above descrip­tion, monotherapy with clopidogrel resulted in signifi­cantly decreased adhesion compared to clopidogrel combined with ASA for Factors 1 and 7. This was also observed for the variable shown as a representative exam­ple of Factor 6 (Figure 3B). The two factors corresponding to flow cytometric measurements (Factors 14 and 15, Fig­ure 4) both showed that ASA-treated platelets were more active than platelets treated with clopidogrel alone or clopidogrel plus ASA. Furthermore, serum TXB2-levels (Figure 2) was significantly decreased by ASA alone or by ASA plus clopidogrel compared with clopidogrel alone. Regarding the other measurements not directly measuring platelet function, significant differences were found for Factor 10 including HDL and for platelet count (Factor 12) but neither for the factor corresponding to inflamma­tion (Factor 9) nor for Factor 11 including LDL. Factor 10 including HDL was found to be elevated by both ASA and clopidogrel monotherapies compared with dual therapy (p = 0.003 for ASA, p = 0.019 for clopidogrel, data not shown). Platelet count were found to be increased after dual therapy compared with both monotherapies (p < 0.001, data not shown). flow chart of patients and controls_Image_2 The influence of ASA and clopidogrel on platelet adhesion. The main figures are representative examples of the varia­bles constituting the respective factors. The insets show the Z-scores for each factor. Also shown in the insets are the compar­isons between the control means of visit 1 and 2 and treatment with ASA (A), clopidogrel (C) and the combination of ASA and clopidogrel (A+C). The respective figures show the effect of platelet inhibiting treatment on ADP-induced adhesion (Factor 1, Fig A), ristocetin-induced adhesion to albumin (Factor 6, Fig B), LPA-induced adhesion to albumin (Factor 7, Fig C) and adhe­sion to collagen (Factor 8, Fig D) for patients (n = 29) and healthy controls (n = 29). All values are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. flow chart of patients and controls_Image_4 The influence of ASA and clopidogrel on platelet activity measured by flow cytometry. The effects of platelet inhibiting treatment on platelet activation detected by flow cytometry induced by ADP (Factor 14, Fig A) and SFLLRN (Factor 15, Fig B) on patients (n = 29). The main figures are representative examples of the variables constituting the respective fac­tors. The insets show the Z-scores for each factor. All values are presented as mean + SEM. ***p < 0.001, ns = not significant. Platelets from patients (n = 29) were activated in vitro with adenosine 5′-diphosphate (ADP; 0.1 and 0.6 μmol/L) or SFLLRN (5.3 μmol/L) followed by flow cytometric measurements of fibrinogen-binding or expression of P-selectin. Presented results are the mean-% of fibrinogen-binding and P-selectin expression ± SEM. Reference values (obtained earlier during routine analysis at the accredited Dept. of Clinical Chemistry at the University hospital in Linköping) are shown as mean with reference interval within parenthesis. Stars indicate significant differences for patients compared to reference values. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.  (Table not shown)


With the aim of finding variables sensitive to clopidogrel and ASA-treatment, this study used a screening approach and measured several different variables simultaneously. To reduce the complexity of the material we performed PCA in order to find correlating variables that measured the same property. In this way the 54 measurements of platelet adhesion were reduced to 8 factors. Visual inspec­tion revealed that each factor represented a separate entity of platelet adhesion and the factors could therefore be renamed according to the aspect they measured. We thus conclude that future studies must not involve all 54 adhe­sion variables, but instead, one variable from each factor should be enough to cover 8 different aspects of platelet adhesion. In addition to the adhesion data, the remaining 15 variables also formed distinct factors that were possible to rename according to measured property. It is notable that serum TXB2 formed a distinct group not correlated to any of the other measurements.

It is important that laboratory assays used for clinical pur­poses are reproducible and that they measure parameters that are not confounded by other variables. Some of the measurements performed in this study (clinical chemistry variables and platelet function measured by flow cytome-try) are used for clinical analysis at accredited laboratories at the University hospital in Linköping. However, the reproducibility of the platelet adhesion assay was mostly unknown before this study. Our initial results suggested that the factors corresponding to ADP-induced adhesion and adhesion to fibrinogen were not reproduci­ble. We therefore excluded the most varied variables con­stituting these factors, which resulted in no intra-individual effects for healthy controls in the platelet adhe­sion assay. From this we conclude that many, but not all, measures of platelet adhesion are reproducible. Moreover, the static condition might limit the possibilities for trans­lating the results from the adhesion assay into in vivo platelet adhesion occurring during flow conditions. How­ever, platelet adhesion to collagen and fibrinogen is dependent on α2131– and αIIb133-receptors respectively in the current assay. This suggests that the static platelet adhesion assay can measure important aspects of platelet function despite its simplicity. Furthermore, vWf depend­ent adhesion is not directly covered in the present assay although ristocetin-induced adhesion appears to be dependent on GPIb-IX-V and vWf . From this discussion it is evident that the adhesion assay as well as flow cytometry can measure effects of clopidog-rel when using ADP as activating stimuli. It is also evident that serum-TXB2 levels measure the effects of ASA. How­ever, these measures focus on the primary interaction between the drugs and the platelets, which could be prob­lematic when trying to evaluate the complex in vivo treat­ment effect. It has previously been found that only 12 of 682 ASA-treated patients (≈ 2%) had residual TXB2 serum levels higher than 2 standard deviations from the popula­tion mean. Measurements of the effect of arachidonic acid on platelet aggregometry have also led to the conclu­sion that ASA resistance is a very rare phenomenon. Thus, our study supports these previous findings that assays measuring the pharmacodynamic activity of ASA (to inhibit the COX-enzyme) seldom recognizes patients as ASA-resistant. This suggests that the cause of ASA-resistance is not due to an inability of ASA to act as a COX-inhibitor.

We suggest that direct measurements of ADP and TXA2-effects (in our case ADP-induced activation measured by adhesion or flow cytometry and serum TXB2-levels) must be combined with measures that are only partly dependent on ADP and TXA2 respectively. For instance, an adhesion variable partly dependent on TXA2 might be able to detect ASA resistance caused by increased signalling through other activating pathways. Such a scenario would be character­ized by serum TXB2 values showing normal COX-inhibi­tion while platelet adhesion is increased. This study employed a screening procedure in order to find such indirect measures of the effects of ASA and clopidogrel. Our results show inhibiting effects of clopidogrel com­pared to ASA on adhesion to albumin in the presence of LPA or ristocetin. This was also observed for our flow cytometric measurements with SFLLRN as activator, which confirms that SFLLRN is able to induce release of granule contents in platelets. SFLLRN- and ADP-induced platelet activation, as measured by flow cytometry, was moderately correlated to each other and adhesion induced by LPA as well as ristocetin showed weak correla­tions with ADP-induced adhesion. These results further confirm that these measures of platelet activity are partly dependent on ADP. We have earlier shown that adhesion to albumin induced by simultaneous stimulation by LPA and adrenaline (a variable belonging to the LPA-factor in the present study) can be inhibited by inhibition of ADP-signalling in vitro. This strengthens our conclusion that the effect on LPA-induced adhesion observed for clopidogrel is caused by inhibition of ADP-signalling. Also, the presence of LPA in atherosclerotic plaques and its possible role in thrombus formation after plaque rup­ture makes it especially interesting for the in vivo set­ting of myocardial infarction. Assays of static platelet adhesion that have been used in previous studies aimed at investigating treatment effects of platelet inhibiting drugs. Importantly, this study shows that the static platelet adhesion assay is reproducible over time. We also showed that the static platelet adhesion assay as well as flow cytometry detected the ability of clopidogrel to inhibit platelet activation induced by ADP. Our results further suggest that other measures of platelet adhesion and platelet activation measured by flow cytometry are indirectly dependent on secreted ADP or TXA2. One such measure is adhesion to a collagen surface, which should be more thoroughly investigated for its ability to detect effects of clopidogrel and ASA. Likewise, due to its connection to atherosclerosis and myocardial infarction, the LPA-induced effect should be further evaluated for its ability to detect effects of clopidogrel. In conclusion, the screening procedure undertaken in this study has revealed suggestions on which measures of platelet activity to com­bine in order to evaluate platelet function.

Effect of protein kinase C and phospholipase A2 inhibitors on the impaired ability of human platelets to cause vasodilation

*,1Helgi J. Oskarsson, 1Timothy G. Hofmeyer, 1Lawrence Coppey & 1Mark A. Yorek 1Department of Internal Medicine, University of Iowa and VA Medical Center, Iowa City, IA British Journal of Pharmacology (1999) 127, 903-908

1   The aim of this study was to examine the mechanism of impaired platelet-mediated endothelium-dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium-dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo-oxygenase pathway, the signal transduction pathway mediating this effect is unknown. 2 Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A2 (PLA2) activity on vasodilation in rabbit carotid arteries. 3 We found that two aldose-reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused significant impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA2 activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4 These results suggest that the impairment of platelet-mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA2 activity. Keywords: Glucose; signal-transduction; platelet; vasodilation; diabetes Abbreviations: ADP, adenosine diphosphate; DAG, diacyglycerol; DEDA, dimethyleicosadienoic acid; EDNO, endothelium-derived nitric oxide; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PKC, protein kinase C; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate


Activated normal platelets produce vasodilation via release of platelet-derived adenosine diphosphate (ADP), which in turn stimulates the release of endothelium-derived nitric oxide (EDNO) . EDNO causes vascular smooth muscle relaxation and inhibits platelet aggregation and excessive thrombus formation. Recent reports suggest that platelets from patients with diabetes mellitus lack the ability to produce EDNO-dependent vasodilation. This platelet defect can be reproduced in vitro by exposure of normal human platelets to high glucose concentrations, in a time and concentration dependent manner. This glucose-induced platelet defect appears to involve activation of the cyclo-oxygenase pathway, including thromboxane synthase. However, it remains unknown how exposure of platelets to high concentrations of glucose in vivo or in vitro, leads to increased activity of these enzymes. Previous studies indicate that high glucose concentrations mediate some of their adverse biologic effects via the polyol pathway high glucose increases intracellular diacylglycer-ol (DAG) levels, upregulates protein kinase C (PKC) activity and can lead to increased arachidonic acid release via PKC-mediated increase in phospholipase A2 activity, which in turn increases activity of cyclo-oxygenase. In this study we explore the possible role of these metabolic pathways in mediating the inability of diabetic and hyperglycaemia-induced platelets to produce vasodilation. In this study we show that in vitro incubation of normal human platelets in high glucose causes a significant increase in platelet DAG levels, which is evident after 30 min.

