Platelets play a central role in the development of thrombi and subsequent ischemic events. The process of platelet-mediated thrombus formation involves adhesion, activation, and aggregation. Within seconds of injury, platelets adhere to collagen fibrils through glycoprotein (GP) Ia/IIa receptors. An adhesive glycoprotein, von Willebrand factor (vWF) allows platelets to stay attached to the subendothelial vessel wall (via GP Ib) despite high shear forces. Following adhesion, platelets are activated to secrete a variety of agonists including thrombin, serotonin, adenosine diphosphate (ADP), and thromboxane A2 (TXA2). These agonists, which further augment the platelet activation process, bind to specific receptor sites on the platelets to activate the GP IIb/IIIa receptor complex, the final common pathway to platelet aggregation. Once activated, the GP IIb/IIIa receptor undergoes a conformational change that enables it to bind with fibrinogen.[1,2] Handin RI. Bleeding and thrombosis. In: Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s Principles of Internal Medicine. Vol 1. 14th ed. New York, NY: McGraw-Hill; 1998:339-345. Schafer AI. Antiplatelet therapy. Am J Med. 1996;101:199-209.
The physiological processes of thrombosis and inflammation should not be viewed in isolation because they are interrelated in many respects. Some of the first-response mechanisms to vascular injury, such as secretion of Weibel-Palade bodies, are shared in thrombosis and inflammation. As Weibel-Palade bodies release their contents, von Willebrand factor (vWF) and P-selectin are expressed. These adhesion molecules mediate platelet and leukocyte rolling on the vascular wall, a step that is critical for leukocyte extravasation and that probably helps form the platelet plug. P-selectin can activate leukocytes that produce procoagulant microparticles containing tissue factor; these particles are recruited into the growing thrombus, where they promote thrombin and fibrin generation. Activated platelets also express the cytokine CD40L, which enhances platelet activation by binding to the major platelet integrin. CD40L can also stimulate inflammatory responses in the surrounding endothelium. Evidence has shown that platelets are involved in inflammation and, similarly, that leukocytes are involved in hemostasis.
As the platelet emerged as a pivotal entity in CVD, a number of antithrombotic agents have been developed that prevent platelet aggregation and activation at critical points in the thrombotic cascade, and they have been shown in large-scale randomized trials to improve patient outcomes in ACS and PCI. The antiplatelet agents include aspirin, the thienopyridine clopidogrel, and its predecessor ticlopidine. Intravenous GP IIb/IIIa inhibitors block the final common pathway of platelet activation and aggregation, and the GP IIb/IIIa receptor is therefore an attractive therapeutic target. GP IIb/IIIa inhibitors include abciximab, tirofiban, and eptifibatide. Thrombin is blocked by unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH), notably enoxaparin, and by direct thrombin inhibitors (eg, bivalirudin).
The mechanism of action of clopidogrel is similar to that of ticlopidine but different from that of aspirin. Both clopidogrel and ticlopidine require biotransformation for their pharmacologic activity. Clopidogrel is a potent, noncompetitive inhibitor of ADP-induced platelet aggregation. Clopidogrel inhibits the binding of ADP to platelet membrane receptors. The effect of clopidogrel on ADP binding is irreversible and lasts for the duration of platelet life, about 7 to 10 days. The inhibition is also specific and does not significantly affect cyclooxygenase or arachidonic acid metabolism. Both low- and high-affinity ADP receptors are present on platelets, and the active metabolite of clopidogrel binds to the low-affinity receptors. ADP binding to this site is necessary for activation of the GP IIb/IIIa receptor, which is the binding site for fibrinogen. Fibrinogen links different platelets together to form the platelet aggregate. Clopidogrel thus ultimately inhibits the activation of the GP IIb/IIIa receptor and its binding with fibrinogen. Aspirin inhibits the cyclooxygenase enzyme, preventing the production of prostaglandin and thromboxane A2 (TXA2) from arachidonic acid. TXA2 activates the GP IIb/IIIa binding site on the platelet, allowing fibrinogen to bind. Aspirin also exerts its effects on other parts of the body system. Paradoxically, aspirin blocks synthesis of prostacyclin by endothelial cells, resulting in an effect that promotes platelet aggregation. Dipyridamole has been suggested to act as an antiplatelet drug by several possible mechanisms. It directly stimulates prostacyclin synthesis, potentiates the platelet inhibitory actions of prostacyclin, and inhibits phosphodiesterase to raise platelet cyclic AMP (cAMP) levels. However, these effects may not occur at therapeutic levels of the drug; hence the mechanism of action of dipyridamole remains to be elucidated. Schrör K. The basic pharmacology of ticlopidine and clopidogrel. Platelets. 1993;4:252-261. Plavix® (clopidogrel bisulfate) Prescribing Information. Schafer AI. Antiplatelet therapy. Am J Med. 1996;101:199-209.
