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Summary of Genomics and Medicine: Role in Cardiovascular Diseases
Author: Larry H. Bernstein, MD, FCAP
The articles within Chapters and Subchapters you have just read have been organized into four interconnected parts.
Genomics and Medicine
Epigenetics – Modifyable Factors Causing CVD
Determinants of CVD – Genetics, Heredity and Genomics Discoveries
Individualized Medicine Guided by Genetics and Genomics Discoveries
The first part established the
rapidly evolving science of genomics
aided by analytical and computational tools for the identification of nucleotide substitutions, or combinations of them
that have a significant association with the development of
cardiovascular diseases,
hypercoagulable state,
atherosclerosis,
microvascular disease,
endothelial disruption, and
type-2DM, to name a few.
These may well be associated with increased risk for stroke and/or peripheral vascular disease in some cases,
essentially because the involvement of the circulation is systemic in nature.
Part 1
establishes an important connection between RNA and disease expression. This development has led to
the necessity of a patient-centric approach to patient-care.
When I entered medical school, it was eight years after Watson and Crick proposed the double helix. It was also
the height of a series of discoveries elucidating key metabolic pathways.
In the period since then there have been treatments for some of the important well established metabolic diseases of
carbohydrate,
protein, and
lipid metabolism,
such as – glycogen storage disease, and some are immense challenges, such as
Tay Sachs, or
Transthyretin-Associated amyloidosis.
But we have crossed a line delineating classical Mendelian genetics to
multifactorial non-linear traits of great complexity and
involving combinatorial program analyses to resolve.
The Human Genome Project was completed in 2001, and it has opened the floodgates of genomic discovery. This resulted in the identification of
genomic alterations in
cardiovascular disease,
cancer,
microbial,
plant,
prion, and
metabolic diseases.
This has also led to
the identification of genomic targets
that are either involved in transcription or
are involved with cellular control mechanisms for targeted pharmaceutical development.
In addition, there is great pressure on the science of laboratory analytics to
codevelop with new drugs,
biomarkers that are indicators of toxicity or
of drug effectiveness.
I have not mentioned the dark matter of the genome. It has been substantially reduced, and has been termed dark because
this portion of the genome is not identified in transcription of proteins.
However, it has become a lightning rod to ongoing genomic investigation because of
an essential role in the regulation of nuclear and cytoplasmic activities.
Changes in the three dimensional structure of these genes due to
changes in Van der Waal forces and internucleotide distances lead to
conformational changes that could have an effect on cell activity.
Part 2
is an exploration of epigenetics in cardiovascular diseases. Epigenetics is
the post-genomic modification of genetic expression
by the substitution of nucleotides or by the attachment of carbohydrate residues, or
by alterations in the hydrophobic forces between sequences that weaken or strengthen their expression.
This could operate in a manner similar to the conformational changes just described. These changes
may be modifiable, and they
may be highly influenced by environmental factors, such as
smoking and environmental toxins,
diet,
physical activity, and
neutraceuticals.
While neutraceuticals is a black box industry that evolved from
the extraction of ancient herbal remedies of agricultural derivation (which could be extended to digitalis and Foxglove; or to coumadin; and to penecillin, and to other drugs that are not neutraceuticals).
The best examples are the importance of
n-3 fatty acids, and
fiber
dietary sulfur (in the source of methionine), folic acid, vitamin B12
arginine combined with citrulline to drive eNOS
and of iodine, which can’t be understated.
In addition, meat consumption involves the intake of fat that contains
the proinflammatory n-6 fatty acid.
The importance of the ratio of n-3/n-6 fatty acidsin diet is not seriously discussed when
we look at the association of fat intake and disease etiology.
Part 2 then leads into signaling pathways and proteomics.The signaling pathways are
critical to understanding the inflammatory process, just as
dietary factors tie in with a balance that is maintained by dietary intake,
possibly gut bacteria utilization of delivered substrate, and
proinflammatory factors in disaease.
These are being explored by microfluidic proteomic and metabolomic technologies that were inconceivable a half century ago.
This portion extended into the diagnosis of cardiovascular disease, and
elucidated the relationship between platelet-endothelial interaction in the formation of vascular plaque.
It explored protein, proteomic, and genomic markers
for identifying and classifying types of disease pathobiology, and
for following treatment measures.
Part 3
connected with genetics and genomic discoveries in cardiovascular diseases.
Part 4
is the tie between life style habits and disease etiology, going forward with
the pursuit of cardiovascular disease prevention.
The presentation of walking and running, and of bariatric surgery (type 2DM) are fine examples.
It further discussed gene therapy and congenital heart disease. But the most interesting presentations are
in the pharmacogenomics for cardiovascular diseases, with
volyage-gated calcium-channels, and
ApoE in the statin response.
This volume is a splendid example representative of the entire collection on cardiovascular diseases.
Introduction to Genomics and Epigenomics Roles in Cardiovascular Diseases
Author and Curator: Larry H Bernstein, MD, FCAP
This introduction is to a thorough evaluation of a rich source of research literature on the genomic influences, which may have variable strength in the biological causation of atherosclerosis, microvascular disease, plaque formation, not necessarily having expressing, except in a multivariable context that includes the environment, dietary factors, level of emotional stress, sleep habits, and the daily activities of living for affected individuals. The potential of genomics is carried in the DNA, copied to RNA, and this is most well studied in the micro RNAs (miRNA). The miRNA has been explored for the appearance in the circulation of specific miRNAs that might be associated with myocyte or endothelial cell injury, and they are also being used as targets for therapeutics by the creation of silencing RNAs (siRNA). The extent to which there is evidence of success in these studies is limited, but is being translated from animal studies to human disease. There is also a long history of the measurement of circulating enzymes and isoenzymes (alanine amino transferase, creatine kinase, and lactate dehydrogenase, not to leave out the adenylate kinase species specific to myocardium), and more recently the release of troponins I and T, and the so far still not fully explored ischemia modified albumin, or of miRNAs for the diagnosis of myocardial infarction.
There is also a significant disagreement about the value of measuring high sensitivity C reactive protein (hs-CRP), which has always been a marker for systemic inflammatory disease, in both chronic rheumatic and infectious diseases having a broad range, so that procalcitonin has appeared to be better for that situation, and for early diagnosis of sepsis. The hs-CRP has been too easily ignored because of
1. the ubiquitous elevations in the population
2. the expressed concerns that one might not be inclined to treat a mild elevation without other risk factors, such as, LDL cholesterolemia, low HDL, absent diabetes or obesity. Nevertheless, hs-CRP raises an reasonable argument for preventive measures, and perhaps the use of a statin.
There has been a substantial amount of work on the relationship of obesity to both type 2 diabetes mellitus (T2DM) and to coronary vascular disease and stroke. Here we bring in the relationship of the vascular endothelium, adipose tissue secretion of adiponectin, and platelet activation. A whole generation of antiplatelet drugs addresses the mechanism of platelet activation, adhession, and interaction with endothelium. Very interesting work has appeared on RESISTIN, that could bear some fruit in the treatment of both obesity and T2DM.
It is important to keep in mind that epigenomic gene rearrangements or substitutions occur throughout life, and they may have an expression late in life. Some of the known epigenetic events occur with some frequency, but the associations are extremely difficult to pin down, as well as the strength of the association. In a population that is not diverse, epigenetic changes are passed on in the population in the period of childbearing age. The establishment of an epigenetic change is diluted in a diverse population. There have been a number of studies with different findings of association between cardiovascular disease and genetic mutations in the Han and also in the Uyger Chinese populations, which are distinctly different populations that is not part of this discussion.
This should be sufficient to elicit broad appeal in reading this volume on cardiovascular diseases, and perhaps the entire series. Below is a diagram of this volume in the series.
PART 1 – Genomics and Medicine
Introduction to Genomics and Medicine (Vol 3)
Genomics and Medicine: The Physician’s View
Ribozymes and RNA Machines
Genomics and Medicine: Genomics to CVD Diagnoses
Establishing a Patient-Centric View of Genomic Data
VIDEO: Implementing Biomarker Programs P Ridker
PART 2 – Epigenetics – Modifiable
Factors Causing CVD
Diseases Etiology
Environmental Contributors
Implicated as Causing CVD
Diet: Solids and Fluid Intake
and Nutraceuticals
Physical Activity and
Prevention of CVD
Psychological Stress and
Mental Health: Risk for CVD
Correlation between
Cancer and CVD
PART 3 Determinants of CVD – Genetics, Heredity and Genomics Discoveries
Introduction
Why cancer cells contain abnormal numbers of chromosomes (Aneuploidy)
Functional Characterization of CV Genomics: Disease Case Studies @ 2013 ASHG
Leading DIAGNOSES of CVD covered in Circulation: CV Genetics, 3/2010 – 3/2013
Commentary on Biomarkers for Genetics and Genomics of CVD
PART 4 Individualized Medicine Guided by Genetics and Genomics Discoveries
Medical Etiologies: EBM – LEADING DIAGNOSES, Risks
Pharmacogenomics for Cardio-
vascular Diseases
Signaling Pathways
Response to Rosuvastatin in
Patients With Acute Myocardial Infarction:
Hepatic Metabolism and Transporter Gene
Variants Effect http://pharmaceuticalintelligence.com/2014/
01/02/response-to-rosuvastatin-in-patients-
with-acute-myocardial-infarction-hepatic-
metabolism-and-transporter-gene-variants-effect/
Proteomics and Metabolomics
Voltage-Gated Calcium Channel and Pharmaco-
genetic Association with Adverse Cardiovascular
Outcomes: Hypertension Treatment with Verapamil
SR (CCB) vs Atenolol (BB) or Trandolapril (ACE) http://pharmaceuticalintelligence.com/2014/01/02/
voltage-gated-calcium-channel-and-pharmacogenetic-
association-with-adverse-cardiovascular-outcomes-
hypertension-treatment-with-verapamil-sr-ccb-vs-
atenolol-bb-or-trandolapril-ace/
These presentations covered several views of the utilization of cardiac markers that have evolved for over 60 years. The first stage was the introduction of enzymatic assays and isoenzyme measurements to distinguish acute hepatitis and acute myocardial infarction, which included lactate dehydrogenase (LD isoenzymes 1, 2) at a time that late presentation of the patient in the emergency rooms were not uncommon, with the creatine kinase isoenzyme MB declining or disappeared from the circulation. The world health organization (WHO) standard definition then was the presence of two of three:
1. Typical or atypical precordial pressure in the chest, usually with radiation to the left arm
2. Electrocardiographic changes of Q-wave, not previously seen, definitive; ST- elevation of acute myocardial injury with repolarization;
T-wave inversion.