The role of protein kinase-C (PKC)

DAG and OAG are known activators of PKC. Data in Figure 2 show that normal human platelets incubated with the DAG analogue, (OAG), in order to mimic the effect of increased intracellular DAG, lost their ability to cause vasodilation.  Next we tested whether enhanced PKC activity plays a role in the signalling pathway leading to impaired ability of diabetic platelets to cause vasodilation. We found that platelets from patients with diabetes mellitus that were treated with the PKC-inhibitor calphostin-C produced normal vasodilation, while untreated platelets from the same patients lacked the ability to cause vasorelaxation (Figure 3A). Similarly, while normal platelets incubated in high glucose lost their ability to cause vasorelaxation, co-incubation with calphostin-C prevented the glucose-mediated impairment of platelet-mediated vasodila-tion (Figure 3B). Calphostin-C did not affect the ability of normal platelets to mediate vasodilation: 35±3 vs 37±4% increase in vessel diameter, with or without the inhibitor (n=5), respectively. Similar results were obtained with the PKC-inhibitor H7 (50 ILM) (results not shown).  In addition, normal platelets  `primed’ by a 20 min incubation in Tyrode’s buffer containing PMA (80 nM) completely lost their ability to produce vasorelaxation (Figure 4). Figure 3 (A) Platelets were isolated from patients with diabetes mellitus (n=6). Platelets were incubated in Tyrode’s buffer for 2 h with or without calphostin-C (50 nM). Subsequently the platelets were thrombin (0.1 U ml1) activated and perfused through a phenylephrine (10 jIM) preconstricted normal rabbit carotid artery, and the change in vessel diameter measured. *P<0.01. (B) Platelets isolated from healthy donors (n=6) were incubated in Tyrode’s buffer containing either 6.6 mM (118 mg dl1) [NL Plts] or 17 mM (300 mg dl1) [Glucose Plts] glucose for 4 h. For the last 2 h the PKC-inhibitor calphostin-C (50 nM) was added to some of the high glucose treated platelets. Subsequently the three groups of platelets were thrombin (0.1 U ml1) activated and perfused through a phenylephrine (10 jIM) preconstricted normal rabbit carotid artery, and the change in vessel diameter measured. *P<0.01 vs NL-Plts and Gluc-Plts+Calp-C. (noy shown) Figure 4 Platelets from healthy donors (n=8) were isolated separated into two groups and treated with or without phorbol 12-myristate 13-acetate (PMA) (80 nM) for 20 min. After a washout period, treated and untreated platelets were thrombin (0.1 U ml1) activated and perfused through a phenylephrine (10 jIM) precon-stricted rabbit carotid artery, and the change in vessel diameter measured. *P<0.01 for PMA-Plts vs NL-Plts. (not shown)


In summary, the results of this study along with recently published data (Oskarsson & Hofmeyer 1997; Oskarsson et al., 1997) suggest that high glucose levels cause an increase in platelet DAG that upregulates the activity of PKC, which in turn increases the activity of phospholipase A2 that causes release of arachidonic acid which leads to increased activity of cyclo-oxygenase and thromboxane synthase in platelets (Oskarsson et al., 1997). From a clinical perspective this pathway is of considerable interest since it lends itself to therapeutic interventions with inhibitors both at the level of cyclo-oxygenase and the thromboxane-synthase.


OSKARSSON, H.J. & HOFMEYER, T.G. (1996). Platelet-mediated endothelium-dependent vasodilation is impaired by platelets from patients with diabetes mellitus. J. Am. Coll. Cardiol., 27, 1464 – 1470. OSKARSSON, H.J. & HOFMEYER, T.G. (1997). Diabetic human platelets release a substance which inhibits platelet-mediated vasodilation. Am. J. Phys., 273, H371 – H379. OSKARSSON, H.J., HOFMEYER, T.G. & KNAPP, H.R. (1997). Malondialdehyde inhibits platelet-mediated vasodilation by interfering with platelet-derived ADP. JACC, 29 (Suppl A): 304A.

G-Protein−Coupled Receptors as Signaling Targets for Antiplatele t Therapy

Susan S. Smyth, Donna S. Woulfe, Jeffrey I. Weitz, Christian Gachet, Pamela B. Conley, et al. Participants in the 2008 Platelet Colloquium Arterioscler Thromb Vasc Biol. 2009;29:449-457.    Online ISSN: 1524-4636


Platelet G protein–coupled receptors (GPCRs) initiate and reinforce platelet activation and thrombus formation. The clinical utility of antagonists of the P2Y12 receptor for ADP suggests that other GPCRs and their intracellular signaling pathways may represent viable targets for novel antiplatelet agents. For example, thrombin stimulation of platelets is mediated by 2 protease-activated receptors (PARs), PAR-1 and PAR-4. Signaling downstream of PAR-1 or PAR-4 activates phospholipase C and protein kinase C and causes autoamplification by production of thromboxane A2, release of ADP, and generation of more thrombin. In addition to ADP receptors, thrombin and thromboxane A2 receptors and their downstream effectors—including phosphoinositol-3 kinase, Rap1b, talin, and kindlin—are promising targets for new antiplatelet agents. The mechanistic rationale and available clinical data for drugs targeting disruption of these signaling pathways are discussed. The identification and development of new agents directed against specific platelet signaling pathways may offer an advantage in preventing thrombotic events while minimizing bleeding risk. (Arterioscler Thromb Vasc Biol. 2009;29:449-457.) Key Words: platelets . signaling . G proteins . receptors . thrombosis


Since the first observations of agonist-induced platelet aggregation in 1962, remarkable progress has been made in identifying cell surface receptors and intracellular signaling pathways that regulate platelet function. These discoveries have translated into estab­lished, new, and emerging therapeutics to treat and prevent acute ischemic events by targeting platelet signal transduction.  Indeed, antiplatelet therapy is a mainstay of initial management of patients with ACS and those undergoing percutaneous coronary intervention (PCI). Evidence-based refinements in anticoagulant and antiplatelet therapies have played an important role in the progressive decline in the death rate from coronary disease observed from 1994 to 2004. Despite these therapeutic advances, however, ACS patients receiving “optimal” antithrombotic therapy still suf­fer cardiovascular events. Platelet Signaling Pathways

Vascular injury—whether caused by spontaneous rupture of atherosclerotic plaque, plaque erosion, or PCI-related or other trauma—exposes adhesive proteins, tissue factor, and lipids promoting platelet tethering, adhesion, and activation. Once bound and activated, platelets release soluble mediators such as ADP, thromboxane A2, and serotonin and facilitate throm­bin generation. These mediators, in turn, stimulate GPCRs on the platelet surface that are critical to initiation of various intracellular signaling pathways, including activa­tion of phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide (PI)-3 kinase. Both calcium and PKC con­tribute to activation of the small G protein,  Recently, members of the kindlin family of focal adhesion proteins have been identified as integrin activators, perhaps functioning to facilitate talin–integrin interactions. Platelet signaling pathways Figure. Role of G protein–coupled receptors in the thrombotic process. In humans, protease-activated receptors (PAR)-1 and PAR-4 are coupled to intracellular signaling pathways through molecular switches from the Gq, G12, and Gi protein families. When thrombin (scissors) cleaves the amino-terminal of PAR-l and PAR-4, several signaling pathways are activated, one result of which is ADP secretion. By binding to its receptor, P2Y12, ADP activates additional Gi-mediated pathways. In the absence of wounding, platelet activation is counteracted by signaling from PG I2 (PGI2). Adapted from references 26–28 with permission. Ca2 indicates calcium; CalDAG-GEF1, calcium and diacylglcerol-regulated guanine-nucleotide exchange factor 1; GP, glycoprotein; IP, prostacyclin; PKC, pro­tein kinase C; PLC, phospholipase C; RIAM, Rap1-GTP–interacting adapter molecule.

Future Directions: P2Y1 and P2X Inhibition

Given the clinical success of the P2Y12 antagonists, it is worthwhile to investigate other purinergic signaling pathways in platelets. Although platelets have 2 P2Y receptors acting synergistically through different signaling pathways, the overall platelet response to ADP is relatively modest. For example, ADP alone elicits only reversible responses and does not promote platelet secretion. The low number of ADP receptors on the platelet surface also may limit signal­ing.

Thrombin Signaling in Platelets

Thrombin, the most potent platelet agonist, has diverse effects on various vascular cells. For example, thrombin promotes chemotaxis, adhesion, and inflammation through its effects on neutrophils and monocytes. Thrombin also influ­ences vascular permeability through its effects on endothelial cells and triggers smooth muscle vasoconstriction and mitogenesis.54 Thrombin interacts with 2 protease-activated receptors (PARs) on the surface of human platelets—PAR-1 and PAR-4. Signaling through the PARs is triggered by thrombin-mediated cleavage of the extracellular domain of the receptor and exposure of a “tethered ligand” at the new end of the receptor (Figure 1). Signaling through either PAR can activate PLC and PKC and cause autoamplification through the production of thromboxane A2, the release of ADP, and generation of more thrombin on the platelet surface.