This slide shows the mechanism of action of aspirin. Aspirin blocks the conversion of arachadonic acid (AA) to
Clopidogrel is a potent, non-competitive inhibitor of adenosine diphosphate- (ADP) induced platelet aggregation, irreversibly inhibiting the binding of ADP to its platelet membrane receptors. Consequently, platelets exposed to clopidogrel are affected for the remainder of their lifespan (approximately 7–10 days). The inhibition is specific and does not significantly affect cyclo-oxygenase (COX) or arachidonic acid metabolism. Clopidogrel can also indirectly inhibit platelet aggregation induced by agonists other than ADP by blocking the amplification of platelet activation by released ADP: ADP binding is necessary for activation of the GPIIb/IIIa receptor, which is the binding site for fibrinogen. Fibrinogen links different platelets together to form the platelet aggregate. Clopidogrel thus ultimately inhibits the activation of the GPIIb/IIIa receptor, its binding to fibrinogen and further platelet aggregation.
Adenosine diphosphate (ADP) is an important activator of platelet aggregation. ADP acts through binding to low- and high-affinity membrane receptors. These receptors are members of the P2 family of nucleotide receptors.1 They are further classified into P2X (ion-channel receptors) and P2Y (G-protein linked receptors). Recently, three separate ADP receptor subtypes have been identified on the platelets, notably: P2X1: an ion-channel receptor that mediates the early transmembrane flux of calcium, but does not appear to affect shape change or aggregation P2Y1: a receptor coupled to the activation of phospholipase C, which results in increased inosital triphosphate levels, the mobilization of intracellular calcium, and platelet shape change P2Y12 (or previously known as P2TAC or P2T12): a G-protein linked receptor coupled to the ADP-induced inhibition of adenylate cyclase and aggregation, providing a sustained platelet aggregation and secretion. Recent studies show that clopidogrel inhibits this receptor.2
A loading dose of clopidogrel provides rapid and full effect by 3 hours and was very well tolerated. A study of healthy volunteers demonstrated that the rapid inhibition of platelet aggregation was achieved with a 300 mg dose of clopidogrel alone. The full antiplatelet affect (ADP platelet inhibition between 60–80%) was achieved within 3 hours and a significant effect was seen within the first 1.5 hours.* *At 24 hours, both groups received a second dose of 75 mg
CD154 (CD40 ligand) is a cell surface molecule mainly expressed on activated T lymphocyte cells. Interaction of CD40 ligand (CD40L) with its receptor CD40 on B lymphocyte cells regulates B cell proliferation, production of immunoglobulins and other B cell responses. In addition to immunoregulatory functions, CD40L-CD40 interactions are also involved in atherogenesis. Both CD40 and CD40L have been found in atheroma-associated cells and interaction has been shown to regulate smooth muscle and endothelial cell functions relevant for the pathogenesis of atherosclerosis.1 Recent work has established that platelets can express CD154 (CD40L), which has a role in regulating tissue factor gene expression in macrophage and smooth muscle cells.1 In healthy volunteers treated with clopidogrel (75 mg/day for 7 days), ADP-induced expression of CD40L was completely abolished, while ASA (100 µM in vitro) was ineffective. Similarly, ASA did not inhibit ADP-induced expression of CD62P or CD63, while clopidogrel did. These data indicate that stimulation of the ADP-receptor is required for both platelet granule secretion and CD40L externalization.2 The authors concluded that this effect may contribute to the overall beneficial effects of clopidogrel and that the possible modulation of platelet-mediated inflammatory responses in vascular cells represents a novel aspect of action of this antiplatelet compound.2