3. The release into the circulation of myocardial derived enzymes –
creatine kinase – MB (which was adapted to measure infarct size), LD-1,
both of which were replaced with troponins T and I, which are part of the actomyosin contractile apparatus.
The research on infarct size elicited a major research goal for early diagnosis and reduction of infarct size, first with fibrinolysis of a ruptured plaque, and this proceeded into the full development of a rapidly evolving interventional cardiology as well as cardiothoracic surgery, in both cases, aimed at removal of plaque or replacement of vessel. Surgery became more imperative for multivessel disease, even if only one vessel was severely affected.
So we have clinical history, physical examination, and emerging biomarkers playing a large role for more than half a century. However, the role of biomarkers broadened. Patients were treated with antiplatelet agents, and a hypercoagulable state coexisted with myocardial ischemic injury. This made the management of the patient reliant on long term followup for Warfarin with the international normalized ratio (INR) for a standardized prothrombin time (PT), and reversal of the PT required transfusion with thawed fresh frozen plasma (FFP). The partial thromboplastin test (PPT) was necessary in hospitalization to monitor the heparin effect.
Thus, we have identified the use of traditional cardiac biomarkers for:
1. Diagnosis
2. Therapeutic monitoring
The story is only the beginning. Many patients who were atypical in presentation, or had cardiovascular ischemia without plaque rupture were problematic. This led to a concerted effort to redesign the troponin assays for high sensitivity with the concern that the circulation should normally be free of a leaked structural marker of myocardial damage. But of course, there can be a slow leak or a decreased rate of removal of such protein from the circulation, and the best example of this would be the patient with significant renal insufficiency, as TnT is clear only through the kidney, and TNI is clear both by the kidney and by vascular endothelium. The introduction of the high sensitivity assay has been met with considerable confusion, and highlights the complexity of diagnosis in heart disease. Another test that is used for the diagnosis of heart failure is in the class of natriuretic peptides (BNP, pro NT-BNP, and ANP), the last of which has been under development.
While there is an exponential increase in the improvement of cardiac devices and discovery of pharmaceutical targets, the laboratory support for clinical management is not mature. There are miRNAs that may prove valuable, matrix metalloprotein(s), and potential endothelial and blood cell surface markers, they require
1. codevelopment with new medications
2. standardization across the IVD industry
3. proficiency testing applied to all laboratories that provide testing
4. the measurement on multitest automated analyzers with high capability in proteomic measurement (MS, time of flight, MS-MS)
This article is a segment in several articles about platelets, platelet function, and advances in applying the surge of knowledge to therapy. In acute coronary syndromes, plaque rupture leads to thrombotic occlusion. We have also seen that the development of a plaque occurs in 3 stages, only the last of which involves plaque rupture. Platelets interact with the vascular endothelium, and platelet-endothelial as well as platelet-platelet interactions are known to be important in atherogenesis. We learned that platelets are derived from megakaryocytes that break up and these elements are released into the blood stream. It has recently been discovered that platelets can replicate in the circulation. The turnover of platelets is rapid, and platelets sre stored at room temperature with shaking, and are viable for perhaps only 3-4 days once they are received in the blood bank for use. In cardiology, the identification, isolation, and characterization of GPIIb/IIIa from the platelet was a huge advance in the potential for coronary intervention, and that potential became of paramount importance with the introduction of GPIIb/IIIa inhibitors as a standard in coronary vascular therapeutic procedures. The following manuscript by Barry Coller, at Rockefeller University, is a presentation of the GPIIb/IIIa story as an excellent example of Translational Medicine.
Search for GPIIb/IIIa inhibitor of the (anti-αIIb133 (GPIIb/IIIa) receptor)
The deliberate search for drugs to inhibit the αIIb133 (GPIIb/IIIa) receptor ushered in the era of rationally designed antiplatelet therapy and thus represents an important milestone in the evolution of antiplatelet drug development. The selection of the αIIb133 receptor as a therapeutic target rested on a broad base of basic and clinical research conducted by many investigators in the 1960s and 1970s working in the fields of platelet physiology, the rare bleeding disorder Glanzmann thrombasthenia, platelet membrane glycoproteins, integrin receptors, coronary artery pathology, and experimental thrombosis. Thus, αIIb133 was found to mediate platelet aggregation by virtually all of the physiology agonists (e.g., ADP, epinephrine, and thrombin) through a mechanism in which platelet activation by these agents results in a change in the conformation of the receptor. This is followed by increased affinity of the receptor for the multivalent ligands fibrinogen and von Willebrand factor, both of which are capable of binding to receptors on two platelets simultaneously, producing platelet crosslinking and aggregation. At about the same time, experimental studies demonstrated platelet thrombus formation at sites of vascular injury, and biochemical studies in humans demonstrated evidence of platelet activation during acute ischemic cardiovascular events.
Our own studies initially focused on platelet-fibrinogen interactions using an assay in which normal platelets agglutinated fibrinogen-coated beads. The agglutination was enhanced with platelet activators. Platelets from patients with Glanzmann thrombasthenia, who lack the αIIb133 receptor, did not agglutinate the beads. We adapted this assay to a microtiter plate system to identify monoclonal antibodies that inhibited platelet-fibrinogen interactions and then demonstrated that these antibodies bound to αIIb133. They were also more potent inhibitors of platelet aggregation than any known antiplatelet agent and produced a pattern of aggregation that was virtually identical to that found using platelets from patients with Glanzmann thrombasthenia.
I recognized the theoretical potential of using an antibody to inhibit platelets in vivo but also recognized the challenges and limitations. Since experimental models of thrombosis had been developed in the dog, and since the antibody we initially worked with did not react with dog platelets, we had to go back to our original samples to identify an antibody (7E3) that reacted with dog platelets in addition to human platelets. Since coating platelets with immunoglobulins results in their rapid elimination of the platelets from the circulation, and since the clearance is mediated by the immunoglobulin Fc region, we prepared F(ab’)2 fragments of 7E3 for our in vivo studies. Additional challenges included preparing large quantities of antibody on a very limited budget and purifying the antibodies so they contained only minimal amounts of endotoxin. With the small amount of 7E3-F(ab’)2 we initially prepared, we were able to show dose response inhibition of platelet aggregation in three dogs, achieving greater inhibition than with aspirin or ticlopidine, the only antiplatelet agents approved for human use at that time. We also devised an assay using radiolabeled 7E3 to quantify the percentage of platelet αIIbβ3 receptors that were blocked when a specific dose of 7E3-F(ab’)2 was administered in vivo. This allowed us to directly measure the effect of the agent on its target receptor on its target cell.
I considered two criteria most important in selecting the initial animal models in which to test the efficacy and safety of administering 7E3-F(ab’)2:
1) the model had to convincingly simulate a human vascular disease, and
2) aspirin had to have failed to produce complete protection from thrombosis.
The latter criterion was particularly important because I planned to stop this line of research if the 7E3-F(ab’)2 was not more efficacious than aspirin.
Ultimately, we collaborated with Dr. John Folts of the University of Wisconsin, who had developed a dog model of unstable angina by attaching a short cylindrical ring to partially occlude a coronary artery and using a hemostat to induce vascular injury. Pretreatment of the animal with 7E3-F(ab’)2 was more effective than aspirin or any other compound Dr. Folts had previously tested in preventing platelet thrombus formation,as judged by its effects on the characteristic repetitive cycles of platelet deposition and embolization. Electron microscopy of the vessels confirmed the reduction in platelet thrombi by 7E3-F(ab’)2, with only a monolayer of platelets typically deposited.
Dr. Chip Gold and his colleagues at Massachusetts General Hospital had developed a dog model to assess the effects of tissue plasminogen activator (t-PA) on experimental thrombi induced in the dog coronary artery. Although t-PA was effective in lysing the thrombi, the blood vessels rapidly reoccluded with new thrombi that were rich in platelets. Aspirin could not prevent reocclusion, whereas 7E3-F(ab’)2 not only prevented reocclusion, but also increased the speed of reperfusion by t-PA.
The next steps in drug development could not be performed in my laboratory because they required resources far in excess of those in my grant from the National Heart, Lung, and Blood Institute to study basic platelet physiology. As a result, in 1986 the Research Foundation of the State University of New York licensed the 7E3 antibody to Centocor, Inc., a new biotechnology company specializing in the diagnostic and therapeutic application of monoclonal antibodies.
