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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 ashuldin@medicine.umaryland.edu

Abstract

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 Information-clinicaltrials.gov; Identifiers:NCT00799396 and NCT00370045

SOURCE:

http://www.ncbi.nlm.nih.gov/pubmed/23392654

http://circgenetics.ahajournals.org/content/6/2/184.short?rss=1

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

 

 

 

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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:/dx.doi.org/10.1186/1479-5876-7-42   http://www.translational-medicine.com/content/7/1/42

Abstract

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)

Discussion

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   http://www.stockton-press.co.uk/bjp

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

Introduction

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)

Conclusions

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.

References

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.     http://dx.doi.org/10.1161/ATVBAHA.108.176388    Online ISSN: 1524-4636    http://atvb.ahajournals.org/content/29/4/449

Abstract

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

Introduction

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.

Conclusions

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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Can Resolvins Suppress Acute Lung Injury?

Reporter: Larry H Bernstein, MD, FCAP

Putting the brakes on acute lung injury: can resolvins suppress acute lung injury?

Cox RR Jr., Phillips O,and Kolliputi N

http://www.frontiersin.org Front.Physio. 2012;3:445.        http://dx.doi.org/10.3389/fphys.2012.00445
http://www.FrontPhysiol.com/putting_the_brakes_on_acute_lung_injury_can_resolvins_suppress_acute_lung_injury?/

The presence of resolvins, proresolving lipid mediators: their role in the resolution of ALI
Eickmeier et al.,
Mucosal Immunity 2012

conversion of DHA to RvD1 by

  • activation of RvD1 Receptor (ALX/FPR2) in pulmonary mucosa alleviates effects of inflammation in APALI

endogenous attenuation of inflammation in APALI
http://www.MucosalImmunity.com/Eikmeier/The_presence_of_resolvins:_their_role_in_the_resolution_of_ALI

 Putting the brakes on acute lung injury: can resolvins suppress acute lung injury?
Ruan R. Cox Jr., Oluwakemi Phillips and Narasaiah Kolliputi*
Front Physiol. 2012;3:445.   doi: 10.3389/fphys.2012.00445. Epub 2012 Nov 29.       http://dx.doi.org/10.3389/fphys.2012.00445
http://fphys.com/Putting the brakes on acute lung injury: can resolvins suppress acute lung injury?

A commentary on

Aspirin-triggered resolvin D1 reduces mucosal inflammation and promotes resolution in a murine model of acute lung injury

by Eickmeier, O., Seki, H., Haworth, O., Hilberath, J. N., Gao, F., Uddin, M., et al. (2012). Mucosal Immunol. doi: 10.1038/mi.2012.66

Acute lung injury (ALI), a syndrome of respiratory failure, is a major clinical problem in the United States. With a high incidence rate, affecting nearly 200,000 annually and a significant morbidity and mortality rate, ALI represents a significant source of health care expenditure with a cost of 3.5–6 billion dollars annually (Treggiari et al., 2004; Rubenfeld et al., 2005; Raghavendran et al., 2011). Pneumocytes, unique cells in the alveolar epithelium, are responsible for

  • facilitating gas exchange,
  • regulating fluid transport, and
  • secreting surfactant to reduce alveolar surface tension.

When the alveolar barrier is disrupted, proteinaceous exudates and extracellular components of necrotic pneumocytes activate resident alveolar macrophages causing massive cytokine release (Ware and Matthay, 2000).  The inflammatory response, if left uncontrolled, can lead to further deterioration of the lung epithelium and the development of a fibroproliferative environment (Raghavendran et al., 2011).

In the July 2012 issue of Mucosal Immunity, Eickmeier et al., discuss the presence of

  1. resolvins,
  2. proresolving lipid mediators, and
  3. present exciting findings on their role in the natural resolution of ALI (Eickmeier et al., 2012).