Clopidogrel significantly inhibited intimal proliferation after arterial injury in rabbits. The mechanisms underlying these effects are not really known.1 This study examined the effects of clopidogrel treatment (75 mg/day for 7 days; n = 10, healthy volunteers) and ASA (100 µM in vitro) on platelet dependent mitogenesis of vascular smooth muscle cells after stimulation with ADP at physiological Ca2+ concentration. Platelet analyses were performed at baseline and at the end of the treatment period (1 hour after last clopidogrel dose).2 Stimulation of smooth muscle cells with ADP-stimulated platelets induced a marked increase in DNA synthesis. This effect was significantly reduced by clopidogrel. Clopidogrel treatment almost completely inhibited aggregation and surface expression of the activation marker CD62P. In contrast, no significant inhibition was observed with ASA.2 The authors concluded that these effects may provide an explanation for the inhibitory effects of clopidogrel on restenosis in the rabbit injury model and might help to explain the efficacy of clopidogrel in reducing cardiovascular events as observed in the CAPRIE study.1
The clinical benefit of clopidogrel has been demonstrated in trials involving more than 30,000 patients: CAPRIE* study: clopidogrel was significantly more effective than ASA in patients with myocardial infarction, stroke or peripheral arterial disease1 CLASSICS†: clopidogrel was at least as effective and safer than ticlopidine2 CURE‡ study: highly significant 20% additional relative risk reduction with clopidogrel on top of standard therapy (including ASA) in patients with ACS (without ST segment elevation).3 Ongoing trials are recruiting up to 50,000 patients. Additional planned clinical trials target more than 30,000 patients. *Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events †CLopidogrel ASpirin Stent International Cooperative Study ‡Clopidogrel in Unstable angina to prevent Recurrent Events
The key to understanding atherothrombosis is that the benefit in event reduction from clopidogrel is additive not duplicative of the ASA benefit. As ASA and clopidogrel inhibit different pathways of platelet activation, there is potential for synergy between the two agents. Clopidogrel is a potent, non-competitive inhibitor of ADP-induced platelet aggregation, irreversibly inhibiting the binding of ADP to its platelet membrane receptors.1,2 The inhibition is specific and does not significantly affect cyclo-oxygenase or arachidonic acid metabolism.3 ADP binding is necessary for activation of the GPIIb/IIIa receptor, which is the binding site for fibrinogen. Fibrinogen links different platelets together thus forming the platelet aggregate.1 Therefore, clopidogrel ultimately inhibits the activation of the GPIIb/IIIa receptor and its binding to fibrinogen. ASA inhibits the cyclo-oxygenase enzyme, preventing the production of prostaglandin and thromboxane A2 (TXA2) from arachidonic acid. TXA2 activates the GPIIb/IIIa binding site on the platelet, allowing fibrinogen to bind.1
The synergistic effects of clopidogrel and ASA have been demonstrated in animal models. In a rabbit model of thrombus formation after electrical stimulation of the carotid artery, thrombus formation was significantly reduced (p < 0.05) by clopidogrel 10 mg/kg administered orally 2 hours before the stimulation. ASA at 10 mg/kg did not significantly reduce thrombus formation. However, simultaneous administration of clopidogrel and ASA (10 mg/kg plus 10 mg/kg) resulted in greater antithrombotic activity (p < 0.05) compared with either clopidogrel or ASA administered alone, and greater than the additive effect of the two alone.