Subsequent Development of 7E3
The subsequent development of 7E3 as a therapeutic agent required extensive collaboration among myself, a large number of outstanding scientists at Centocor, and many leading academic cardiologists. Many decisions and hurdles remained for us, including the decision to develop a mouse/human chimeric 7E3 Fab (c7E3 Fab); the design and execution of the toxicology studies; the assessment of the potential toxicity of 7E3 crossreactivity with αVβ3; the development of sensitive and specific assays to assess immune responses to c7E3 Fab; the design, execution, and analysis of the Phase I, II, and III studies; and the preparation, submission, and presentation of the Product Licensing Application to the Food and Drug Administration, and comparable documents to European and Scandinavian agencies.
Based on the results of the 2,099 patient EPIC trial, in which conjunctive treatment with a bolus plus infusion of c7E3 Fab significantly reduced the risk of developing an ischemic complication (death, myocardial infarction, or need for urgent intervention) after coronary artery angioplasty or atherectomy in patients at high risk of such complications, the Food and Drug Administration approved the conjunctive use of c7E3 Fab (generic name, abciximab) in high-risk angioplasty and atherectomy on December 22, 1994. Since then it has been administered to more than 2.5 million patients in the U.S., Europe, Scandinavia, and Asia. Its optimal role in treating cardiovascular disease continues to evolve in response to the introduction of new anticoagulants, antiplatelet agents, stents, and procedures.
Extended Investigations
We have also been able to apply the monoclonal antibodies we prepared to αIIb33 to the prenatal detection of Glanzmann thrombasthenia, and have used the antibodies as probes for characterizing both the biogenesis of the receptor and the conformational changes that the receptor undergoes with activation. We have been able to precisely map the 7E3 epitope on 33, providing additional insights into the mechanism by which it prevents ligand binding. We have also exploited the ability of another antibody to αIIb33 to stabilize the receptor complex in order to facilitate production of crystals of the αIIb33 headpiece; the x-ray diffraction properties of these crystals were studied in collaboration with Dr. Timothy Springer’s group at Harvard and provide the first structural information on the receptor.
In landmark studies in the 1980s, Pierschbacher and Ruoslahti demonstrated the importance of the arginine-aspartic acid (RGD) sequence in the interaction of the integrin α531 with fibronectin, and they went on to show that peptides with the RGD sequence could inhibit this interaction. Subsequent studies by many groups demonstrated that these peptides could also inhibit the interaction of platelet αIIb33 with fibrinogen and von Willebrand factor. Dr. David Phillip and Dr. Robert Scarbrough led the team at Cor Therapeutics that made a cyclic pentapeptide with high selectivity for αIIb33 over αV33 by patterning their compound on the KGD sequence in the snake venom barbourin. The resulting antiplatelet agent, eptifibatide, received FDA approval in May 1998. At Merck, Dr. Robert Gould led the team that developed the nonpeptide RGD-mimetic tirofiban, which also is selective for αIIb33 compared to αV33. It also received FDA approval in May 1998. Our recent x-ray crystallographic studies in collaboration with Dr. Springer’s group provided structural information on the mechanisms and sites of binding of these drugs with αIIb33.
Translation of Basic Science into Therapy
Many important elements and an enormous amount of good fortune were needed for the translation of the basic science information about platelet aggregation into the drug abciximab, including, but not limited to:
1) the support of basic studies of platelet physiology by the National Institutes of Health in my laboratory and many other laboratories,
2) the creation and ongoing funding of a core facility available to all faculty members to prepare monoclonal antibodies at the State University of New York at Stony Brook under the direction of Dr. Arnold Levine,
3) the 1988 Bayh-Dole Act and its subsequent amendments, and the expertise of the Technology Transfer Office at Stony Brook in licensing 7E3 to Centocor, which then provided the capital and additional expertise required for its development, and
4) the expert and enthusiastic collaboration by two large and disciplined cooperative groups of interventional cardiologists (TAMI, EPIC) under the dynamic leadership of Drs. Eric Topol and Rob Califf,
tirofiban, that were eager to test the safety and efficacy of the 7E3 derivatives. Although the translation of each new scientific discovery into improved health via novel preventive, diagnostic, or therapeutic strategies requires the blazing of a unique path, optimizing these elements and similar ones may allow the path to be shorter and/or to be traversed more easily, at a lower cost, or in a shorter period of time.
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 1 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/10.1186/1479-5876-7-42http://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). 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). 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. 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 ml—1) 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 dl—1) [NL Plts] or 17 mM (300 mg dl—1) [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 ml—1) 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 ml—1) 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
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. 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( (33 activation. 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.
This article is one of a 2 part presentation posted as an example of a central role of platelet biology in translational medicine investigations leading to the prevention and control of hemolytic and coagulopathic conditions, and to an understanding of atherosclerotic cardiovascular disease. The study of coagulation traces back to the early work on Warfarin in bleeding, and even earlier than that to the geneological evidence of inherited hemophilia in the Royal family of 18th Century Victoria. The amount of work has been voluminous, and the conceptual framework has been difficult to put into practice over generations of postgraduate physicians. No wonder, considering the clotting proteins and the amazing platelet.
Part I of Platelets in Translaional Research is a comprehensive coberage of the signaling and control involved in platelet-endothelial reactions, platelet-platelet reactions, and platelet transciptomics, all of which have a significant bearing on atherosclerotic plaque buildup, plaque rupture, and acute coronary syndrome as well as chronic ischemic heart disease.
Part II will cover a range of studies pointing to anti-platelet therapeutic targets.
Related work in Pharmaceutical Intelligence
Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
David Gregg, MD; Pascal J. Goldschmidt-Clermont, MD
Duke University Medical Center, Durham, NC.
Platelets are specialized disk-shaped cells in the blood stream that are involved in the formation of blood clots that play an important role in heart attacks, strokes, and peripheral vascular disease. In most people, the more than 200 million platelets in a milliliter of blood act as tiny building blocks to form the basis of a clot to stop bleeding from cuts or injuries. Platelets can detect a disruption in the lining of a blood vessel and react to build a wall to stop bleeding.
Figure 1. Platelets form a platelet plug to stop bleeding from an injured blood vessel.
In cardiovascular disease, abnormal clotting occurs that can result in heart attacks or stroke. Blood vessels injured by smoking, cholesterol, or high blood pressure develop cholesterol-rich build-ups (plaques) that line the blood vessel; these plaques can rupture and cause the platelets to form a clot. Even though no bleeding is occurring, platelets sense the plaque rupture and are confused, thinking that an injury has taken place that will cause bleeding. Instead of sealing the vessel to prevent bleeding as would occur with a cut, a clot forms in an intact blood vessel, causing a blockage of blood flow (Figure 2). Without blood, a portion of the heart muscle can die, leading to a heart attack.
Figure 2. Plaque rupture results in clot formation to block blood flow, which may result in a heart attack or stroke
Platelet Disorders
Platelets may be abnormal either quantitatively (too many or too few) or qualitatively (the right number but they do not work correctly). The number of platelets is routinely tested as part of the complete blood count (CBC). Normal counts range from 150 000 to 450 000. A decrease in the number of platelets indicates a condition known as thrombocytopenia and may result in increased bleeding, the first signs of which may include gum bleeding, nose bleeds, and increased bruising. In cardiology, the most frequent cause of a low platelet count is an abnormal immune response caused by drug therapy, particularly with the intravenous blood thinner heparin (heparin-induced thrombocytopenia), and rarely with other drugs to control high blood pressure or symptoms of congestive heart failure (diuretics), to control diabetes (antidiabetic medications), or to regulate your blood clotting (antiplatelet drugs). Elevated platelet counts can also occur, usually in association with diseases in the elderly, and can result in either excess clotting or even abnormal bleeding.
Because platelets are so important in stopping bleeding from everyday injuries such as cuts or bruises, severe inherited disorders of platelets are quite rare. Researchers, however, have discovered more subtle genetic variations in platelets called polymorphisms that may alter platelets in subtle ways to raise the risk of cardiovascular disease when combined with other risk factors, but which on their own do not result in overt disease. These polymorphisms may also be important in understanding who may gain the greatest benefit from anti-platelet drugs.
The most commonly used antiplatelet agent is aspirin, although you may also be prescribed other oral agents, such as ticlopidine, clopidogrel, or dipyridamole, or intravenous antiplatelet drugs such as abciximab or eptifibatide while you are in the hospital or undergoing angioplasty procedures. Each agent affects platelets in slightly different ways and may have unique side effects, but all either cause the platelets to stick together or induce them to clot less well. Your doctor will choose the drug that best suits your situation. Table 1 shows some of the unique features of commonly used oral antiplatelet drugs
TABLE 1. Commonly Used Oral Antiplatelet Drugs: Uses and Side Effects
Aspirin
Clopidogrel
Ticlopidine
Dipyridamole + Aspirin
Uses
Heart disease and stroke; inexpensive
Heart disease and stroke, particularly after stenting
Mostly in stroke; requires blood count monitoring
Stroke; may not be suitable for patients with heart disease
Side effects
Gastrointestinal (GI) intolerance; GI bleeding
Diarrhea (much less common than with ticlopidine); rash and itching
Diarrhea and GI upset (usually resolves in 2 weeks); may decrease blood counts, particularly white blood cells
Headache; GI bleeding; GI intolerance
Antiplatelet drugs are different than blood thinners or anticoagulants such as warfarin (Coumadin, Bristol-Myers Squibb) or heparin. Anticoagulants block a second step in clotting known as coagulation but do not directly affect the platelets.
Eur J Cardiovasc Nurs. 2002 Dec;1(4):273-88.
Platelets and cardiovascular disease
Willoughby S, Holmes A, Loscalzo J.