PAR-1 Antagonists as Antithrombotic Therapy

The expression profiles of PARs on platelets differ between humans and nonprimates. Mouse platelets lack PAR-1 and largely signal through PAR-4 in response to thrombin, with PAR-3 serving a cofactor function. Platelets from cynomol-gus monkeys contain primarily PAR-1 and PAR-4, and a peptide-mimetic PAR-1 antagonist extends the time to throm­bosis after carotid artery injury. The nonpeptide antagonist SCH 530348 (described below) inhibits thrombin- and PAR-1 agonist peptide (TRAP)-induced platelet aggregation (inhibitory concentrations of 47 nmol/L and 25 nmol/L, respectively), but it has no effect on ADP, collagen, U46619, or PAR-4 agonist peptide stimulation of platelets. SCH 530348 has excellent bioavailability in rodents and monkeys (82%; 1 mg/kg) and completely inhibits ex vivo platelet aggregation in response to TRAP within 1 hour of oral administration in monkeys with no effect on prothrombin or activated partial thromboplastin times. Of the PAR-1 antagonists, SCH 530348 and E5555 are the compounds farthest along in development and clinical testing. SCH 530348 is an oral reversible PAR-1 antagonist de­rived from himbacine, a compound found in the bark of the Australian magnolia tree. In clinical trials, 68% of patients showed ~80% inhibition of platelet aggregation in response to thrombin receptor activating peptide (TRAP; 15 mol/L) 60 minutes after receiving a 40-mg loading dose of SCH 530348. By 120 minutes, the proportion had risen to 96%. In a Phase 2 trial of SCH 530348, 1031 patients scheduled for angiography and possible stenting were randomized to re­ceive SCH 530348 or placebo plus aspirin, clopidogrel, and antithrombin therapy (heparin or bivalirudin). Major and minor bleeding did not differ substantially between the placebo and individual or combined SCH 530348 groups.

Future Directions: PAR-4 Inhibition

Activation and signaling of PAR-1 and PAR-4 provoke a biphasic “spike and prolonged” response, with PAR-1 acti­vated at thrombin concentrations 50% lower than those required to activate PAR-4. A 4-amino acid segment, YEPF, on the extracellular domain of PAR-1 appears to account for the receptor’s high-affinity interactions with thrombin. The YEPF sequence has homology to the COOH-terminal of hirudin and its synthetic GEPF analog, bivaliru-din, which can interact with exosite-1 on thrombin. Thus, thrombin may interact in tandem with PAR-1 and PAR-4, with the initial interactions involving exosite-1 and PAR-1, and subsequent docking at PAR-4 via the thrombin active site.56 PAR-1 and PAR-4 may form a stable heterodimer that enables thrombin to act as a bivalent functional agonist, rendering the PAR-1–PAR-4 heterodimer complex a unique target for novel antithrombotic therapies. Pepducins, or cell-permeable peptides derived from the third intracellular loop of either PAR-1 or PAR-4, disrupt signaling between the receptors and G proteins and inhibit thrombin-induced platelet aggregation. In mice, a PAR-4 pepducin has been shown to prolong bleeding times and attenuate platelet activation. Combining bivalirudin with a PAR-4 pepducin (P4pal-i1) inhibited aggregation of human platelets from 15 healthy volunteers, even in response to high concentrations of thrombin. In addition, although bivaliru-din and P4pal-i1 each delayed the time to carotid artery occlusion after ferric chloride-induced injury in guinea pigs, their combination prolonged the time to occlusion more than did bivalirudin alone. Additional blockade of the PAR-4 receptor may confer a benefit beyond that achieved by inhibition of thrombin activity.

Targeting Thromboxane Signaling

Thromboxane A2 acts on the thromboxane A2/prostaglandin (PG) H2 (TP) receptor, causing PLC signaling and platelet activation. Several drugs have been tested and developed that prevent thromboxane synthesis—most notably, aspirin. Be­yond the documented success of aspirin, however, results have been uniformly disappointing with a wide variety of thromboxane synthase inhibitors.  Likewise, a multitude of TP receptor antagonists have been developed, but few have progressed beyond Phase 2 trials because of safety concerns. More recently, the thromboxane A2 receptor antagonist terutroban (S18886) showed rapid, potent inhibition of platelet aggregation in a porcine model of in-stent thrombosis that was comparable to the combination of aspirin and clopidogrel but with a more favorable bleeding profile. Ramatroban, another TP inhibitor approved in Japan for treatment of allergic rhinitis, has shown antiaggre-gatory effects in vitro comparable to those of aspirin and cilostazol.

Novel Downstream Signaling Targets

Signaling pathways stimulated by GPCR activation are es­sential for thrombus formation and may represent potential targets for drug development. One pathway involved in platelet activation is signaling through lipid kinases. PI-3 kinases transduce signals by generating lipid second­ary messengers, which then recruit signaling proteins to the plasma membrane. A principal target for PI-3K signaling is the protein kinase Akt (Figure 1). Platelets contain both the Akt1 and Akt2 isoforms.28 In mice, both Akt1 and Akt2 are required for thrombus formation. Mice lacking Akt2 have aggregation defects in response to low concentrations of thrombin or thromboxane A2 and corresponding defects in dense and a-granule secretion. The Akt isoforms have multiple substrates in platelets. Glycogen synthase kinase (GSK)-3(3 is phosphorylated by Akt in platelets and sup­presses platelet function and thrombosis in mice. Akt-mediated phosphorylation of GSK-3(3 inhibits the kinase activity of the enzyme, and with it, its suppression of platelet function. Akt activation also stimulates nitric oxide produc­tion in platelets, which results in protein kinase G–dependent degranulation. Finally, Akt has been implicated in activa­tion of cAMP-dependent phosphodiesterase (PDE3A), which plays a role in reducing platelet cAMP levels after thrombin stimulation.67 Each of these Akt-mediated events is expected to contribute to platelet activation. Rap1 members of the Ras family of small G proteins have been implicated in GPCR signaling and integrin activation. Rap1b, the most abundant Ras GTPase in platelets, is activated rapidly after GPCR stimulation and plays a key role in the activation of integrin aIIb(3) Stimulation of Gq-linked receptors, such as PAR-4 or PAR-1, activates PLC and, with consequent increases in intracellular calcium, PKC. These signals in turn activate calcium and diacylglcerol-regulated guanine-nucleotide exchange factor 1 (CalDAG-GEF1), which has been implicated in activation of Rap1 in plate-lets. Experiments in CalDAG-GEF1-deficient platelets indicate that PKC- and CalDAG-GEF1–dependent events represent independent synergistic pathways leading to Rap1-mediated integrin aIIb(33 activation. Consistent with this concept, ADP can stimulate Rap1b activation in a P2Y12– and PI-3K-dependent, but calcium-independent, manner. A final common step in integrin activation involves bind­ing of the cytoskeletal protein talin to the integrin-(33-subunit cytoplasmic tail. Rap1 appears to be required to form an activation complex with talin and the Rap effector RIAM, which redistributes to the plasma membrane and unmasks the talin binding site, resulting in integrin activation. Mice that lack Rap1b or platelet talin have a bleeding disorder with impaired platelet aggregation because of the lack of integrin aIIb( (3activation. In contrast, mice with a integrin-(33 subunit mutation that prevents talin binding have impaired agonist-induced platelet aggregation and are protected from throm­bosis, but do not display pathological bleeding, suggest­ing that this interaction may be an attractive therapeutic target. Recently, members of the kindlin family of focal adhesion proteins, kindlin-2 and kindlin-3, have been identi­fied as coactivators of integrins, required for talin activation of integrins. Kindlin-2 binds and synergistically en­hances talin activation of aIIb. Of note, deficiency in kindlin-3, the predominant kindlin family member found in hematopoietic cells, results in severe bleeding and protection from thrombosis in mice.


Antiplatelet therapy targeting thromboxane production, ADP effects, and fibrinogen binding to integrin aIIb(33 have proven benefit in preventing or treating acute arterial thrombosis. New agents that provide greater inhibition of ADP signaling and agents that impede thrombin’s actions on platelets are currently in clinical trials. Emerging strategies to inhibit platelet function include blocking alternative platelet GPCRs and their intracellular signaling pathways. The challenge remains to determine how to best combine the various current and pending antiplatelet therapies to maximize benefit and minimize harm. It is well documented that aspirin therapy increases bleeding compared with pla­cebo; that when clopidogrel is added to aspirin therapy, bleeding increases relative to the use of aspirin therapy alone; and that when even greater P2Y12 inhibition with prasugrel is added to aspirin therapy, bleeding is further increased com­pared with the use of clopidogrel and aspirin combination therapy. Does this mean that improved antiplatelet efficacy is mandated to come at the price of increased bleeding? Not necessarily, but it will require a far better understanding of platelet signaling pathways and what aspects of platelet function must be blocked to minimize arterial thrombosis. One of the best clinical examples of the disconnect between antiplatelet-related bleeding and antithrombotic ef­ficacy is the case of the oral platelet glycoprotein (GP) IIb/IIIa antagonists. The use of these agents uniformly led to significantly greater bleeding compared with aspirin but no greater efficacy; in fact, mortality was increased among patients receiving the oral glycoprotein IIb/IIIa inhibitors.77 Through an improved understanding of platelet signaling pathways, antiplatelet therapies likely can be developed not based on their ability to inhibit platelets from aggregating, as current therapies are, but rather based on their ability to prevent the clinically meaningful consequences of platelet activation. What exactly these are remains the greatest obstacle.