The effects of clopidogrel and ASA alone and in combination were evaluated in a pig model of stent thrombosis, under high-shear conditions. Clopidogrel administration resulted in rapid and dose-dependent inhibition of stent thrombosis, whereas ASA alone had minimal effects. When administered together, there was synergy between ASA and clopidogrel, and the combination resulted in 95–98% inhibition of stent thrombosis.
GP IIb-IIIa: the final common pathway to platelet aggregation Numerous agonists, including adenosine diphosphate (ADP), epinephrine, collagen, thrombin, and thromboxane A2 (TxA2), can activate platelets, but the final common pathway leading to platelet aggregation and thrombus formation is the expression of GP IIb-IIIa and the cross-linking of platelet receptors GP IIb-IIIa by fibrinogen molecules.
Vascular injury with ensuing platelet thrombus formation is the underlying pathophysiologic process in acute coronary syndromes (ACS) and in the development of complications of percutaneous coronary intervention (PCI). The injury may occur as a result of atherosclerotic plaque rupture or secondary to endothelial damage from balloon expansion or stent implantation. In either instance, the result is platelet adhesion to the damaged endothelium, leading to activation and aggregation. This process is mediated through the glycoprotein (GP) IIb/IIIa receptor and may be blocked by agents that bind to the receptor.
Platelet activation causes changes in the shape of platelets and conformational changes in GP IIb/IIIa receptors, transforming the receptors from a ligand-unreceptive to a ligand-receptive state. Ligand-receptive GP IIb/IIIa receptors bind fibrinogen molecules, which form bridges between adjacent platelets and facilitate platelet aggregation. Inhibitors of GP IIb/IIIa receptors also bind to GP IIb/IIIa receptors, blocking the binding of fibrinogen and thus preventing platelet aggregation.
Our present state of understanding is shown on this slide: 1. The platelet is at first in a resting/inactive state. 2. Under the right stimulus, the platelet becomes active and the GP IIb/IIIa receptors become available for binding with fibrinogen. 3. Under normal conditions this results in crosslinking of the platelets by fibrinogen with platelet aggregation and clot formation. 4. Blockade of the GP IIb/IIIa receptor, shown in the upper right hand corner of the slide, now provides a therapeutic opportunity to prevent platelet aggregation by blocking this receptor and preventing crosslinking of the platelets with fibrinogen.
When platelets are activated, CD40L is translocated and expressed on the platelet surface where it initiates various inflammatory responses. Surface-expressed CD40L is cleaved and shed from the platelet surface as sCD40L. GP IIb/IIIa antagonists block sCD40L release from activated platelets in vitro even in the absence of aggregation, demonstrating that the GP IIb/IIIa receptor plays a direct role in the cleavage mechanism.
When a patient undergoes PCI after receiving a dose of GP IIb/IIIa inhibitors sufficient to occupy ≥80% of the GP IIb/IIIa receptors for a prolonged period of time, CD40L release is inhibited and platelet activation decreases, lowering the risk of adverse events. During the course of infusion for patients who are not receiving surgical therapy, if GP IIb/IIIa receptor occupancy is <80% for >12 hours, the GP IIb/IIIa inhibitors, paradoxically, can increase the risk of thrombosis, potentially by raising levels of serum CD40L, as well as various other mechanisms that are not well understood. To achieve the benefits of GP IIb/IIIa inhibitor therapy, doses must be sufficient to block more than 80% of GP IIb/IIIa receptors.
GP IIb/IIIa inhibition has been showed to reduce sCD40L and circulating leukocyte-platelet aggregates. Reduction in sCD40L with GP IIb/IIIa inhibitors is independent of preprocedural use of clopidogrel. Studies with tirofiban show that GP IIb/IIIa inhibition blunts the rise in C-reactive protein (CRP) after NSTEMI and after PCI in patients with stable CAD. GP IIb/IIIa blockade also reverses endothelial dysfunction induced by PCI in patients with stable CAD.