Queen Elizabeth Hospital, Adelaide University, South Australia, Adelaide, Au
Platelets play an important, but often under-recognized role in cardiovascular disease. For example, the normal response of the platelet can be altered, either by increased pro-aggregatory stimuli or by diminished anti-aggregatory substances to produce conditions of increased platelet activation/aggregation and occur in active cardiovascular disease states both on a chronic (e.g. stable angina pectoris) and acute basis (e.g. acute myocardial infarction). In addition, platelet hyperaggregability is also associated with the risk factors for coronary artery disease (e.g. smoking, hypertension, and hypercholesterolaemia). Finally, the utility of an increasing range of anti-platelet therapies in the management of the above disease states further emphasizes the pivotal role platelets play in the pathogenesis of cardiovascular disease. This paper provides a comprehensive overview of the normal physiologic role of platelets in maintain homeostasis, the pathophysiologic processes that contribute to platelet dysfunction in cardiovascular disease and the associated role and benefits of anti-platelet therapies. PMID: 14622657
Triggering of Plaque Disruption and Arterial Thrombosis in an Atherosclerotic Rabbit Model
GS Abela, PD Picon, SE Friedl, OC Gebara, A Miyamoto, et al.
Deaconess Hospital, Harvard Medical School, Boston, Mass; Federal Univ and Univ Passo Fundo (P.D.P.), Rio Grande de Sul, Brazil; Heart Institute, Univ São Paulo (O.C.G.), São Paulo, Brazil; National Defense Medical College (A.K.), Saitama, Jp.
It is now recognized that plaque disruption and thrombosis, a process often triggered by activities of the patient, is generally the cause of the onset of acute coronary syndromes. Plaque disruption and subsequent arterial thrombosis are now recognized as critical to the onset of acute coronary ischemic syndromes. It is hypothesized that occurrence of thrombotic coronary occlusion has three components. First, a plaque that is vulnerable to disruption must be present. Second, acute physiological events are required to induce plaque disruption and thrombosis. Third, a relatively hypercoagulable state and heightened vasomotor tone increase the likelihood that arterial thrombosis will produce complete lumen occlusion.
In human patients, the opportunity to study factors responsible for acute onset of myocardial infarction is limited because coronary angiography performed before the event cannot prospectively identify plaques vulnerable to disruption. After the event, angiography cannot distinguish the features of the plaque responsible for the disruption from those resulting from the disruption. Moreover, plaque disruptions producing total vascular occlusion and death may be more severe than those occurring in asymptomatic individuals or in patients with unstable angina or nonfatal myocardial infarction. These difficulties, inherent in the study of plaque disruption and thrombosis in human patients, create a great need for an animal model of the process. An atherosclerotic rabbit model of triggering of arterial thrombosis that was introduced by Constantinides and Chakravarti more than 30 years ago but not subsequently used. Aortic plaques were induced by a high-cholesterol diet, by mechanical balloon injury of the artery, or by a combination of the two. Triggering was attempted by injection of Russell’s viper venom (RVV), which is a proteolytic procoagulant, and histamine. A recent review of the animal models of thrombosis currently in use noted that “thus far, it has not been possible to duplicate in a model the most common clinical cause of thrombosis—an ulcerated atherosclerotic plaque.” The advantage of the Constantinides model over other animal models used to study thrombosis is that it uses a biological intervention to trigger localized atherosclerotic plaque disruption and formation of platelet-rich arterial thrombi. Disadvantages of the Constantinides model are (1) the low yield of triggering (only about one third of the rabbits developed thrombosis) and (2) the long (8-month) preparatory period. In addition, there is a need to replicate the findings of Constantinides and Chakravarti13 from 30 years ago because of the biological variability of rabbit strains and RVV. It cannot be assumed that the rabbits and RVV currently available will produce the results obtained in the 1960s.
A total of 53 New Zealand White rabbits were exposed to one of four preparatory regimens: rabbits in group I (n=9) were fed a regular diet for 8 months; rabbits in group II (n=13) were fed a diet of 1% cholesterol for 2 months alternated with 2 months of a regular diet for a total of 8 months; rabbits in group III (n=5) underwent balloon-induced arterial wall injury, then were given a regular diet for 8 months; and rabbits in group IV (n=14) underwent balloon-induced arterial wall injury, then were given a diet of 1% cholesterol for 2 months followed by a regular diet for 2 months for a total of 4 months. After completion of the preparatory regimen, triggering of plaque disruption and thrombosis was attempted by injection of RVV (0.15 mg/kg IP) and histamine (0.02 mg/kg IV). In group I, normal control rabbits without atherosclerosis, only one small thrombus was noted in 1 of 9 rabbits. In group II, cholesterol-fed rabbits, thrombosis occurred in 3 of 13 rabbits. Thrombus occurred in all rabbits in group III (5 of 5) and in 10 of 14 rabbits in group IV. Although the frequency of thrombosis was not significantly different between groups I and II, possibly due to a small sample size, it was significantly different among all four groups (P<.001). Also, the frequency and amount of thrombus formation were significantly different among all four groups (P<.001; P<.0001) but not between groups I and II.Rabbits with atherosclerosis (those in groups II and IV) demonstrated plaque disruption and overlying platelet-rich thrombus formation similar to that observed in patients with acute coronary syndromes. The surface area covered by thrombus was 2 mm2 in group I, 15.3±19.2 mm2 in group II, 223±119 mm2 in group III, and 263±222 mm2 in group IV. Rabbits in groups III and IV had the greatest amount of thrombus, and this amount was significantly greater than in rabbits in groups I and II (P<.001 and P<.03, respectively).
The intima in group I rabbits appeared normal by gross inspection. In group II rabbits, white-yellow plaque was widely distributed over the arterial surface, with focal punctate ulceration occasionally noted under a dissecting microscope. In group III rabbits, the intima was smooth and widely covered with white plaque.Group IV rabbits had extensive sheets of elevated white-yellow plaque. By gross visualization, ulceration of the surface was present without superimposed thrombus in two rabbits in group IV. In sections from groups II and IV, some areas of plaque directly adjacent to the thrombi had marked thinning of the connective tissue cap and areas of dehiscent foam cells. These observations were rare and were noted in <0.5% of the examined lesions. In most cases, the arterial thrombus was not located at a site of obvious plaque rupture. Foam cell infiltration was also noted adjacent to sites of thrombosis. Scanning electron microscopy demonstrated fissures of various lengths below areas from which overlying thrombi were removed. Endothelial cells could be seen lining the intimal surface of the aorta in the rabbits that had undergone balloon-induced arterial wall injury 8 months earlier. Surface blebs and focal endothelial breakdown with ulcer formation, without grossly visible thrombosis, were occasionally seen in samples from groups II and IV. The base of these ulcers was layered with platelets, fibrin, and red blood cells. Transmission electron microscopy of areas with thrombosis confirms that the thrombi were platelet rich.
In the two groups that received cholesterol feeding, the total cholesterol content in tissue samples pooled from the thoracic and abdominal aorta was significantly higher in group IV (16±7.2 mg/g) than in group II (2.8±1.6 mg/g) (P<.0001). Rabbits that were maintained on a regular diet (groups I and III) had equally low levels of tissue cholesterol (0.05±0.04 versus 0.06±0.02 mg/g, P=NS). The average fibrinogen level before triggering in the 27 rabbits in which fibrinogen was measured was 210±119 mg/dL; it rose to 403±168 mg/dL 48 hours after triggering (P<.001). Plasma fibrinolytic activity did not change after triggering (85.5±37.8 versus 94.8±33.5 arbitrary units). Platelet counts (measured in only 19 rabbits in groups II and IV) decreased from 350±84×103 to 215±116×103 per cubic millimeter after triggering (P<.001).
Conclusions
The results demonstrate that vulnerable plaques can be produced and that plaque disruption and platelet-rich arterial thrombus formation may be triggered pharmacologically in an animal model of arterial plaque. This finding documents that the New Zealand White rabbit strains and the RVV currently available can be used to obtain the same results observed by Constantinides and Chakravarti13 more than 30 years ago. This animal model is suitable for the study of plaque disruption and arterial thrombosis. Hypercholesterolemia and mechanical arterial wall injury seemed to produce plaques vulnerable to triggering of disruption and thrombosis, whereas normal arteries were relatively resistant to triggering. The model provides a method to evaluate agents that might decrease the occurrence of vulnerable plaques or the amount of thrombus formed after triggering. Most important, the model can be used to identify the features of vulnerable plaques and the pharmacological stressors that trigger plaque disruption and thrombus formation.
Certain features of the lesions seen in this model are similar to those of human lesions seen at autopsy of patients with fatal myocardial infarction, ie, a lesion with a fissured collagen cap overlying a lipid mass of amorphous and crystalline lipid. However, most of the lesions in the model did not have these features and were more consistent with a recent pathological study of fatal coronary thrombosis, which revealed that in approximately half the cases, the plaque was relatively intact but an inflammatory infiltrate was present. Perhaps the incidence of plaque rupture causing thrombus may be even lower in patients with nonfatal coronary thrombosis, as suggested from angioscopic studies of coronary arteries that have shown plaque ulceration of various severities. Analyses of human plaques have demonstrated that disrupted plaques have significantly less collagen, glycosaminoglycans, and smooth muscle cells and more extracellular lipid and macrophagesthan do nondisrupted plaques. This is consistent with findings in our study that rabbits in group II had more connective tissue and a lower rate of disruption and thrombosis than those in groups III and IV.
references
Herrick JB. Clinical features of sudden obstruction of the coronary arteries. JAMA. 1912;59:2015-2020.
Chapman I. Morphogenesis of occluding coronary artery thrombosis. Arch Pathol. 1965;80:256-261.