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UPDATED on 10/4/2016

Soriot’s $3.5B Brilinta dream is dashed by yet another big trial flop for AstraZeneca

by john carroll
October 4, 2016 09:00 AM EDT
Updated: 09:33 AM

Brilinta, the drug failed to demonstrate a benefit over generic Plavix (clopidogrel) for peripheral artery disease. Back in March, the heart drug flopped in a large stroke study, unable to prove that it could beat aspirin. And Soriot can chalk up those expensive studies to proving Brilinta’s serious deficiencies.

“We don’t believe the goal of $3.5 billion is attainable. I think it would be unrealistic to believe that,” Ludovic Helfgott, head of AstraZeneca’s Brilinta business, told Reuters.

Brilinta brought in a total of $619 million last year after disappointing analysts repeatedly with lower-than-expected quarterly revenue.

Heart studies aren’t cheap. AstraZeneca recruited 13,500 patients for the EUCLID study, and it had enrolled close to that number for the earlier SOCRATES trial.


UPDATED on 9/4/2014

Prehospital Ticagrelor in ST-Segment Elevation Myocardial Infarction

Gilles Montalescot, M.D., Ph.D., Arnoud W. van ‘t Hof, M.D., Ph.D., Frédéric Lapostolle, M.D., Ph.D., Johanne Silvain, M.D., Ph.D., Jens Flensted Lassen, M.D., Ph.D., Leonardo Bolognese, M.D., Warren J. Cantor, M.D., Ángel Cequier, M.D., Ph.D., Mohamed Chettibi, M.D., Ph.D., Shaun G. Goodman, M.D., Christopher J. Hammett, M.B., Ch.B., M.D., Kurt Huber, M.D., Magnus Janzon, M.D., Ph.D., Béla Merkely, M.D., Ph.D., Robert F. Storey, M.D., D.M., Uwe Zeymer, M.D., Olivier Stibbe, M.D., Patrick Ecollan, M.D., Wim M.J.M. Heutz, M.D., Eva Swahn, M.D., Ph.D., Jean-Philippe Collet, M.D., Ph.D., Frank F. Willems, M.D., Ph.D., Caroline Baradat, M.Sc., Muriel Licour, M.Sc., Anne Tsatsaris, M.D., Eric Vicaut, M.D., Ph.D., and Christian W. Hamm, M.D., Ph.D. for the ATLANTIC Investigators

September 1, 2014DOI: 10.1056/NEJMoa1407024


The direct-acting platelet P2Y12 receptor antagonist ticagrelor can reduce the incidence of major adverse cardiovascular events when administered at hospital admission to patients with ST-segment elevation myocardial infarction (STEMI). Whether prehospital administration of ticagrelor can improve coronary reperfusion and the clinical outcome is unknown.


We conducted an international, multicenter, randomized, double-blind study involving 1862 patients with ongoing STEMI of less than 6 hours’ duration, comparing prehospital (in the ambulance) versus in-hospital (in the catheterization laboratory) treatment with ticagrelor. The coprimary end points were the proportion of patients who did not have a 70% or greater resolution of ST-segment elevation before percutaneous coronary intervention (PCI) and the proportion of patients who did not have Thrombolysis in Myocardial Infarction flow grade 3 in the infarct-related artery at initial angiography. Secondary end points included the rates of major adverse cardiovascular events and definite stent thrombosis at 30 days.


The median time from randomization to angiography was 48 minutes, and the median time difference between the two treatment strategies was 31 minutes. The two coprimary end points did not differ significantly between the prehospital and in-hospital groups. The absence of ST-segment elevation resolution of 70% or greater after PCI (a secondary end point) was reported for 42.5% and 47.5% of the patients, respectively. The rates of major adverse cardiovascular events did not differ significantly between the two study groups. The rates of definite stent thrombosis were lower in the prehospital group than in the in-hospital group (0% vs. 0.8% in the first 24 hours; 0.2% vs. 1.2% at 30 days). Rates of major bleeding events were low and virtually identical in the two groups, regardless of the bleeding definition used.


Prehospital administration of ticagrelor in patients with acute STEMI appeared to be safe but did not improve pre-PCI coronary reperfusion. (Funded by AstraZeneca; ATLANTIC number, NCT01347580.)




UPDATED on 2/7/2014

PLATO Controversy Hits the Wall Street Journal

February 05, 2014

NEW YORK, NY – The controversy surrounding the PLATO trial of ticagrelor (Brilinta, AstraZeneca) continues unabated, according to a story published in the Wall Street Journal. Specifically, a sealed complaint filed in US district court in the District of Columbia by a researcher contends that the cardiovascular events in the study “may have been manipulated” [1] .

Dr Victor Serebruany (HeartDrug Research Laboratories, Johns Hopkins University, Towson, MD), who has long been a thorn in the side of AstraZeneca and the PLATO investigators, filed the complaint under the False Claims Act, reports theWall Street Journal. The Journal notes that the US attorney’s office in Washington, DC, has contacted Serebruany and is currently investigating the clinical trial.As reported by heartwire in October 2013, the US Department of Justice issued a civil investigative demand from its civil division “seeking documents and information regarding PLATO.” AstraZeneca is complying with the request.

First reported by heart wire in 2009 , the PLATO trial was a positive study involving more 18 000 patients from 43 countries. PLATO investigators, led by Dr Lars Wallentin (Uppsala Clinical Research Center, Sweden), showed that treating acute coronary syndrome patients with ticagrelor significantly reduced the rate of MI, stroke, and cardiovascular death compared with patients taking clopidogrel. Results were presented at the European Society of Cardiology 2009 Congress and reported in the New England Journal of Medicine.

PLATO has been dogged by questions, including prior to approval. In the sealed complaint, Serebruany takes issue with a number of things, many of which have been reported previously. He alleges that the

  • number of clinical events among those taking clopidogrel was high compared with other studies, pointing out that the rate of all-cause death was 5.9% among clopidogrel-treated patients—nearly twice as high as earlier studies. In addition,
  • the sealed complaint documents the geographic discrepancies in the trial, noting there was a trend toward worse outcomes with ticagrelor at North American sites. The complaint also alleges that
  • an initial count of clinical events suggested the two drugs were equivalent, but adjudication by the Duke Clinical Research Institute attributed another 45 MIs to the clopidogrel group, which tipped the results in favor of ticagrelor. Other questions raised about the study include
  • site monitoring and timing of clinical events. Serebruany also alleges that
  • the trial may have unintentionally been unblinded because of the shape of clopidogrel’s “split capsules,” which would have enabled doctors and nurses to know which drug patients received.

AstraZeneca rebutted these issues, telling the Journal that it is cooperating with the government. It said it is confident in the integrity of the trial and noted the overall study showed the superiority of ticagrelor over clopidogrel. There is no evidence the trial was unblinded and researchers used the same standards when qualifying all clinical events, including MIs, they noted. In addition, the company said it is not possible to compare event rates with clopidogrel in PLATO with other studies because the patient populations differ.

The Journal reports that Serebruany became embroiled in the controversy when asked by the FDA‘s Dr Thomas Marciniak to advise the agency about the PLATO data in 2010. Marciniak, who led the FDA’s review of PLATO, called AstraZeneca’s submission on serious adverse events the “worst submission” he ever encountered. According to the submission, he noted, 12 patients reported their own deaths by telephone. Before approving ticagrelor, the FDA requested an additional analysis of PLATO, and it was eventually approved in the US in July 2011. Ticagrelor was approved in Europe in December 2010 and is authorized for use in more than 100 countries.

The Journal called Serebruany an expert in the antiplatelet field but said he is a “controversial figure,” partly because of his financial ties to industry and repeated criticisms of new drug approvals. Through HeartDrug Research, Serebruany has worked on prasugrel (Effient, Lily/Daiichi-Sankyo), a competing antiplatelet agent, but has also done work for AstraZeneca.


Burton TM. Doctor challenges testing of AstraZeneca’s Brilinta. Wall Street Journal, February 2, 2014. Available here.


UPDATED 3/28/2013

How AstraZeneca Will Use A Diagnostic To Market Its Blood Thinner

by Matthew Harper, Forbes Staff on 3/21/2013

Earlier today I wrote about how AstraZeneca is telling investors that its blood-thinner Brilinta, used to prevent second heart attacks, could be a multi-billion dollar drug, at least twice as big as Wall Street analysts expect. So far the drug has been a disappointment.

I wrote:

Another key data point Astra presented was that blood levels of troponin, a muscle protein released by the heart during a heart attack, predict which patients get the most benefit from Brilinta. This data is not in AstraZeneca’s label, but a spokeswoman said that she believed it would be something the company can market to doctors.

via Can Pascal Soriot Turn Around AstraZeneca? It May Come Down To One Drug – Forbes.

But will the Food and Drug Administration allow Astra to tell doctors that? Stratification using troponin is not in Brilinta’s FDA-approved label, and off-label promotion is illegal. But Ferguson says that communications about troponin will be allowed because all patients with high troponin are patients who would be included in the FDA-approved indication. He confirms that use of troponin testing will be part of the new marketing plan for Brilinta.