Resolution phase interaction products (resolvins) are omega-3 polyunsaturated fatty acid derivatives of potent anti-inflammatory precursors, eicosapentaenoic acid (EPA), and docasahexaenoic acid (DHA) (Serhan et al., 2002). “E-series” and “D-series” resolvins are derived from EPA and DHA, respectively. The airway mucosa has been shown to be rich in DHA (Freedman et al., 2004), however, the conversion of DHA to D-series resolvins has not been shown. Resolvin D1 (RvD1), a derivative of DHA, has been found in murine resolving inflammatory peritoneal exudates (Serhan et al., 2002). To investigate the potential role that RvD1 may play in the resolution of ALI, Eickmeier et al. used a murine aspiration pneumonitis acute lung injury (APALI) model induced by hydrochloric acid (HCl) administration into the left lung. Picogram quantities of RvD1 were found using metabolipidomics analysis following HCl instillation. Immunohistochemical analysis showed enhanced expression of RvD1 receptor (ALX/FPR2) as early as 2 h post-APALI. This suggested that there was a conversion of DHA to RvD1 following lung injury. Activation of ALX/FPR2 dampens the inflammatory responses through blockage of proinflammatory MAP kinase and NF-κB signaling (Chiang et al., 2006).  Eickmeier et al. demonstrated that the conversion of DHA in pulmonary mucosa alleviates the effects of inflammation in APALI. AT-RvD1 showed therapeutic effects, and bronchio-alveolar lavage fluid (BALF) collected from AT-RvD1 treated mice contained decreased leukocytes and proinflammatory cytokines in comparison to control. AT-RvD1 treated mice demonstrated decreased lung resistance and improved lung mechanics in comparison to controls. The authors showed that AT-RvD1 restored barrier integrity in APALI mice in comparison to control. The anti-inflammatory effects of ALX/FPR2 activation were shown to be a result of

  • reduced activation and nuclear translocation of the transcription factor NF-κB.

Eickmeier et al., demonstrated that mice treated with AT-RvD1 demonstrated reduced

  • NF-κB phosphorylation, which is necessary for the activation,
  • translocation and DNA binding functions of this proinflammatory molecule.

The work of Eickmeier et al. revealed that RvD1 is a central mediator in the endogenous attenuation of inflammation seen in APALI. In most cases of ALI, the injury is indeed self-limiting and resolves on its own (Dos Santos and Slutsky, 2006). This work gives insight to the mechanism involved in the lung injury resolution process. A recent clinical study demonstrates that,
ALI progression is associated with

  • increased ventilator time and
  • longer intensive care unit (ICU) stays.

These patients show an enhanced proinflammatory cytokine profile which was also correlated with increased morbidity (Dolinay et al., 2012). Previous reports have also demonstrated that ALI/ARDS patients represent 34% of yearly costs for all ICU trauma patients (Treggiari et al., 2004). In the case that the ALI does not resolve, the patient is at risk for developing acute respiratory distress syndrome in as little as 3 days (Marshall et al., 1998). Finding endogenous mediators that may control the ungoverned inflammation seen in ALI is a pivotal step to finding a treatment for this disease that entails more than just supportive care (Marshall et al., 1998). The work of Eickmeier et al. has paved the way for the exploration of the beneficial effects of resolvins in the incidences of other sterile injuries, such as atherosclerosis, gout, Alzheimer’s disease, and diabetes.

Br J Pharmacol. 2008 March; 153(S1): S200–S215.
Published online 2007 October 29. doi:  10.1038/sj.bjp.0707489
PMCID: PMC2268040

Endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus

C N Serhan1,2,* and N Chiang1
This article has been cited by other articles in PMC.

Abstract

Complete resolution of an acute inflammatory response and its return to homeostasis are essential for healthy tissues. We here consider work to characterize cellular and molecular mechanisms that govern the resolution of self-limited inflammation. Systematic temporal analyses of evolving inflammatory exudates using

  1. mediator lipidomics-informatics,
  2. proteomics, and
  3. cellular trafficking with murine resolving exudates demonstrate
    • novel endogenous pathways of local-acting mediators that share both anti-inflammatory and pro-resolving properties.

In murine systems, resolving-exudate leukocytes switch their phenotype to actively generate new families of mediators from major omega-3 fatty acids EPA and DHA termed resolvins and protectins. Recent advances on their biosynthesis and actions are reviewed with a focus on the E-series resolvins (RvE1, RvE2), D series resolvins (RvD1, RvD2) and the protectins including neuroprotectin D1/protectin D1 (NPD1/PD1) as well as their aspirin-triggered epimeric forms.  These endogenous agonists of resolution pathways constitute a novel genus of chemical mediators that possess

  • pro-resolving,
  • anti-inflammatory, and
  • antifibrotic as well as
  • host-directed antimicrobial actions.
    These may be useful in the design of new therapeutics and treatments for diseases with the underlying trait of uncontrolled inflammation and redox organ stress.
Keywords: leukocytes, eicosanoids, resolvins, acute inflammation, ω-3 fatty acids, protectins

Introduction

Acute inflammation has several outcomes that include

  • progression to chronic inflammation,
  • scarring and fibrosis or
  • complete resolution (Cotran et al., 1999).