Friedman M, van den Bovenkamp GJ. The pathogenesis of a coronary thrombus. Am J Pathol. 1966;80:19-44.
This reader sees a validation in this study of the noted cardiologist, Alan Jaffe, at Mayo Clinic, in referring to Type I and Type II myocardial infarcts, which accounts for differences in troponin elevations in patients.
The Platelet in Cardiovascular Disease
1. microthrombi adhering to foam cells
2. Platelets secrete
– Platelet-derived growth factor (PDGF) that promotes smooth muscle cell migration and collagen production
– Plasminogen activator inhibitor-1 (PAI-1) that suppresses fibrinolysis
Davies MJ, Woolf N, Rowles PM, et al. Morphology of the endothelium over atherosclerotic plaques in human coronary arteries. Br Heart J 1988;60:459-64.
– Microhemorrhages attract and activate neighboring platelets, support fibrin generation
Inoue M, Itoh H, Ueda M, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: Possible pathophysiological significance in progression of atherosclerosis. Circulation 1998;98:2108-16.
Systems biology of platelet-vessel wall interactions
Scott L. Diamond*, Jeremy Purvis, Manash Chatterjee and Matthew H. Flamm
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA
Front Phys 26 August 2013 http://dx.doi.org/10.3389/fphys.2013.00229
Blood systems biology seeks to quantify outside-in signaling as platelets respond to numerous external stimuli, typically under flow conditions. Platelets can activate via GPVI collagen receptor and numerous G-protein coupled receptors (GPCRs) responsive to ADP, thromboxane, thrombin, and prostacyclin. A bottom-up ODE approach allowed prediction of platelet calcium and phosphoinositides following P2Y1 activation with ADP, either for a population average or single cell stochastic behavior. The homeostasis assumption (i.e., a resting platelet stays resting until activated) was particularly useful in finding global steady states for these large metabolic networks. Alternatively, a top-down approach involving Pairwise Agonist Scanning (PAS) allowed large data sets of measured calcium mobilization to predict an individual’s platelet responses. The data was used to train neural network (NN) models of signaling to predict patient-specific responses to combinatorial stimulation. A kinetic description of platelet signaling then allows prediction of inside-out activation of platelets as they experience the complex biochemical milieu at the site of thrombosis. Multiscale lattice kinetic Monte Carlo (LKMC) utilizes these detailed descriptions of platelet signaling under flow conditions where released soluble species are solved by finite element method and the flow field around the growing thrombus is updated using computational fluid dynamics or lattice Boltzmann method. Since hemodynamic effects are included in a multiscale approach, thrombosis can then be predicted under arterial and venous thrombotic conditions for various anatomical geometries. Such systems biology approaches accommodate the effect of anti-platelet pharmacological intervention where COX1 pathways or ADP signaling are modulated in a patient-specific manner.
CLOTTING UNDER FLOW CONDITIONS
Collagen is sufficient to capture and activate platelets under venous wall shear rates (ãw 100–200s_1). In the arterial circulation (ãw 1000–2000 s_1), collagen adsorbed von Willebrand factor (vWF) facilitates platelet capture, allowing collagen induced GPVI signaling and subsequent á2â1 and á2bâ3 activation. Under flow conditions, red blood cells help enrich the platelet concentration by 3–8x in the plasma layer near the wall. At pathological high shear exposures (>5000 s_1) encountered in severe stenosis, mechanical heart valves, and continuous LVAD pumps, the plasma vWF may undergo structural changes, such as a transition from a globular to an extended state (Schneider et al., 2007), likely increasing the availability of A1 domains in the vWF polymer for multivalent contacting with platelet GPIb receptors.
GROWTH OF THE PLATELET AGGREGATE VIA AUTOCATALYTIC SIGNALING
Collagen triggers GPVI clustering, leading to rapid phosphorylation of the GPVI-associated Fc receptor by Src family tyrosine kinases. Such phosphotyrosine residues are recognized by Syk, and the binding and activation of Syk activates PLCã2. PLCã2 converts phosphatidylinositol (PI)-4,5-P2 (PIP2) to inositol 1,4,5-trisphosphate (1,4,5-IP3 or IP3) and diacyclglycerol (DAG). IP3 opens Ca2+ channels in the platelet dense tubular system (DTS). Depletion of DTS Ca2+ results in STIM1 activation and binding to Orai1, leading to store operated calcium entry (SOCE). DAG/Ca2+ activates protein kinase C (PKC) in platelets, which in turn governs several serine/threonine phosphorylation events.
Beyond the first monolayer of platelets adherent to colla-gen/VWF, the addition of subsequent layers of platelets to the growing thrombus is strongly potentiated by locally released ADP and thromboxane (TXA2) as well as locally generated thrombin. ADP activates P2Y1 and P2Y12 while TXA2 activates the TP receptor and thrombin cleaves PAR1 and PAR4. Activation of a GPCR causes an exchange of GTP for GDP on the α subunit of the G protein and dissociation of the α and γ subunits. Both these units in turn interact with secondary effectors such as PLC and adenylate cyclase. Human platelets express at least 10 forms of Gα (including members of the Gq, Gi, G12, and Gs families) (Brass et al., 2006; Offermanns, 2006). Thrombin, ADP, and TXA2 activate PLC via Gq. PLC generates IP3 from membrane PIP2. Rising Ca2+ levels activate the Ras family member, Rap1B via Cal-DAG GEF. Rap1B activation is a precursor to αIIb 3 activation and allows the platelets to form aggregates with other platelets through fibrinogen cross-bridging. Ca2+-dependent signaling drives myosin light chain kinase and activation of GTP binding proteins of the Rho family. Rho activation in turn activates kinases like p160ROCK and 5 LIM-kinase that can phosphorylate myosin light chain kinase and cofilin to regulate actin-dependent cytoskeletal shape changes. Endothelial derived prostacyclin (PGI2) binds the IP receptor and causes Gs mediated increase in adenyl cyclase activity. Also, NO from the endothelium and platelets can activate guany-late cyclase resulting in elevated cGMP levels that subsequently inhibit the hydrolysis of cAMP by intracellular phosphodi-esterases. Taken together these mechanisms elevate intracellular cAMP levels, which strongly downregulate platelet signaling. Agonists coupled to Gi family members inhibit cAMP production in platelets, thus allowing activation to proceed unhindered. Additionally the âã subunits of these receptors can activate PLCâ and the ã isoform of PI3K. The effectors for PI3K include Rap1b and Akt.
FIGURE 1 | Detailed reaction schemes for platelet signaling modules. Four interconnected models were defined: (A) Ca2+ module: cytosolic and DTS compartments are separated by the DTS membrane, which contains the IP3R and SERCA. (B) Phosphoinositide (PI) module: Membrane-bound PIs are cleaved by PLC-â to form diffusible inositol phosphates and DAG, which are substrates for resynthesis of PIs. (C) PKC module: Ca2+i and DAG activate PKC, which migrates to the plasmamembrane where it phosphorylates PLC-â. (D) P2Y1 module: extracellular ADP binds to and activates P2Y1. Active P2Y1 accelerates guanine nucleotide exchange on bound Gq. The Gq·GTP binds and activates PLC-â, which increases the GTPase activity of Gq·GTP.
ADP is stored in platelet dense granules and is released upon activation. P2Y1 and P2Y12 are the primary receptors for this agonist. P2Y1 is Gq coupled and signaling through this receptor causes Ca2+ mobilization, shape change, and thromboxane generation. P2Y12 is the target of the commonly used anti-platelet drug Plavix, and is a Gi2 coupled receptor that inhibits cAMP production in platelets. Thrombin is a potent platelet agonist that causes fast mobilization of intracellular Ca2+, and activation of phospholipase A2 and subsequent thromboxane generation (Offermanns et al., 1997). Also, thrombin can trigger Rho dependent signaling pathways in platelets (Moers et al., 2003), that contribute to actin modeling and shape change. Thrombin signals through the protease-activated receptor (PAR) family of GPCRs. PAR1 and PAR4 are expressed on human platelets, while PAR3 and PAR4 are expressed on mouse platelets. Thrombin cleaves the N-terminus of these receptors, exposing a new N-terminus that serves as a tethered ligand for these receptors. Synthetic peptides are able to selectively activate these receptors and mimic the actions of thrombin (for example, SFLLRN for PAR1, and AYPGKF for PAR4). Kinetic studies have shown that the human platelet response to thrombin is biphasic and involves first signaling through PAR1 and subsequent signaling through PAR4 (Covic et al., 2000). In mouse platelets signaling occurs primarily via PAR4, and is facilitated by PAR3. In addition to the PAR receptors, GP1bá has high affinity for thrombin. Absence of GP1bá reduces responses to low doses of thrombin and diminishes PAR1 signaling, suggesting that this receptor facilitates signaling through the PARs (Dormann et al., 2000). Ca2+ mobilization also activates phospholipase A2 (PLA2), which in turn converts membrane phospholipids to arachidonic Acid. TXA2 is produced from membrane arachidonate by the aspirin sensitive cyclooxygenase (COX-1) enzyme. TXA2 causes Ca2+ mobilization, aggregation, secretion, phosphoinositide hydrolysis, and protein phosphoryla-tion. TXA2 can diffuse across the membrane and activate nearby platelets, but its activity is limited by the molecule’s short half life (∼30 s).