Can Pascal Soriot Turn Around AstraZeneca? It May Come Down To One Drug

by Matthew Herper, Forbes Staff on 3/21/2013

This morning in New York, new AstraZeneca chief executive Pascal Soriot is telling investors how he is going to turn around the company that has had the absolute worst track record in research and development among any big pharmaceutical firm. The plan is fairly wide-ranging and involves a lot of the steps one might expect:

  • new layoffs (2,300 jobs);
  • a re-focusing of research and development on three areas: heart disease and diabetes; oncology; and respiratory and inflammation;
  • new R&D initiatives involving Moderna, a biotech company, and the Karolinska Instutet;
  • moving the company’s headquarters to its R&D hub in Cambridge, U.K.;
  • re-focusing on emerging markets, where AZ already gets $6 billion in sales, especially China.

But the short-term key to delivering on his promises today seems to come down to a single drug: Brilinta, the Plavix competitor thatAstraZeneca introduced in 2011 which has so far disappointed, generating  just $324 $89 million in global sales last year. This is a medicine to prevent heart attacks and strokes in patients who suffer acute coronary syndrome, the condition that occurs after a heart attack or serious heart-related chest pain. It works by preventing the formation of blood clots.

Plavix was the second biggest drug in the world, with $6 billion in annual sales, but it is now generic. The conventional wisdom is that it will be difficult to compete with cheap generics. Brilinta is actually trailing Effient, a similar medicine from Eli Lilly, in usage. Wall Street consensus currently sees Brilinta growing to become a moderate-sized drug in 2018, with $1.3 billion in annual sales. But AstraZeneca is saying that it thinks Brilinta can be a multi-billion dollar product. Astra has confirmed that this means Brilinta will have to surpass Effient. The newer drugs also cause more bleeding than Plavix.

What is the company’s argument? In his presentation today, Paul Hudson, Astra’s Executive Vice President, North America, said that the key would be focusing on one key fact: Brilinta reduced cardiovascular deaths by 21% compared to Plavix in a big clinical trial. That would mean that if everyone eligible for Brilinta got it, 100,000 lives would be saved.

But the reality is that doctors have been skeptical of that data because in the part of that trial that was run in North America, the benefit was less clear. AstraZeneca says that this may have been due to an interaction of Brilinta and aspirin and that, according to current cardiovascular guidelines, doctors should be prescribing less aspirin anyway.

Another key data point Astra presented was that blood levels of troponin, a muscle protein released by the heart during a heart attack, predict which patients get the most benefit from Brilinta. This data is not in AstraZeneca’s label, but a spokeswoman said that she believed it would be something the company can market to doctors.

A lot of what Astra will do in the short term on Brilinta will be blocking and tackling. It needs to pay bigger rebates to insurers to make sure that patients can get cheap access to the drug. (This is how discounts happen in the American insurance system: the patient pays a co-payment and the insurer pays full price for the drug, but then the drug maker gives the insurer money back to make the end cost cheaper.) It will also be doing a lot of medical marketing, involving its internal experts or paid, external doctors, to get the word out about the benefits of Brilinta.

Brilinta has other advantages (it stops acting quickly) and disadvantages (it must be given twice a day). But the other big question for expanding results is whether large clinical trials that are now ongoing will show that it works in a broader array of heart patients. Astra is starting a big trial to show Brilinta prevents strokes. These trials are risky and expensive, but there will be a big payoff if they work.

Astra has some other commercial levers to point to. It’s diabetes pill Onglyza, which is sold with Bristol-Myers Squibb, will have results in a big study of its efficacy in preventing heart disease before a similar study of Merck’s top-selling Januvia, which started first. Soriot has smart ideas about which drugs to advance into later testing. But Brilinta is going to be the biggest single indicator of whether Soriot’s new strategies are paying off.


BRILINTA is an antiplatelet medication

Taking BRILINTA is a first step in the treatment your physician has chosen for you. At, you will find helpful information and useful learning tools to help you complete your course of BRILINTA therapy. Make sure you and your loved ones read through all of the sections.


BRILINTA is a type of prescription antiplatelet medication for people who have had a recent heart attack or severe chest pain that happened because their heart wasn’t getting enough oxygen and who are being treated with medicines or procedures to open blocked arteries in the heart. BRILINTA is used with aspirin to stop platelets from sticking together and forming a blood clot that could block blood flow to the heart and cause another, possibly fatal, heart attack. Platelets are small cells in the blood that help with normal blood clotting.

Take BRILINTA and aspirin exactly as instructed by your doctor: BRILINTA twice a day, plus one 81-mg aspirin tablet once a day. You should not take a dose of aspirin higher than 100 mg each day because it can affect how well BRILINTA works. Tell your doctor about any medicines you are taking that contain aspirin. Do not take any new medicines that contain aspirin.


BRILINTA used with aspirin lowers your chance of having another serious problem with your heart or blood vessels such as heart attack, stroke, or blood clots in your stent if you received one. These can be fatal. In fact, in a large clinical study BRILINTA was even better than Plavix® (clopidogrel bisulfate) tablets at lowering your chances of having another heart attack.

BRILINTA is used to lower your chance of having another heart attack or dying from a heart attack, but BRILINTA (and similar drugs) can cause bleeding that can be serious and sometimes lead to death.

Complete the


BRILINTA is used to lower your chance of having another heart attack or dying from a heart attack or stroke, but BRILINTA (and similar drugs) can cause bleeding that can be serious and sometimes lead to death. Instances of serious bleeding, such as internal bleeding, may require blood transfusions or surgery. While you take BRILINTA, you may bruise and bleed more easily and be more likely to have nosebleeds. Bleeding will also take longer than usual to stop.

Call your doctor right away if you have any signs or symptoms of bleeding while taking BRILINTA, including: severe, uncontrollable bleeding; pink, red, or brown urine; vomit that is bloody or looks like coffee grounds; red or black stool; or if you cough up blood or blood clots.

Do not stop taking BRILINTA without talking to the doctor who prescribes it for you. People who are treated with a stent, and stop taking BRILINTA too soon, have a higher risk of getting a blood clot in the stent, having a heart attack, or dying. If you stop BRILINTA because of bleeding, or for other reasons, your risk of a heart attack or stroke may increase. Tell all your doctors and dentists that you are taking BRILINTA. To decrease your risk of bleeding, your doctor may instruct you to stop taking BRILINTA 5 days before you have elective surgery. Your doctor should tell you when to start taking BRILINTA again, as soon as possible after surgery.

Take BRILINTA and aspirin exactly as instructed by your doctor. You should not take a dose of aspirin higher than 100 mg daily because it can affect how well BRILINTA works. Tell your doctor if you take other medicines that contain aspirin. Do not take new medicines that contain aspirin.

Do not take BRILINTA if you are bleeding now, especially from your stomach or intestine (ulcer), have a history of bleeding in the brain, or have severe liver problems.

BRILINTA can cause serious side effects, including bleeding and shortness of breath. Call your doctor if you have new or unexpected shortness of breath or any side effect that bothers you or that does not go away. Your doctor can decide what treatment is needed.

Tell your doctor about all the medicines you take, including prescription and nonprescription medicines, vitamins, and herbal supplements. BRILINTA may affect the way other medicines work, and other medicines may affect how BRILINTA works.

Approved uses
BRILINTA is a prescription medicine for people who have had a recent heart attack or severe chest pain that happened because their heart wasn’t getting enough oxygen and who are being treated with medicines or procedures to open blocked arteries in the heart.

BRILINTA is used with aspirin to lower your chance of having another serious problem with your heart or blood vessels such as heart attack, stroke, or blood clots in your stent if you received one. These can be fatal.

Please read Prescribing Information, including Boxed WARNINGS.

Please read Medication Guide.

You are encouraged to report negative side effects of prescription drugs to the FDA. Visit or call 1-800-FDA-1088.

If you are without prescription coverage and cannot afford your medication, AstraZeneca may be able to help. For more information, please visit

This product information is intended for US consumers only.

BRILINTA is a trademark of the AstraZeneca group of companies.

Plavix® is a registered trademark of sanofi-aventis.

©2012 AstraZeneca.706809-1789005 8/12


BRILINTA (ticagrelor)

Ticagrelor (trade name Brilinta in the US, Brilique and Possia in the EU) is a platelet aggregation inhibitor produced by AstraZeneca. The drug was approved for use in the European Union by the European Commission on December 3, 2010.[1][2] The drug was approved by the US Food and Drug Administrationon July 20, 2011.[3]


Ticagrelor is indicated for the prevention of thrombotic events (for example stroke or heart attack) in patients with acute coronary syndrome or myocardial infarction with ST elevation. The drug is combined with acetylsalicylic acid unless the latter is contraindicated.[4] Treatment of acute coronary syndrome with ticagrelor as compared with clopidogrel significantly reduces the rate of death.[5]


Contraindications for ticagrelor are: active pathological bleeding and a history of intracranial bleeding, as well as reduced liver function and combination with drugs that strongly influence activity of the liver enzymeCYP3A4, because the drug is metabolized via CYP3A4 and excreted via the liver.[4]

Adverse effects

The most common side effects are shortness of breath (dyspnea, 14%)[6] and various types of bleeding, such as hematomanosebleedgastrointestinalsubcutaneous or dermal bleeding. Allergic skin reactions such as rash and itching have been observed in less than 1% of patients.[4]

Physical and chemical properties

Ticagrelor is a nucleoside analogue: the cyclopentane ring is similar to the sugar ribose, and the nitrogen rich aromatic ring system resembles the nucleobase purine, giving the molecule an overall similarity toadenosine. The substance has low solubility and low permeability under the Biopharmaceutics Classification System.[1]

Ticagrelor as a nucleoside analogue

The nucleoside adenosinefor comparison


Ticagrelor is absorbed quickly from the gut, the bioavailability being 36%, and reaches its peak concentration after about 1.5 hours. The main metabolite, AR-C124910XX, is formed quickly via CYP3A4 by de-hydroxyethylation at position 5 of the cyclopentane ring.[7] It peaks after about 2.5 hours. Both ticagrelor and AR-C124910XX are bound to plasma proteins (>99.7%), and both are pharmacologically active. Blood plasma concentrations are linearly dependent on the dose up to 1260 mg (the sevenfold daily dose). The metabolite reaches 30–40% of ticagrelor’s plasma concentrations. Drug and metabolite are mainly excreted via bile and feces.