With the isolation of endogenous anti-inflammatory and pro-resolving mediators and their characterization, it became clear that resolution is an active process involving biochemical circuits that

The resolution phase has emerged as a new terrain for drug design and resolution-directed therapeutics (Gilroy et al., 2004Lawrence et al., 2005). A pro-resolving small molecule can, in addition to serving as

  • an agonist of anti-inflammation, also
  • promote the uptake and clearance of apoptotic neutrophils (polymorphonuclear leukocyte, PMN)

A recent consensus report from investigators at the forefront of this emerging area has addressed these definitions to help delineate this new terrain (Serhan et al., 2007). Some agents such as the widely used COX-2 inhibitors proved to be resolution toxic (Gilroy et al., 1999Bannenberg et al., 2005Serhan et al., 2007), whereas others can possess pro-resolving actions, such as

Interest in natural resolving mechanisms has been heightened in recent years (Henson, 2005Luster et al., 2005Serhan and Savill, 2005) because inflammation (characterized by the cardinal symptoms dolor, calor, rubor and loss of function) is now recognized as a central feature in the pathogenesis of many prevalent diseases in modern Western civilization, such as

  1. stroke,
  2. Alzheimer’s and
  3. Parkinson’s diseases (Majno and Joris, 1996;Nathan, 2002Erlinger et al., 2004Hansson et al., 2006).

Resolution of inflammation is required for the return from inflammatory disease to health, that is, catabasis (Bannenberg et al., 2005). New evidence from this laboratory and others indicates that the catabasis from inflammation to the ‘normal’ noninflamed state is not merely passive termination of inflammation but rather an actively regulated program of resolution (Serhan et al., 2007). This event is accompanied by lipid mediator class switching from pro-inflammatory prostaglandins (PGs) and leukotrienes (LT) to the biosynthesis of anti-inflammatory mediators, such as lipoxins (LXs) (Levy et al., 2001), as well as the appearance of new families of pro-resolving mediators biosynthesized in exudates from ω-3 polyunsaturated fatty acid (PUFA) precursors (Serhan et al., 20002002Hong et al., 2003) (Figure 1a).   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268040/bin/0707489f1.gif

The essential roles of omega-3 PUFAs in preventing disease in rodents were established in 1929 (Burr and Burr, 1929). In humans, the beneficial actions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the major omega-3 PUFA, remains a topic of interest because structure–activity relationships remained to be established.  One theory suggests that the omega-3 PUFA compete with the storage of arachidonic acid (AA),

  • replacing it and blocking the production of pro-inflammatory eicosanoids (Lands, 1987).

Along with the pro-inflammatory PGs and LT, the n−6 essential fatty acid AA is precursor to LX and aspirin-triggered LX, which possess potent anti-inflammatory and pro-resolving actions. Therefore, the popular view of essential n−6 and n−3 PUFA actions in inflammation and homeostasis was incomplete.   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268040/table/tbl1/?report=thumb

The evidence available to date  indicates that

The term resolvins (resolution-phase interaction products) was first introduced to signify that the new structures were endogenous mediators possessing potent anti-inflammatory and immunomodulatory actions demonstrated in the nanogram dose range in vivo(Serhan et al., 2002). These include

  • reducing neutrophil traffic and pro-inflammatory cytokines, as well as
  • lowering the magnitude of the inflammatory response in vivo (Serhan et al., 20002002).

The terms protectin and neuroprotectin (when generated in neural tissues) (Serhan et al., 2006a) were introduced given the anti-inflammatory (Hong et al., 2003) as well as the protective actions of the
DHA-derived mediator NPD1/PD1 in neural systems (Mukherjee et al., 2004),

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268040/bin/0707489f2.gif

RvE1 possesses an interesting and novel distinct structure consisting of

  • a conjugated diene plus
  • conjugated diene chromophore present within the same molecule.