These modules use previously validated or data-consistent kinetic networks for SERCA, IP3-Receptor, PKC translocation, and GPCR signaling (Figures 1E–H). Assembling the four modules together results in a global ODE model that has 77 reactions, 132 fixed kinetic rate constants, and 70 species. Since the reaction network (Figure 1) and the kinetic parameters are fixed, the reaction topology of the model is also fixed. Such a model takes the general form: dc/dt = F(c) and c(t = 0) = co where c is a vector of all species concentrations and co is a specified initial condition vector at t = 0. To determine appropriate sets of co that are suitable for use in modeling platelets, a challenge exists that the copy number of each species in a resting platelet is not known. Imposing a homeostasis assumption results in powerful tool to define a set of acceptable co vectors. The homeostasis assumption states that a resting platelet remains resting until activated. This means that an acceptable initial condition co also represents a steady state for the system and will satisfy the equation dc/dt = 0. Finding a global co involves assembling the steady state solutions of each module (Figure 2).
FIGURE 2 | Homeostasis requirement: Assembly of full model from steady-state modules using principle component analysis (PCA). The full model is assembled by combining PCA-reduced, steady-state solution spaces from each module into a combined steady state solution space. This global space is searched for full-length, steady-state solution vectors that satisfy both the steady state requirements of each module and the desired time-dependent properties when the steady-state is perturbed. A simple linear constraint is imposed for every pair of modules that share a common molecule ci to ensure that steady state solutions are Keywords: platelet, thrombosis, hemodynamic, ADP, thromboxane consistent. To assemble the platelet signaling model, a set of 16 PC vectors representing all 72 unknown variables in the model were used as search directions in a global optimization routine. The global solution space was searched for models with accurate dynamic behavior using experimental time-series data for ADP-stimulated Ca2+ release. Species are grouped according to compartment. Color values correspond to molar concentrations (mol/L or mol/m2) or as indicated: ∗DTS species (mol L−1). †Extracellular species (mol L−1). ‡DTS volume (L). §PM leak conductance/area (S m−2).
The first phase of the method involves generating a compact representation of the steady-state solutions for each module. First, conservative bounds are chosen for c based on physiological and practical considerations. Also, because molecular concentrations can span several orders of magnitude, it is most efficient to delineate this range of values on a logarithmic scale rather than a linear scale. Once the sampling distribution for c has been defined, steady-state solutions (co = c55) for each module are calculated using fixed kinetic parameters for each reaction in the module. For non-oscillating systems, steady-state solutions may be obtained by simulating the system until equilibrium is reached (i.e., until dc/dt = 0). In the third step, a large collection of steady-state solutions for each module is subjected to principal component analysis (PCA) (Purvis et al., 2009). PCA is then used to transform these points to a new coordinate set that optimally covers the space of steady-state solutions using the fewest number of dimensions. For example, if two molecule concentrations in the steady-state space are highly correlated due to participation in the same reaction, PCA will locate a single dimension to represent each pair of points in the transformed space. Ultimately, these new dimensions will be combined across all modules to search for global solutions that lie in the steady-state space for the fully combined network. Since PCA is a linear method, a steady-state solution space that is highly nonlinear may require more principal component vectors to accurately estimate the solutions. The reduction procedure is shown for the human platelet model comprising 4 interlinked signaling modules (Figure 2). For this step, we generated more than 109 sets of initial guesses (co) for each module, computed the initial value problem for each co until a steady state was reached (dc/dt ≈ 0), and selected only those steady states (c55) that were consistent with known concentrations (i.e., [Ca2+]o ∼100 nM). Interestingly, only a small fraction of initial guesses produce steady-state solutions that are also consistent with known concentration values. For example, it was shown that only 50,000 of 109 initial guesses (0.005%) in the Ca2+ balance module (Figure 1A) met both requirements and were suitable for further analysis. This observation shows that the kinetic topology of these molecular networks places very strong constraints on the range of concentrations that can exist at steady state. In biological terms, this suggests that fixed kinetic properties at the molecular level (e.g., IP3R and SERCA kinetics) can affect not only the dynamical features of a biochemical system but can also determine the abundance of chemical species and the compartmental structures that contain them. A fully assembled initial condition vector results (bottom, Figure 2) results in new hypotheses about allowable concentrations and ratios of concentrations (i.e., IP3/SERCA ratio is very small). The allowed co = css is consistent with the known resting levels of Ca2+, IP3, P2Y1, DAG, PA, PI, PIP2, and PIP (bottom, Figure 2) as well as the stimulated response of platelets to increasing amounts of ADP (right, Figure 2). With a global simulation of P2Y1 signaling, it is possible to simulate the ADP dose-response of calcium mobilization and IP3 generation in platelets as well as the mobilization of intracellular calcium in a single platelet due to stochastic fluctuations (Figure 3).
FIGURE 3 | Tests of P2Y1 signaling model. ADP dose response for the full platelet model from 100 nM to 10 ìM ADP for calcium mobilization (A) or IP3 generation (B). Stochastic simulation of a single platelet (C). A single, fura-2-loaded platelet was immobilized on a fibrinogen-coated coverslip and activated with 40 ìM ADP at t = 90 [Ca2+ trace from Heemskerk et al. (2001)]. After 90 s of simulated rest, the platelet model was activated by setting extracellular [ADP] to 40ìM. Simulated interval times were binned in 2s increments for direct comparison with experiment (inset).
Since many initial condition vectors can be found to allow a resting platelet to remain resting and then respond appropriately to stimulation, investigation of these multiple steady states and associated cell responses can allow an ad-hoc sensitivity analysis. Some species (flexible nodes) may vary widely in the allowed initial condition vectors but have little effect on system response. In contrast, other species (rigid nodes) may be forced to take on values in a very narrow range due to the kinetic constraints of the problem.
To examine the changes in steady-state properties caused by kinetic perturbations in the P2Y1 model, we altered the rates of important regulatory reactions and observed the system response to each perturbation. Each perturbation cause a brief adjustment phase lasting ∼200 s followed by a more gradual phase characterized by a new steady-state profile. After 1 h of simulated time, steady-state concentrations and reaction fluxes were quantified relative to their original steady-state levels (Figure 4). In a computational perturbation, the inhibition of phospholipase C-β (PLC-β) activity by PKC was reduced 10-fold. Since PKC has a negative-feedback role in suppressing the platelet-stimulating activity of PLC-β, this perturbation caused a 2-fold increase
in steady-state PIP2 hydrolysis, elevated IP3 concentration, and accelerated Ca2+ release. This was a compensatory effect caused by the negative feedback loop involving Ca2+-regulated activity of PKC, a resulting new hypothesis that can be probed experimentally. In another example, increasing the hydrolytic activity of PLC-â for the substrate PIP2 by 10-fold caused an expected stimulatory effect, raising intracellular calcium and steady-state levels of cytosolic inositol phosphates (IP3, IP2, and IP) between 2- and 3-fold. Interestingly, reaction fluxes for phosphoinositide hydrolysis were diminished, possibly due to substrate depletion. Taken together, these examples illustrate the system-wide effects of perturbations in the kinetic rate processes. The procedure could easily be extended to examine multiple simultaneous perturbations in both reaction rates and steady-state concentrations. In future applications of this approach, genomic or proteomic information of multiple perturbations could be used to help predict platelet signaling phenotypes.
FIGURE 4 | Shifts in steady-state profiles caused by kinetic perturbations. The steady-state platelet model was perturbed by changing selected kinetic parameters (±10-fold) and simulating for 1 h. After approaching a new steady state, the model concentrations and fluxes were determined relative to their original steady-state values and colored according to fold-change. Green indicates no change (NC) relative to initial flux/concentration. Red indicates a relative increase and blue indicates a relative decrease. Note that the color scale in each panel is normalized separately to maximize distinctions in fold change. New steady states were achieved after (top) 10-fold decrease in PKC-mediated inhibition of PLC-β, and (bottom) 10-fold increase in PIP2 hydrolysis (10-fold increase in kcat of hydrolysis). ∗, active state.
FIGURE 5 | Pairwise agonist scanning to predict global calcium response in human platelets. (A) Simplified schematic of signaling pathways examined in this study that converge on intracellular calcium release in human platelets. (B) Dynamic NN model used to train platelet response to combinatorial agonist activation. A sequence of input signals representing agonist concentrations is introduced to the network at each time point. Processing layers integrate input values with feedback signals to predict the next time point. (C) A total of 154 calcium traces were measured for single and pairwise activation using 6 different agonists (“Experiment”) and used for neural network training. The NN training accurately predicted (“NN Prediction”) the training data.
FIGURE 6 | Multiscale modeling. The multiscale model has four main components (A) fluid flow, transport of soluble species, motion and binding of platelets, and the activation state of each platelet. The fluid flow is perturbed by the growing clot and is determined using the lattice Boltzmann method. The released soluble agonists form a boundary layer in the flow, and this process is determined using the finite element method. Platelet motion and bonding are simulated with lattice kinetic Monte Carlo. Platelet activation state is estimated from the history of intracellular calcium concentration, which is determined by a neural network model. (B) Multiscale simulation of patient-specific platelet deposition under flow for a specific donor and PAS-trained neural network of calcium signaling. Platelet activation (black, unactivated; white, activated) and deposition at 500 s (inlet wall shear rate, 200 s−1) showing released ADP (top) and TXA2 (middle) and perturbation of the flow field (bottom). Flow: left to right (streamlines, black lines); surface collagen (250 ìm long): red bar.