Plasma concentrations of ticagrelor are slightly increased (12–23%) in elderly patients, women, patients of Asian ethnicity, and patients with mild hepatic impairment. They are decreased in patients that described themselves as ‘coloured’ and such with severe renal impairment. These differences are considered clinically irrelevant. In Japanese people, concentrations are 40% higher than in Caucasians, or 20% after body weight correction. The drug has not been tested in patients with severe hepatic impairment.[4]

Mechanism of action

Like the thienopyridines prasugrelclopidogrel and ticlopidine, ticagrelor blocks adenosine diphosphate (ADP) receptors of subtype P2Y12. In contrast to the other antiplatelet drugs, ticagrelor has a binding site different from ADP, making it an allosteric antagonist, and the blockage is reversible.[8] Moreover, the drug does not need hepatic activation, which might work better for patients with genetic variants regarding the enzyme CYP2C19 (although it is not certain whether clopidogrel is significantly influenced by such variants).[9][10][11]

Comparison with clopidogrel

The PLATO trial, funded by AstraZeneca, in mid-2009 found that ticagrelor had better mortality rates than clopidogrel (9.8% vs. 11.7%, p<0.001) in treating patients with acute coronary syndrome. Patients given ticagrelor were less likely to die from vascular causes, heart attack, or stroke but had greater chances of non-lethal bleeding (16.1% vs. 14.6%, p=0.0084), higher rate of major bleeding not related to coronary-artery bypass grafting (4.5% vs. 3.8%, P=0.03), including more instances of fatal intracranial bleeding. Rates of major bleeding were not different. Discontinuation of the study drug due to adverse events occurred more frequently with ticagrelor than with clopidogrel (in 7.4% of patients vs. 6.0%, P<0.001)[5] The PLATO trial showed a statistically insignificant trend toward worse outcomes with ticagrelor versus clopidogrel among US patients in the study – who comprised 1800 of the total 18,624 patients. The HR actually reversed for the composite end point cardiovascular (death, MI, or stroke): 12.6% for patients given ticagrelor and 10.1% for patients given clopidogrel (HR = 1.27). Some believe the results could be due to differences in aspirin maintenance doses, which are higher in the United States.[12] Others state that the central adjudicating committees found an extra 45 MIs in the clopidogrel (comparator) arm but none in the ticagrelor arm, which improved the MI outcomes with ticagrelor. Without this adjudication the trials’ primary efficacy outcomes should not be significant[13]

Consistently with its reversible mode of action, ticagrelor is known to act faster and shorter than clopidogrel.[14] This means it has to be taken twice instead of once a day which is a disadvantage in respect of compliance, but its effects are more quickly reversible which can be useful before surgery or if side effects occur.[4][15]


Inhibitors of the liver enzyme CYP3A4, such as ketoconazole and possibly grapefruit juice, increase blood plasma levels and consequently can lead to bleeding and other adverse effects. Conversely, drugs that are metabolized by CYP3A4, for example simvastatin, show increased plasma levels and more side effects if combined with ticagrelor. CYP3A4 inductors, for example rifampicin and possibly St. John’s wort, can reduce the effectiveness of ticagrelor. There is no evidence for interactions via CYP2C9.

The drug also inhibits P-glycoprotein (P-gp), leading to increased plasma levels of digoxinciclosporin and other P-gp substrates. Ticagrelor and AR-C124910XX levels are not significantly influenced by P-gp inhibitors.[4]

In the US a boxed warning states that use of ticagrelor with aspirin doses exceeding 100 mg/day decreases the effectiveness of the medication.[16]


  1. a b “Assessment Report for Brilique”European Medicines Agency. January 2011.
  2. ^ European Public Assessment Report Possia
  3. ^ “FDA approves blood-thinning drug Brilinta to treat acute coronary syndromes”. FDA. 20 July 2011.
  4. a b c d e f Haberfeld, H, ed. (2010) (in German). Austria-Codex (2010/2011 ed.). Vienna: Österreichischer Apothekerverlag.
  5. a b Wallentin, Lars; Becker, RC; Budaj, A; Cannon, CP; Emanuelsson, H; Held, C; Horrow, J; Husted, S et al. (August 30, 2009). “Ticagrelor versus Clopidogrel in Patients with Acute Coronary Syndromes”NEJM 361 (11): 1045–57. doi:10.1056/NEJMoa0904327PMID 19717846.
  6. ^ Brilinta: Highlights of prescribing information
  7. ^ Teng, R; Oliver, S; Hayes, MA; Butler, K (2010). “Absorption, distribution, metabolism, and excretion of ticagrelor in healthy subjects”. Drug metabolism and disposition: the biological fate of chemicals 38 (9): 1514–21. doi:10.1124/dmd.110.032250PMID 20551239.
  8. ^ Birkeland, Kade; Parra, David; Rosenstein, Robert (2010). “Antiplatelet therapy in acute coronary syndromes: focus on ticagrelor”Journal of Blood Medicine 1: 197–219.
  9. ^ H. Spreitzer (February 4, 2008). “Neue Wirkstoffe – AZD6140” (in German). Österreichische Apothekerzeitung (3/2008): 135.
  10. ^ Owen, RT, Serradell, N, Bolos, J (2007). “AZD6140”. Drugs of the Future 32 (10): 845–853. doi:10.1358/dof.2007.032.10.1133832.
  11. ^ Tantry, Udaya S; Bliden, Kevin P (2010). “First Analysis of the Relation Between CYP2C19 Genotype and Pharmacodynamics in Patients Treated With Ticagrelor Versus Clopidogrel”. Circulation: Cardiovascular Genetics 3: 556–566. doi:10.1161/CIRCGENETICS.110.958561.
  12. ^ Bernardo Lombo, José G Díez. Ticagrelor: the evidence for its clinical potential as an oral antiplatelet treatment for the reduction of major adverse cardiac events in patients with acute coronary syndromes Core Evid. 2011; 6: 31–42.
  13. ^ Serebruany VL, Atar D. Viewpoint: Central adjudication of myocardial infarction in outcome-driven clinical trials—Common patterns in TRITON, RECORD, and PLATO? Thromb Haemost 2012; DOI: 10.1160/TH12-04-0251.
  14. ^ Miller, R (24 February 2010). “Is there too much excitement for ticagrelor?”.
  15. ^ H. Spreitzer (17 January 2011). “Neue Wirkstoffe – Elinogrel” (in German). Österreichische Apothekerzeitung (2/2011): 10.
  16. ^ July 20, 2011 AstraZeneca: Ticagrelor (Brilinta) Gains FDA Approval Larry Husten


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Reporter and Curator: Aviva Lev-Ari, PhD, RN

WOEST (What is the Optimal Antiplatelet and Anticoagulant Therapy in Patients with Oral Anticoagulantion and Coronary Stenting): Get Rid Of The Aspirin In Triple Therapy

According to current guidelines and clinical practice, PCI patients already taking an oral anticoagulant generally end up on triple therapy comprising the anticoagulant plus clopidogrel and aspirin. However, there is no supporting evidence base for this approach and the triple therapy regimen is known to increase bleeding complications. Now a new study– the first randomized trial to address this situation, according to the investigators–  may have a large impact on clinical practice by demonstrating that the omission of aspirin in this context appears to be safe and may reduce adverse events.

Results of the WOEST (What is the Optimal Antiplatelet and Anticoagulant Therapy in Patients with Oral Anticoagulantion and Coronary Stenting) trial were presented by Willem Dewilde at the ESC in Munich today. Investigators in the Netherlands and Belgium randomized 573 patients to triple therapy or dual therapy of an anticoagulant plus clopidogrel for at least one month after implantation of a bare-metal stent or one year after a drug-eluting stent. Two-thirds of the patients were receiving oral anticoagulation for atrial fibrillation.

The primary endpoint, the total number of bleeding events, was dramatically reduced in the dual therapy group at one year:

  • 44.9% in the triple therapy group versus 19.5% (HR 0.36, CI 0.26-0.50)

There were 3 intracranial bleeds in each group. Most of the difference in bleeding occurred in TIMI minor and minimal bleeding. The difference in TIMI major bleeding (3.3% versus 5.8%) did not achieve statistical significance.

Clinical events, the trials’s secondary endpoint, were numerically lower in the dual therapy group. The difference in mortality achieved statistical significance.

  • Mortality: 7 deaths (2.6%) in the dual therapy group versus 18 deaths (6.4%) in the triple therapy group, p=0.027
  • MI: 3.3% versus 4.7%, p=0.382
  • TVR: 7.3% versus 6.8%, p=0.876
  • Stroke: 1.1% versus 2.9%, p=0.128)
  • Stent thrombosis: 1.5% versus 3.2%, p=0.165

“The WOEST study demonstrates that omitting aspirin leads to less bleedings but does not increase the risk of stent thrombosis, stroke or myocardial infarction,” said Dewilde in an ESC press release. “Although the number of patients in the trial is limited, this is an important finding with implications for future treatment and guidelines in this group of patients known to be at high risk of bleeding and thrombotic complications.”

David Holmes said the trial addressed “an incredibly important issue” and predicted that it would “change the way we practice medicine, it will change practice right away.” Keith Fox said that the evidence base prior to WOEST was extremely limited and that the trial showed that there was no hazard in doing without aspirin. The ESC discussant, Marco Valgimigli, said the trial showed it was safe to drop aspirin and provided another demonstration that “we have hit the wall” with anticoagulation.