Both biogenic (Serhan et al., 2000) and total organic syntheses were achieved and its complete stereochemical assignment was established along with that of several related natural isomers (Arita et al., 2005a). RvE1 proved to be 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoicacid.

Human recombinant 5-LOX generates resolvin E2 (RvE2) from a common precursor of E-series resolvins, namely 18-HEPE. RvE2, which is 5S,18-dihydroxyeicosapentaenoic acid, stopped zymosan-induced PMN infiltration, displaying potent anti-inflammatory properties in murine peritonitis (Tjonahen et al., 2006). In addition, RvE1 and RvE2, when given together, displayed additive action in controlling PMN infiltration. These results demonstrate that RvE2, together with RvE1, may contribute to the beneficial actions of ω-3 fatty acids in human diseases. Moreover, they indicate that the 5-LOX, in human leukocytes, is a pivotal enzyme that is temporally and spatially regulated in vivo to produce either pro- or anti-inflammatory local chemical mediators.

Resolvins of the E-series comprise several molecules. Among them, RvE1 was the first isolated and studied in depth. RvE1 displayed potent stereoselective actions in vivo and with isolated cells.
At nanomolar levels in vitro, RvE1 dramatically reduced

  1. human PMN transendothelial migration,
  2. dendritic cell (DC) migration and
  3. interleukin (IL)-12 production
    (Serhan et al., 2002Arita et al., 2005a).

These new findings provide evidence for

  • endogenous mechanism(s) that may account for some of the widely touted beneficial actions noted with dietary supplementation with ω-3 PUFA (EPA and DHA),
  • thereby providing new approaches for the treatment of gastrointestinal mucosal and oral inflammation.

The new families of EPA- and DHA-derived chemical mediators, namely the resolvins and protectins, qualify as ‘resolution agonists’ along with the n−6 derived agonists of resolution, the LX, in this new arena of immunomodulation and tissue protection. These are conserved structures in evolution, because rainbow trout biosynthesize resolvins and protectins, which are present in their neural and hematopoietic tissues (Hong et al., 2005). Their functional roles in fish and lower phyla remain to be established, but are likely to involve

  1. cell trafficking,
  2. motility and
  3. protection.

Additionally, they now open new avenues to design ‘resolution-targeted’-based therapies where aberrant uncontrolled inflammation and/or impaired resolution are components of the disease pathophysiology.

Lipoxin A4 Regulates Natural Killer Cell and Type 2 Innate Lymphoid Cell Activation in Asthma
C Barnig, M Cernadas, S Dutile,…BR Levy.
Sci Transl Med 27 Feb 2013: 5(174) 174ra26   http://dx.doi.org/10.1126/scitranslmed.3004812   http://www.scitranslmed.com//LipoxinA4_Regulates_Natural_Killer_Cell_and_Type2_Innate_Lymphoid_Cell_Activation_in_Asthma/


Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation.
Both NK cells and ILC2s expressed

Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly

Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation

Neutrophil granulocyte migrates from the blood...

Neutrophil granulocyte migrates from the blood vessel to the matrix, sensing proteolytic enzymes, in order to determine intercellular connections (to the improvement of its mobility) and envelop bacteria through Phagocytosis. (Photo credit: Wikipedia)

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Aspirin a Day Tied to Lower Cancer Mortality

Reporter: Aviva Lev-Ari, PhD, RN

Aspirin a Day Tied to Lower Cancer Mortality

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Daily aspirin use is associated with a modest decrease in mortality from cancer, particularly for malignancies of the gastrointestinal tract, a large retrospective study confirmed.

Individuals who were current daily users for 5 years or more at baseline had an 8% decrease in cancer mortality compared with non-users (RR 0.92, 95% CI 0.83 to 1.02), according to Eric J. Jacobs, PhD, and colleagues from the American Cancer Society in Atlanta.

The association was stronger, with a 16% decrease for those with daily use for 5 years or more, when the analysis included data collected periodically during 2 decades of follow-up (RR 0.84, 95% CI 0.75 to 0.95), the researchers reported in the Journal of the National Cancer Institute.

A recent pooled analysis of more than 50 trials involving aspirin use for cardioprotection found a 37% reduction in deaths from cancer among users, which was considerably greater than in observational studies and trials of alternate-day aspirin.

To clarify the magnitude of the association between aspirin use and overall cancer mortality, Jacobs and colleagues analyzed data from the Cancer Prevention Study II, which began in 1992 and included 100,139 participants who completed questionnaires with information on demographics, medical history, and behavioral influences.