PLATELET INTERACTIONS WITH THE VESSEL WALL
The multiscale systems biology model accommodates platelet signaling, platelet adhesion to collagen and other activated platelets, release of soluble agonists, thrombus growth, and distortion of the prevailing flow field (Figure 6A). The lattice Boltzmann (LB) method is used to solve for the velocity field of the fluid. Platelets in the growing aggregate release ADP and TXA2 into the fluid, and a boundary layer is formed with the flow. The dynamics of this process are determined with a finite element method solution of the convection-diffusion-reaction equation for each of the soluble species, ADP and TXA2. Platelets move in the fluid by convection and RBC-augmented dispersion. They also bind to the collagen surface as well as previously bound platelets. The motion and binding of platelets is simulated using the convective lattice kinetic Monte Carlo (LKMC) algorithm validated for stochastic convective-diffusive particle transport (Flamm et al., 2009, 2011, 2012). The level of integrin activation and associated adhesiveness for each platelet is related to the cumulative intracellular calcium concentration. The intracellular calcium concentration is determined using a NN trained on a specifc patient’s platelet PAS phenotyping experiment. Using this multiscale approach, Multiscale simulations predicted the density of platelets adherent to the surface, platelet activation states, as well as the spatiotemporal dynamics of ADP and TXA2 release, morphology of the growing aggregate, and the distribution of shear along the solid-fluid boundary (Figure 6B). Platelets stick to the collagen surface and release ADP and TXA2 which forms a boundary layer extending up to 10 pm from the thrombus. Boundary layer concentrations of up to 10 pM ADP and 0.1 pM TXA2 were found by simulation. TXA2 concentrations were found to be sub-physiological (<0.0067 pM or <0.1 xEC50) until a sufficient platelet mass accumulated at the surface after ∼250 s. Boundary layer ADP concentrations were within the effective dynamic range (0.1–10 pM) throughout the simulation. The strong temporal and spatial fluctuations in the concentration of ADP were predominately driven by the short release time (5 s), whereas the longer release time of TXA2 (100 s) smoothed fluctuations. The shear rate along the solid-fluid boundary became nonuniform during the simulation (5–10-fold increase above 200 s−1) due to surface roughness. At 500 s, the platelet deposit was characterized by platelet clusters 20–30 pm in length, fully consistent with microfluidic measurements of platelet cluster size on collagen at this shear rate.
Developing tools to define platelet variations between patients and the relationship of platelet phenotype to prothrombotic or bleeding traits will have significant impact in stratifying patients according to risk. This multiscale approach also makes feasible patient-specific prediction of platelet deposition and drug response in more complex in vivo geometries such as stenosis, aneurysms, stented vessels, valves, bifurcations, or vessel rupture (for prediction of bleeding risks) or in geometries encountered in mechanical biomedical devices.
Platelet–Leukocyte–Endothelial Cell Interactions After Middle Cerebral Artery Occlusion
Summary: The adhesion of both leukocytes and platelets to microvascular endothelial cells has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds. The objectives of this study were to (1) assess the platelet–leukocyte–endothelial cell interactions induced in the cerebral microvasculature by middle cerebral artery occlusion (MCAO)/reperfusion, and (2) define the molecular determinants of the prothrombogenic and inflammatory responses in this model of focal I/R. MCAO was induced for 1 hour in wild-type (WT) mice, WT mice treated with a monoclonal antibody (mAb) to either P-selectin or GPIIb/IIIa, and in P-selectin−/−(P-sel−/−) chimeras. Isolated platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) were administered intravenously and observed with intravital fluorescence microscopy. Leukocytes were observed after intravenous injection of rhodamine 6G. One hour of MCAO followed by 1 hour of reperfusion resulted in the rolling and adhesion of leukocytes in venules, and after 4 hours of reperfusion, the adhesion of both leukocytes and platelets was detected. Although both the P-selectin and GPIIb/IIIa mAbs significantly reduced the adhesion of leukocytes and platelets at 4 hours of reperfusion, the antiadhesive effects of the P-selectin mAb were much greater. The leukocyte and platelet adhesion responses were significantly attenuated in both P-sel−/−-WT and WT-P-sel−/− bone marrow chimeras, compared with WT-WT chimeras. Neutropenia, induced by antineutrophil serum treatment, also reduced the recruitment of leukocytes and platelets after cerebral I/R. These findings implicate a major role for both platelet-associated and endothelial cell– associated P-selectin, as well as neutrophils in the inflammatory and prothrombogenic responses in the microcirculation after focal cerebral I/R.
Key Words: Platelet—Leukocyte—P-selectin—GPIIb/IIIa—Cerebral ischemia—Reperfusion.
Adhesion of leukocytes and platelets after treatment with mAb against P-selectin or GPIIIb/IIIa
The I/R-induced recruitment of rolling and adherent leukocytes was significantly attenuated in P-selectin mAb-treated mice, compared with the responses noted in untreated mice exposed to 1-hour MCAO and 4-hour reperfusion (Figs. 3A and 3B). However, the number of adherent leukocytes after P-selectin mAb treatment remained elevated above the level detected in sham experiments. Both the rolling and firm adhesion of platelets was reduced to sham levels in the P-selectin mAb-treated mice. Although treatment with a GPIIb/IIIa mAb significantly reduced the adhesion of both platelets and leukocytes after I/R, the reductions noted were relatively small compared with the responses seen with the P-selectin mAb.
Leukocyte and platelet adhesion in P-selectin–deficient bone marrow chimeras
Our findings related to the role of platelet-associated and endothelial cell–associated P-selectin in mediating the I/R-induced rolling and adhesion of leukocytes and platelets are summarized in Fig. 4. In P-sel / -WT chimeras, the number of rolling and adherent leukocytes were significantly but not completely reduced compared with WT—WT chimeras. However, compared with WT—WT chimeras, the rolling and firm adhesion of platelets was virtually abolished after I/R. In WT—P-sel−/− chimeras, the number of rolling and adherent leukocytes and platelets also decreased significantly compare with WT—WT chimeras; however, some adhesion of leukocytes and platelets was still detected after I/R, similar to the responses noted in the group treated with the P-selectin blocking mAb.
Platelet–leukocyte interaction
Platelets were noted to adhere directly onto adherent leukocytes and platelet-bearing leukocytes were occasionally observed rolling in postischemic venules. Some free-flowing platelets were seen to suddenly bind (with-out rolling) on adherent leukocytes. Some of these platelets detached from the adherent leukocyte whereas others adhered firmly on the leukocyte. Other platelets were seen to roll and adhere directly on venular endothelium. To quantify the contribution of leukocytes to I/R-induced platelet recruitment, some mice were rendered neutropenic with antineutrophil serum. Although leukocyte rolling and adherence were still observed in cerebral venules of serum-treated mice after I/R, the responses were dramatically reduced. The cerebral venules of neutropenic mice also exhibited large and significant reductions in rolling and adherent platelets after I/R (Fig. 5).
FIG. 4. Role of platelet-associated and endothelial cell–associated P-selectin in mediating I/R-induced rolling and adhesion of leukocytes (A) and platelets (B). Four or five animals were studied in each group. Mice in all groups were exposed to 1 hour of MCAO followed by 4 hours of reperfusion. WT—*WT and WT—*P-sel−/− chimeras received CFDASE-labeled platelets from WT mice. P-sel−/−—*WT chimeras received CFDASE-labeled platelets from P-sel−/− mice. *P < 0.05 relative to the WT*WT (control) chimeras.
Signal-Dependent Protein Synthesis by Activated Platelets: New Pathways to Altered Phenotype and Function
New biologic activities of platelets continue to be discovered, indicating that concepts of platelet function in hemostasis, thrombosis, and inflammation require reconsideration as new paradigms evolve. Studies done over 3 decades ago demonstrated that mature circulating platelets have protein synthetic capacity, but it was thought to be low level and inconsequential. In contrast, recent discoveries demonstrate that platelets synthesize protein products with important biologic activities in a rapid and sustained fashion in response to cellular activation. This process, termed signal-dependent translation, uses a constitutive transcriptome and specialized pathways, and can alter platelet phenotype and functions in a fashion that can have clinical relevance. Signal-dependent translation and consequent protein synthesis are examples of a diverse group of posttranscriptural mechanisms in activated platelets that are now being revealed. (Arterioscler Thromb Vasc Biol. 2008;28:s17-s24) Key Words: platelets . translation . protein synthesis . transcriptome . proteome . thrombosis
This article is part of a multi-part CME-certified activity titled Translational Therapeutics at the Platelet Vascular Interface.
New Paradigms at the Vascular Interface
The acute hemostatic functions of platelets are well known, have dominated the attention of the field for decades, and have been the foundation for discoveries that generated new molecular therapies. Rapid, immediate activation responses mediate platelet-dependent thrombosis in a variety of pathologic conditions, and pharmacological antiplatelet strategies are largely aimed at these events. Nevertheless, the focus on adhesion, aggregation, and secretion, and the view that platelets have a repertoire of activities primarily restricted to these acute processes, have also generated a central dogma that may inappropriately limit our view of their actions at the vascular interface and in other settings in health and disease. Clearly, our understanding of the molecular mechanisms by which platelets influence hemostasis, thrombosis, regulated and dysregulated inflammation, and neoplasia remains incomplete and continues to evolve. New paradigms are emerging as previously unrecognized pathways in platelets are identified, and unanticipated activities are characterized. In this regard, the current state of the field of platelet biology may be akin to that of endothelial cells several decades ago, when endothelium was thought by most investigators and physicians to have a limited range of responses; on the contrary, however, when this dogma was reexamined using new approaches that included primary culture of human endothe-lium, active participation of these cells in interactions with leukocytes and a variety of other previously unrecognized functions were discovered.If the comparison is accurate, new paradigms relevant to activities of platelets at the vascular interface are likely to be reported with some frequency.