Republished with permission from CardioExchange, a NEJM group publication.

European Society of Cardiology: Prasugrel Can’t Top Clopidogrel in ACS

 By Todd Neale, Senior Staff Writer, MedPage Today

Published: August 26, 2012

Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco

MUNICH — For patients with unstable angina or non-ST-segment elevation myocardial infarction (non-STEMI) who do not undergo revascularization, increasing platelet inhibition may not improve outcomes, a randomized trial showed.

Added to a background of low-dose aspirin, prasugrel (Effient) did not significantly reduce the rate of MI, stroke, or cardiovascular death compared with clopidogrel (13.9% versus 16%, HR 0.91, 95% CI 0.79 to 1.05), according to Matthew Roe, MD, of Duke University in Durham, N.C.

The risk of severe bleeding was similar with both drugs, although minor and moderate bleeding were increased with prasugrel, Roe reported at the European Society of Cardiology meeting here. The findings were published simultaneously online in the New England Journal of Medicine.

“I think the outcome is a bit surprising because we think usually that more aggressive antiplatelet therapy, conceivably, in the face of an acute coronary syndrome and non-ST-elevation would lead to lesser adverse outcome from acute myocardial infarction or death,” said William Zoghbi, MD, from Methodist DeBakey Heart Center in Houston and president of the American College of Cardiology.

But he said clinicians need to respect the data “and start thinking about pathogenesis and what we’re trying to do with any of our new interventions.”

In patients with unstable angina or non-STEMI, practice guidelines call for angiography within 48 to 72 hours with provisional revascularization. Many of these patients do not ultimately undergo revascularization, placing them at greater risk compared with those who have their arteries opened with percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG).

Recommended medical therapy is with clopidogrel and aspirin, which is an approach that will not change from the current findings, Zoghbi said.

The purpose of the TRILOGY ACS trial was to explore whether using a more powerful platelet inhibitor — prasugrel — would improve outcomes compared clopidogrel (Plavix) in this high-risk patient subset.

The primary analysis involved 7,243 patients younger than 75 (mean age 62) who were receiving aspirin and were randomized to prasugrel 10 mg daily (or 5 mg daily for those weighing less than 132 pounds) or to clopidogrel 75 mg daily. The researchers recommended a daily aspirin dose of 100 mg or less.

A secondary, exploratory analysis involved 2,083 patients, 75 or older, who were randomized to prasugrel 5 mg daily or to clopidogrel 75 mg daily.

The lack of efficacy seen in the primary analysis of patients younger than 75 remained when patients of all ages were combined. There were no between-group differences for any of the components of the primary endpoint.

A prespecified secondary analysis taking multiple recurrent ischemic events into consideration showed a lower risk of MI, stroke, and cardiovascular death with prasugrel in the younger patients (HR 0.85, 95% CI 0.72 to 1.00, P=0.04), a finding consistent with the main results of the TRITON-TIMI 38 trial, which involved patients treated with PCI. The apparent benefit appeared after 12 months of treatment.

“Although this observation is exploratory, it raises the question of whether investigation of the multiplicity of ischemic events is warranted in future secondary-prevention trials, rather than solely analyzing the time to the first event, as has been traditional in studies involving patients who have had an acute coronary event,” the researchers wrote.

Rates of GUSTO severe or life threatening bleeding and TIMI major bleeding — as well as intracranial hemorrhage — were similar in the two groups in both the younger patients and in the overall study population. When minor and moderate bleeding events were added, the bleeding rate was higher with prasugrel.

There were no widespread differences between the groups in rates of nonhemorrhagic serious adverse events, but heart failure was more frequent with clopidogrel (1.8% versus 1.3%, P=0.045).

Douglas Weaver, MD, of Henry Ford Health System, said that he does not think the findings will have any impact on the use of prasugrel, which is not indicated for the patient population included in the study.

“It just doesn’t pass muster in improving value over clopidogrel,” said Weaver, a past president of the American College of Cardiology.

From a clinical perspective, he said, an important message from the study is the evidence of the safety of a reduced dose of prasugrel in the patients 75 and older, which is a consideration when prescribing prasugrel for patients undergoing PCI.

In comments following Roe’s presentation, Raffaele De Caterina, MD, PhD, of the G. d’Annunzio University in Chieti, Italy, provided context about how the findings fit in with the rest of the literature.

He compared the current results to those of a substudy of the PLATO trial, which involved ticagrelor (Brilinta).

In that trial, ticagrelor significantly reduced vascular death, MI, and stroke (HR 0.85, 95% CI 0.73 to 1.00, P=0.045) — the primary endpoint — and all-cause death (HR 0.75, 95% CI 0.61 to 0.93).

He then highlighted the ESC guidelines on treating patients with acute coronary syndromes without persistent ST-segment elevation.

In those, ticagrelor is recommended for all patients at moderate-to-high risk of ischemic events, regardless of initial treatment strategy and including those pre-treated with clopidogrel, and prasugrel is recommended for those who have not taken another P2Y12 inhibitor, who have a known coronary anatomy, and who are proceeding to PCI.

“I believe such statements and recommendations of the guidelines should not be changed,” De Caterina said.

TRILOGY ACS was funded by Eli Lilly and Daiichi Sankyo.

Roe reported relationships with Daiichi Sankyo, Eli Lilly, AstraZeneca, Bristol-Myers Squibb, Janssen Pharmaceuticals, Merck, Hoffmann-La Roche, and sanofi-aventis. The other authors reported numerous relationships with industry.

Primary source: New England Journal of Medicine

Prematurely halted ALTITUDE trial showed When added to monotherapy with either an ACE inhibitor or an angiotensin receptor blocker (ARB), aliskiren (Tekturna) did not improve outcomes in patients with type 2 diabetes who had high cardiovascular and renal risk

ESC: Aliskiren Onboard No Help in T2D

By Todd Neale, Senior Staff Writer, MedPage Today

Published: August 26, 2012

Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco

MUNICH — When added to monotherapy with either an ACE inhibitor or an angiotensin receptor blocker (ARB), aliskiren (Tekturna) did not improve outcomes in patients with type 2 diabetes who had high cardiovascular and renal risk, the prematurely halted ALTITUDE trial showed.

Through an average follow-up of 32 months, a composite of various cardiovascular and renal outcomes occurred in 17.9% of patients receiving the direct renin inhibitor and 16.8% of those receiving placebo (HR 1.08, 95% CI 0.98 to 1.20), according to Hans-Henrik Parving, MD, DMSc, of the University of Copenhagen and Aarhus University in Denmark.

As a Hot Line presentation European Society of Cardiology meeting here, Parving reported that there were no significant differences on any of the individual components of the endpoint — cardiovascular death, resuscitated sudden death, MI, stroke, unplanned hospitalization for heart failure, doubling of baseline serum creatinine, and onset of end-stage renal disease — or all-cause death.

The rate of stroke — mostly ischemic stroke — was numerically higher with aliskiren, although the result fell short of statistical significance (3.4% versus 2.8%; HR 1.25, 95% CI 0.98 to 1.60,P=0.07).

Thus, Parving said, using aliskiren with ACE inhibitors or ARBs in these high-risk patients “is not recommended and may even be harmful.”

The data monitoring committee for the ALTITUDE trial decided to stop the study early in December 2011 both for futility and for adverse events. Then, earlier this year, the FDA issued a warning about using aliskiren with ACE inhibitors or ARBs and changed the drug label to reflect a contraindication for such combinations in patients with diabetes or renal impairment.

The trial included 8,561 patients with type 2 diabetes who had a high risk of cardiovascular or renal disease who were randomized to aliskiren — at 150 mg daily for 1 month followed by 300 mg daily thereafter — or placebo in addition to monotherapy with either an ACE inhibitor or an ARB (but not both).

Adding aliskiren did not improve outcomes, and in fact, may have caused harm, Parving said, as indicated by the apparent increase in stroke risk.

He said that could be explained by the impaired autoregulation of patients with diabetes or by chance, as there are no indications of a stroke risk in other studies of the drug.

Johannes Mann, of Friedrich Alexander University in Erlangen, Germany, and McMaster University in Hamilton, Ontario, who served as the discussant following Parving’s presentation, agreed that it could be a chance finding, but said that it could also be a direct effect of aliskiren itself.

He concluded that the stroke risk was not explained, however, by dual renin system inhibition, because such a signal was not seen in the ONTARGET trial, which compared the combination of ramipril (an ACE inhibitor) and telmisartan (an ARB) with each drug as monotherapy.

As noted when the trial was halted last year, adverse events were more frequent in the aliskiren group.

The percentage of patients who had a potassium level of 5.5 to less than 6.0 mmol/L was greater with active treatment (21% versus 16%), as was the percentage of those with a potassium level of 6.0 mmol/L or greater (8.8% versus 5.6%).

Aliskiren carried higher risks of hyperkalemia (38.7% versus 28.6%), hypotension (12.1% versus 8%), diarrhea (9.6% versus 7.2%), and falls (2.8% versus 2.6%). There was one death caused by hyperkalemia.

Douglas Weaver, MD, of the Henry Ford Health System in Detroit, said that the findings were disappointing, but that they likely wouldn’t change how aliskiren is used in practice.

“I don’t think this is going to have a negative or a positive effect on it,” said Weaver, who is a past president of the American College of Cardiology.

ALTITUDE was sponsored by Novartis Pharma AG.

The executive committee and other investigators or their institutions received a consultancy fee. Some of the authors are employees of Novartis and therefore eligible for stock and stock options.