This meta-analysis, which combined data from 10 clinical trials that evaluated the effect of GP IIb/IIIa inhibitors in >30,000 patients with NSTE ACS, showed an overall risk reduction of 21% for death/MI at 30 days in patients who received GP IIb/IIIa inhibitors compared with those who received placebo. The wide variation among outcomes in the 10 trials is at least partly related to whether patients received PCI and medical therapy, rather than medical therapy alone. The outcomes reflect treatment with three different GP IIb/IIIa inhibitors (abciximab, eptifibatide, and tirofiban) and the results have been used to formulate updated ACC/AHA guidelines for management of patients with UA/NSTEMI.
Diabetic thrombocytopathy refers to differences in platelet function between diabetic and nondiabetic individuals. Both diabetic and prediabetic conditions are associated with platelet and coagulation derangements. Increased platelet aggregability and adhesiveness in diabetes is due to a series of alterations in platelet functions. Because of the importance of platelet aggregation in thrombus formation, treatment strategies have focused on using antiplatelet agents to prevent recurrent events.
In the PURSUIT trial, which compared the effect of eptifibatide vs placebo in patients with NSTEMI, diabetes was present in 4035 (26.4%) of the patients enrolled in the United States. Results of the PURSUIT study showed that both diabetic and nondiabetic patients had a reduction in death or MI at 30 days with eptifibatide. The reduction was somewhat greater in the diabetic subgroup, although the confidence interval was wide.
COX-2 induced by
shear stress and
Targets for anti-platelet therapy
GPIIb - IIIa
Points of action for
GP IIb/IIIa activation
von Willebrand factor
Curran MP, Keating GM. Drugs. 2005;65:2009-35.
Inhibition of platelet
GP IIb/IIIa receptors occupied
Felix Hoffmann produced acetylsalicylic acid.
Bayer distributed aspirin to physicians.
Aspirin available without prescription.
Dr. Lawrence Craven observed aspirin prevented
heart attacks in 400 patients prescribed aspirin.
FDA approved aspirin for Secondary MI prevention.
AHA and US Preventative Services Task Force recommends
Over 26 million Americans use aspirin routinely to
reduce heart attack risk.
100 Billion Aspirin consumed per year.
aspirin to prevent first MI in at-risk
Mechanism of Action of Aspirin
ASA (low dose)
Platelet cyclooxygense -1
Aspirin and COX-2
• Aspirin also inactivates COX-2 (PGH-2-synthase) but is 50 to
100 times less potent at inhibiting COX-2 than COX –1.
• COX-2 is induced in monocytes in response to inflammatory
stimuli and in endothelial cells in response to shear stress.
• COX-2 is present in megakaryocytes and young platelets, but not
in mature platelets.
• COX-2 is not inhibited by low “antithrombotic” doses of aspirin.
Definition of Aspirin Resistance
• Clinical event despite taking aspirin
• Failure to show adequate level of platelet inhibition
• Failure of low dose aspirin to inhibit a test of platelet
function that can be inhibited by higher doses of
• Generation of thromboxane A2 despite treatment with
Definition of Aspirin Resistance
Clinical event despite aspirin
• ASA produces a 25% risk reduction, therefore
75% of patients with vascular disease ‘fail’
• Not surprising because ASA only inhibits one of
a number of mechanisms of platelet activation
• 10% - 40% of patients appear to be resistant
• Variability due to several factors:
– Method of measuring platelet function
– Clinical status of patients
Aspirin resistance exists!
Aspirin resistance is measurable!
-pharmcodynamically & clinically
Aspirin resistance has clinical consequence!