Alternative and traditional views of selected features of platelet biology are listed in the Table. There is already considerable evidence for some of the alternative themes, such as inflammatory and immune activities of platelets,10–16 whereas others are less well explored and more speculative. The remainder of this review summarizes evidence for one such functional capability not generally recognized in platelets until recent discoveries revealed it: synthesis of new protein products in response to cellular activation (reviewed in references5,17).
Table. New Biology of Platelets: Traditional Paradigms May Be Insufficient to Understand Platelet Activities at the Vascular Interface
Traditional View Alternate View
Platelets are biologically simple because they are anucleate Platelets have specializations and biologic activities that are novel and complex. Some
and have a limited repertoire of responses. activities are yet to be discovered.
Platelets do not express new gene products. Platelets have diverse posttranscriptional mechanisms and use a transcriptome and
specialized pathways to modify their proteome, phenotype, and functions.
Platelets are short-acting cells in clots and damaged tissue. Platelets can be relatively long-lived and can mediate cell-cell interactions for many
hours after initial adhesion, aggregation, and secretion.
Platelets operate exclusively in the intravascular Platelets can influence critical events in the extravascular milieu in direct
compartment. and indirect fashions.
Observations from a number of laboratories now demonstrate that physiologically relevant activation signals induce translation of proteins with important functions from constitutive or posttranscriptionally processed messenger RNAs (mRNAs) in human and murine platelets, a process that we have termed signal-dependent translation. These and other studies indicate that the platelet has intricate posttranscriptional mechanisms that allow it to alter its proteome, phenotype, and functions by accomplishing new protein synthesis in response to cellular activation. This capacity may allow platelets to modify the complex milieu of the vascular interface in ways that were previously unrecognized.
Essentially, all of the platelets isolated from normal subjects incorporated radiolabeled amino acids into new protein, demonstrating that this function is not a property of a subset of immature cells. Platelets from splenectomized subjects with idiopathic throm-bocytopenic purpura had increased levels of amino acid incorporation into protein, indicating that the physiological state of the subject or the age and maturity of the platelets influence protein synthesis. Extracellular factors were reported to alter protein synthesis by human platelets under some conditions. This provided evidence suggesting that the synthetic mechanisms involved are regulated.
The Platelet Transcriptome
Circulating human platelets have a substantial and diverse transcriptome, in addition to protein synthetic machinery. RNA-selective fluorescent dyes stain the entire population of platelets isolated from normal subjects, indicating the presence of RNA species transcribed by parent megakaryocytes. Messenger RNAs with 5′-methylguanosyl (m7G) caps and 3′ untranslated region polyadenylated tails are present, as are 18S and 28S ribosomal proteins, which are integral to the structure of ribo-somes. Early experiments with intact platelets from normal subjects indicated that some of the mRNA transcripts are competent to serve as templates for proteins and have relatively long functional half lives that correlate with the lifespan of platelets in the circulation. This observation then lay fallow, for the most part, until the advent of reverse transcriptase polymer-ase chain reaction (RT-PCR) analysis and cDNA cloning meth-odologies. This infusion of new technology resulted in construction of cDNA libraries from platelet transcripts. Most recently, transcript profiling by microarray analysis and serial analysis of gene expression (SAGE) have been applied to platelets, identifying 1500 to 3000 unique transcripts in platelets from normal subjects, depending on the approach. Both cytoplasmic and mitochondrial transcripts are represented.35 There is substantial consistency between data generated by microarray analysis and SAGE, and in platelets isolated from different normal donors.
Multiple Proteins Are Synthesized by Activated Human Platelets
Although early studies indicated that platelets have protein synthetic capacity, the general concept in the field has been that it is low level, vestigial, and likely inconsequential. Several texts of hemostasis and platelet biology do not mention this function, and some commentaries conclude that platelets are simply incapable of any new protein synthesis. Consistent with the notion that platelets have low basal protein synthesis, little incorporation of the radiolabeled amino acid is detectable when freshly isolated human platelets are incubated with [35S] methionine under resting conditions in the absence of activation. However, when an activating signal is delivered to platelets incubated in parallel, multiple labeled proteins are synthesized when lysates and soluble fractions are analyzed by 1-dimensional or 2-dimensional gel electrophoresis (Lindemann S, Weyrich AS, Zimmerman GA, 2001). Some of these newly synthesized proteins have been identified and mechanisms of their signal-dependent translation determined.
Recent findings provided clear evidence for signal-dependent (that is, induced by activating signals) translation of Bcl-3 from mRNA that is transcribed in parent megakaryocytes but is repressed, or “silenced,” in circulating platelets under resting, basal conditions. Immunocytochemical detection of Bcl-3 in platelets in inflamed and thrombosed human vessels in surgical specimens (Figure 1D) provided in situ evidence that the experimental observations have physiological and clinical relevance. We subsequently found that collagen, platelet-activating factor, ADP, and epinephrine are also agonists for signal-dependent translation in plate-lets. Collagen was recently reported to induce Bcl-3 synthesis by platelets in experiments by other investigators. The time course of Bcl-3 synthesis in response to thrombin yielded additional important insights: newly synthesized Bcl-3 could be detected in activated platelets within 15 to 30 minutes in some experiments, consistent with translation of constitutively present mRNA without a requirement for new transcription. This feature is also consistent with the biology of platelets as rapid response cells. Nevertheless, synthesis of Bcl-3 is also prolonged over many hours, indicating that platelets may have important functions in thrombi and injured vessels well beyond the first few minutes of acute activation.
We examined the effect of rapamycin and found that it completely and selectively inhibited Bcl-3 synthesis in thrombin-stimulated platelets, and also inhibited phosphorylation of 4E-BP1 assayed as a marker of mTOR activation in parallel. Pharmacological inhibition of phosphatidylinositol-3-kinase, which lies upstream from
mTOR in signaling cascades linking surface receptors to mTOR activation,49 also blocked both 4E-BP1 phosphoryla-tion and Bcl-3 synthesis.52 Together, these studies demonstrated that synthesis of Bcl-3 is controlled by mTOR and provided evidence for a new and previously unrecognized activity of mTOR as a regulator of expression of specific protein products and phenotypic changes in terminally differentiated cells in response to signals delivered via G protein– coupled receptors and integrins.52 This observation in platelets contributed to a parallel set of discoveries demonstrating that mTOR has similar roles in myeloid leukocytes.69–71 The findings also suggest that inhibition of mTOR by rapamycin may have novel therapeutic effects on gene expression by platelets and leukocytes independent of inhibition of proliferation of other cell types when this agent is applied in antiangiogenic strategies and in “drug-eluting” vascular stents in the clinic.72
Although Bcl-3 provided an index example of specialized, signal-dependent translation of a protein product in activated platelets the functional relevance of this event was not immediately obvious and was initially perplexing because the activity assigned to Bcl-3 at that time was as a transcriptional regulator. A clue lay in the domain structure of Bcl-3, which includes ankyrin repeats and proline-rich N and C termini, suggesting the possibility of multiple protein-protein interactions. Based on this information, we designed experiments to determine whether newly synthesized Bcl-3 interacts with other intracellular proteins. We found that Bcl-3 specifically binds to the tyrosine kinase Fyn via the Fyn SH2 domain in activated platelets and transfected COS cells. Bcl-3 also associates with the actin cytoskeleton in platelets.53
Because Fyn and related intracellular tyrosine kinases influence contractile responses of activated platelets, we examined the contributions of Bcl-3 and mTOR to fibrin clot retraction. Clot retraction is proposed to stabilize thrombi and to modify thrombus remodeling and resolution. It can be modeled in vitro, where activated platelets retract and condense fibrin strands in a fashion that can be examined macroscopically and microscopically (Figure 1E). In parallel loss-of-function and gain-of-function strategies, inhibition of mTOR activity in human platelets using rapamycin under conditions that block Bcl-3 synthesis inhibited clot retraction,
Common and Specialized Elements in Platelet Translational Pathways and Transcripts
Biologic Advantages of Signal-Dependent Translation, and Potential Roles in Disease
Novel Pathways to Signal-Dependent Translation in Activated Human Platelets
Activated Platelets Synthesize Additional Proteins Under Signal-Dependent Control
Translation in Activated Human Platelets
Discovery of synthesis of Bcl-3 by activated platelets sparked a search for the identities of other protein products, yielding IL-1J3 and TF. It also led to the unexpected discovery that their synthesis is preceded by signal-dependent cytoplasmic splicing of IL-1J3 and TF pre-mRNAs, yielding mature transcripts that are translated into precursor (IL-1J3) and active (TF) proteins.24,43,44 This identified a novel mechanism not previously recognized in activated mammalian cells. The splicing capacities of activated platelets are intricate and will be reviewed separately. Signal-dependent splicing, together with the mTOR-dependent translational control mechanism and other regulatory pathways discussed here, indicate that platelets have unexpected diversity in posttranscriptional control. Previous and ongoing studies add to this conclusion and suggest that platelets may also use ribosomal “stalling” or polypeptide termination, participation of micro RNAs (Denis MM, Trask B, Schwertz H, Weyrich AS, Zimmerman GA, 2004) and, potentially, other modes of control.
References
Lindemann S, McIntyre TM, Prescott SM, Zimmerman GA, Weyrich AS. Platelet signal-dependent protein synthesis. In: Quinn M, Fitzgerald D, eds. Platelet Function: Assessment, Diagnosis, and Treatment. Totowa, NJ: Humana Press Inc.;2005:149-74.
Weyrich AS, Lindemann S, Tolley ND, Kraiss LW, Dixon DA, Mahoney TM, Prescott SP, McIntyre TM, Zimmerman GA. Change in protein phenotype without a nucleus: translational control in platelets. Semin Thromb Hemost. 2004;30:491–498.
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