Primary source: European Society of Cardiology
Source reference:
Parving H-H, et al “The Aliskiren Trial in Type 2 Diabetes Using Cardio-Renal Endpoints (ALTITUDE)” ESC 2012; Abstract 399.


From Wikipedia, the free encyclopedia
Systematic (IUPAC) name
Aliskiren (INN) (trade names Tekturna, U.S.; Rasilez, U.K. and elsewhere) is the first in a class of drugs called direct renin inhibitors. Its current licensed indication is essential (primary) hypertension.

Aliskiren was co-developed by the Swiss pharmaceutical companies Novartis and Speedel.[1][2] It was approved by the U.S. Food and Drug Administration in 2007 for the treatment of primary hypertension.[3]

In December 2011, Novartis had to halt a clinical trial of the drug after discovering increased incidence of non-fatal stroke, renal complications, hyperkalemia and hypotension in patients with diabetes and renal impairment.[4]

The following recommendations are being added to the drug labels for aliskiren-containing products as of 4/20/12:

I) A new contraindication against the use of aliskiren with ARBs or ACEIs in patients with diabetes because of the risk of renal impairment, hypotension, and hyperkalemia. II) A warning to avoid use of aliskiren with ARBs or ACEIs in patients with moderate to severe renal impairment (i.e., where glomerular filtration rate [GFR] < 60 mL/min).

Mechanism of Action

Renin is the first enzyme in the renin-angiotensin-aldosterone system which plays a role in blood pressure control. Renin cleaves angiotensinogen to angiotensin I, which is in turn converted by angiotensin-converting enzyme (ACE) toangiotensin II. Angiotensin II has both direct and indirect effects on blood pressure. It directly causes arterial smooth muscle to contract, leading to vasoconstriction and increased blood pressure. Angiotensin II also stimulates the production of aldosterone from the adrenal cortex, which causes the tubules of the kidneys to increase reabsorption of sodium, with water following thereby increasing plasma volume and blood pressure.

Aliskiren binds to the S3bp binding pocket of renin, essential for its activity.[5] Binding to this pocket prevents the conversion of angiotensinogen to angiotensin I.
Aliskiren is also available as combination therapy with hydrochlorothiazide.[6]

Many drugs control blood pressure by interfering with angiotensin or aldosterone. However, when these drugs are used chronically, the body increases renin production, which drives blood pressure up again. Therefore, doctors have been looking for a drug to inhibit renin directly. Aliskiren is the first drug to do so.[7][8]

Aliskiren may have renoprotective effects that are independent of its blood pressure−lowering effect in patients with hypertension, type 2 diabetes, and nephropathy who are receiving the recommended renoprotective treatment. According to the AVOID study, researchers found that treatment with 300 mg of aliskiren daily, as compared with placebo, reduced the mean urinary albumin-to-creatinine ratio by 20% (95% confidence interval, 9 to 30; P<0.001), with a reduction of 50% or more in 24.7% of the patients who received aliskiren as compared with 12.5% of those who received placebo (P<0.001). Furthermore, the AVOID trial shows that treatment with 300 mg of aliskiren daily reduces albuminuria in patients with hypertension, type 2 diabetes, and proteinuria who are receiving the recommended maximal renoprotective treatment with losartan and optimal antihypertensive therapy. Therefore, direct renin inhibition will have a critical role in strategic renoprotective pharmacotherapy, in conjunction with dual blockade of the renin−angiotensin−aldosterone system with the use of ACE inhibitors and angiotensin II–receptor blockers, very high doses of angiotensin II−receptor blockers, and aldosterone blockade.[9]

Adverse effects

  • Angioedema
  • Hyperkalemia (particularly when used with ACE inhibitors in diabetic patients)
  • Hypotension (particularly in volume-depleted patients)
  • Diarrhea and other GI symptoms
  • Headache
  • Dizziness
  • Cough
  • Rash
  • Elevated uric acidgout, and renal stones
  • Rarely: allergic swelling of the face, lips or tongue and difficulty breathing


  • Pregnancy: other drugs such as ACE inhibitors, also acting on the renin-angiotensin system have been associated with fetal malformations and neonatal death[10]
  • Breast feeding: during animal studies, the drug has been found present in milk.[10]

Aliskiren has not yet been evaluated in patients with significantly impaired renal function.

Drug interactions

Aliskiren is a minor substrate of CYP3A4 and, more important, P-glycoprotein:

  • Reduces furosemide blood concentration.
  • Atorvastatin may increase blood concentration, however no dose adjustment needed.
  • Possible interaction with ciclosporin (the concomitant use of ciclosporin and aliskiren is contraindicated).
  • Caution should be exercised when aliskiren is administered with ketoconazole or other moderate P-gp inhibitors (itraconazole, clarithromycin, telithromycin, erythromycin, amiodarone).
  • Doctors should stop prescribing aliskiren-containing medicines to patients with diabetes (type 1 or type 2) or with moderate to severe kidney impairment who are also taking an ACE inhibitor or ARB, and should consider alternative antihypertensive treatment as necessary.[11]


  1. ^ Gradman A, Schmieder R, Lins R, Nussberger J, Chiang Y, Bedigian M (2005). “Aliskiren, a novel orally effective renin inhibitor, provides dose-dependent antihypertensive efficacy and placebo-like tolerability in hypertensive patients”. Circulation 111 (8): 1012–8. doi:10.1161/01.CIR.0000156466.02908.EDPMID 15723979.
  2. ^ Straessen JA, Li Y, and Richart T (2006). “Oral Renin Inhibitors”Lancet 368 (9545): 1449–56. doi:10.1016/S0140-6736(06)69442-7PMID 17055947.
  3. ^ “First Hypertension Drug to Inhibit Kidney Enzyme Approved”CBC. 2007-03-06. Retrieved 2007-03-14.[dead link]
  4. ^ Blood-pressure drug reviewed amid dangerous side effects
  5. ^ “Chemistry & Biology : Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin”. ScienceDirect. Retrieved 2010-01-20.
  6. ^ Baldwin CM, Plosker GL.[1]doi:10.2165/00003495-200969070-00004. Drugs 2009; 69(7):833-841.
  7. ^ Ingelfinger JR (June 2008). “Aliskiren and dual therapy in type 2 diabetes mellitus”N. Engl. J. Med. 358 (23): 2503–5. doi:10.1056/NEJMe0803375.PMID 18525047.
  8. ^ PharmaXChange: Direct Renin Inhibitors as Antihypertensive Drugs
  9. ^ Parving HH, Persson F, Lewis JB, Lewis EJ, Hollenberg NK. “Aliskiren Combined with Losartan in Type 2 Diabetes and Nephropathy,” N Engl J Med 2008;358:2433-46.
  10. a b Tekturna
  11. ^ European Medicines Agency recommends new contraindications and warnings for aliskiren-containing medicines.

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Reporter: Aviva Lev-Ari, PhD, RN

Moving Beyond Plavix PGx

Before it lost patent protection this year, clopidogrel was known under the brand name Plavix and marketed by Bristol-Myers Squibb. The Food and Drug Administration first updated the label for Plavix in 2009 to inform doctors that CYP2C19 poor metabolizers experienced diminished response to the drug and that PGx tests could be used to identify genotypes linked to variable treatment response. Then, in 2010, the FDA added a “black box” warning to Plavix’s label to highlight that poor metabolizers, or patients with the CYP2C19*2/*2 genotype, “exhibit higher cardiovascular event rates following acute coronary syndrome or percutaneous coronary intervention than patients with normal CYP2C19 function.” (PGx Reporter 3/17/2010)

Despite FDA’s vote of confidence in the association between certain CYP2C19 loss-of-function alleles and reduced response to Plavix, there is disagreement among healthcare providers about whether PGx testing in this setting is ready for broad implementation.

Scripps Health was an early adopter of PGx testing for Plavix. When in 2009, Scripps Health and Quest Diagnostics inked a deal to offer CYP2C19 testing to patients undergoing stent procedures, many doctors felt the program was premature given the evolving nature of the science (PGx Reporter 10/28/2009). The controversy has only gotten more contentious as several published meta-analyses have yielded conflicting results as to the validity of the association between genotype and drug response (PGx Reporter 3/28/2012).

The FDA has maintained that the available evidence supports its genetic testing recommendation for Plavix. In this regard, it is perhaps fitting that a forward-looking genetic testing program for Plavix is being launched at UF. Lawrence Lesko, former director of the Office of Clinical Pharmacology at FDA’s Center for Drug Evaluation and Research, who played a leadership role in adding PGx information to Plavix’s label, currently heads UF’s Center for Pharmacometrics and Systems Pharmacology and plays a leadership role in the university’s personalized medicine activities.

According to Johnson, UF launched its personalized medicine program with Plavix PGx testing because the black box warning on the drug’s label provided regulatory backing for implementing such testing. Additionally, “the things you potentially can impact with testing, such as major cardiovascular events, are clinically important,” she added. “We also felt that [since] the CYP2C19-clopidogrel effect is strongest in patients who are post percutaneous coronary interventions, that would allow us to focus on a very small patient population and a small number of physicians.”

Although UF’s genetic testing program is currently focused on cardiac patients who could potentially be treated with Plavix, the university has much bigger personalized medicine plans. “As we begin to roll out other pharmacogenomic indications [for cardiology patients] … we will also move past the cath lab … to the heart failure or electrophysiology clinic,” Johnson said, adding that the university intends to eventually implement genetic testing programs for gastroenterology patients.

“CYP2C19 testing for Plavix is just our starting point, so we can really work out the kinks, figure out how to educate the clinicians, figure out the barriers in a relatively confined setting,” she said. “But really, our goal is that we would run this chip on everybody presenting to the health system.”

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