Beginning in 1997, participants also provided information about aspirin use, and continued to provide updates every 2 years.

The 1997 questionnaire was considered the baseline for the analysis, at which time 23.8% of participants were using either low-dose or adult-strength aspirin.

More than half of participants were older than 60 and female, and almost all were white.

During the 20 years of follow-up, there were 5,138 deaths from cancer.

Among those who reported aspirin use in 1997, three-quarters said they were still taking it in 2003, while among those who were non-users at baseline, 25% had begun doing so.

Baseline aspirin users tended to be more educated, former smokers, and obese, as well as to have a history of cardiovascular disease and diabetes.

Male users also were more likely to have a history of prostate specific antigen (PSA) testing, and women users were more likely to have a history of mammography.

Overall mortality was slightly lower even for individuals who had been users for less than 5 years (RR 0.84, 95% CI 0.76 to 0.94).

Relative risks were similar for users of low-dose and full-strength aspirin, and for those with and without a history of cardiovascular disease, ranging from 0.82 (95% CI 0.72 to 0.91) to 0.95 (95% CI 0.86 to 1.04).

Current users who had never smoked had considerably lower mortality (RR 0.68, 95% CI 0.57 to 0.81), a reduction that was not seen for former smokers (RR 0.92, 95% CI 0.82 to 1.04) or those currently smoking (RR 0.91, 95% CI 0.70 to 1.19).

Even after discounting lung cancer deaths, the only lower mortality among aspirin users was for never-smokers (RR 0.67, 95% CI 0.56 to 0.81).

A possible explanation for the lack of effect on cancers other than those in the lung among ever-smokers is that smoking may attenuate the antiplatelet activity of aspirin, and activated platelets are thought to promote tumor metastases, the researchers explained.

Aspirin use at the 1997 baseline was not significantly associated with mortality from specific cancers, but differences were seen when data through 2008 were included in the analysis:

  • Cancers within the gastrointestinal tract, RR 0.61 (95% CI 0.44 to 0.84)
  • Cancers outside the gastrointestinal tract, RR 0.88 (95% CI 0.78 to 1)
  • Colorectal cancer, OR 0.64 (95% CI 0.42 to 0.98)
  • Esophageal and stomach cancer, RR 0.56 (95% CI 0.37 to 0.86)

“The reduction in overall cancer mortality was driven by both a substantial reduction in mortality from gastrointestinal tract cancers and a small, but statistically significant, reduction in mortality from cancers outside the gastrointestinal tract,” they stated.

They noted that their study was observational, which was an important limitation, in that confounding factors could have resulted in either an underestimate or an overestimate of the effects of aspirin on mortality.

Also, the absolute risk for cancer mortality between non-users and daily long-term aspirin users — approximately 100 per 100,000 person-years for men and about 40 per 100,000 person-years for women — would represent an important benefit of aspirin use if it were causal, the authors stated.

“However, even if causal, differences in absolute rates are likely to differ between our predominantly elderly population and younger populations at much lower risk of cancer mortality,” they warned.

They concluded that the “relatively modest benefit” seen in their analysis could “meaningfully influence the balances of risks and benefits of prophylactic aspirin use.”

In an accompanying editorial, John Baron, MD, of the University of North Carolina in Chapel Hill, offered a word of caution. Baron was the lead author of the meta-analysis on aspirin use and cancer risk.

“Just because aspirin is effective does not mean it necessarily should be used,” he argued.

“Aspirin is a real drug, with definite toxicity. As for any preventative intervention, the benefits must be balanced against the risks, particularly when the benefits are delayed whereas the risks are not,” Baron stated.

The American Cancer Society funds the Cancer Prevention Study II cohort.

The authors are employees of the American Cancer Society.

Editorialist Baron has been a consultant for Bayer, and holds a use patent for aspirin chemoprevention.

Primary source: Journal of the National Cancer Institute
Source reference:
Jacobs E, et al “Daily aspirin use and cancer mortality in a large US cohort” JNCI 2012; DOI: 10.1093/jnci/djs318.

Additional source: Journal of the National Cancer Institute
Source reference:
Baron JA, et al “Aspirin and cancer: trials and observational studies” JNCI 2012; DOI: 10.1093/jnci/djs318.

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