Mode of Action of Clopidogrel 1
ADP (adenosine diphosphate)
TXA2 (thromboxane A2)
1. Jarvis B, Simpson K. Drugs 2000; 60: 347–77.
Effects of ADP-Receptor
ADP / ATP
No effect on
Platelet shape change
Fibrinogen receptor activation
Thromboxane A2 generation
Sustained aggregation response
Adapted from Savi P et al. Biochem Biophys Res Commun 2001; 283: 379–83, and Ferguson JJ.
The Physiology of Normal Platelet Function. In: Ferguson JJ, Chronos N, Harrington RA (Eds).
Antiplatelet Therapy in Clinical Practice. London: Martin Dunitz; 2000: pp.15–35.
A Loading Dose of Clopidogrel
Provides Rapid and Full Effect by
3 Hours 1
Mean inhibition (%)
(n = 20/group)
*p < 0.002 vs clopidogrel 75 mg
1. Data on file, Sanofi-Synthélabo, 1999, internal report PDY 3494.
Effects of Clopidogrel on a Key
Effects ex vivo in healthy volunteers
CD40L (Mn X)
*p < 0.05 versus ADP-stimulated controls
1. Hermann A et al. Platelets 2001; 12: 74–82.
DNA synthesis (x fold increase)
Effects of Clopidogrel on PlateletDependent Mitogenesis of Smooth
Muscle Cells 1,2
*p < 0,05 versus control
1. Hermann A et al. Thromb Res 2002; 105: 173–5. 2. Hermann A et al. Arch Pharmacol 2001;
363(suppl 4): 442.
Clinical Efficacy of Clopidogrel
Clinical Benefit of Clopidogrel in more than
30,000 Patients – from CAPRIE to CURE
On top of standard therapy (including ASA)
Without ST segment elevation
1. CAPRIE Steering Committee. Lancet 1996; 348: 1329–39. 2. Bertrand NE et al. Circulation
2000; 102: 624–9 3. The CURE Trial Investigators. N Engl J Med 2001; 345: 494–502.
Synergistic Mode of Action with
Clopidogrel and ASA 1
ADP (adenosine diphosphate)
TXA2 (thromboxane A2)
1. Schafer AI. Am J Med 1996; 101: 199–209.
Synergistic Action of Clopidogrel
of ASA in Thrombus Formation 1
Clopidogrel (10 mg/kg)
Blood flow (% decrease)
Clopidogrel plus ASA
(10 mg/kg plus 10 mg/kg)
ASA (10 mg/kg)
1. Herbert JM et al. Thromb Haemost 1998; 80: 512–18.
Synergistic Action of
Clopidogrel on top of ASA in
Thrombus weight 20 mg
ASA 10 mg/kg IV
Thrombus weight 18 mg
Clopidogrel 5 mg/kg IV
Thrombus weight 8 mg
Clopidogrel 5 mg/kg IV plus ASA
10 mg/kg IV Thrombus weight 1 mg
1. Makkar RR et al. Eur Heart J 1998; 19: 1538–46.
GP IIb-IIIa Receptor
White HD. Am J Cardiol. 1997; 80(4A):2B-10B.
Platelet GP IIb/IIIa Receptor in
Vascular Injury: Adhesion and
von Willebrand factor
Coller. Heart Disease, Update 4. 1995.
Platelet GP IIb/IIIa Receptor in
Vascular Injury: Aggregation
(or von Willebrand factor)
Coller. Heart Disease, Update 4. 1995.
Receptors in ligandreceptive state
GP IIb/IIIa receptors occupied by fibrinogen
which forms bridges between adjacent platelets
GP IIb/IIIa antagonists block
sCD40L release from platelets
André P et al. Circulation. 2002;106:896-9.
Proposed model for optimal use
of GP IIb/IIIa inhibitors
GP IIb/IIIa + PCI
GP IIb/IIIa + No PCI
Antman EM. Am Heart J. 2003;146(suppl):S18-22.
of thromboinflammation with GP IIb/IIIa
• Inhibit platelet activation
• Reduce sCD40L in ACS and PCI
• Blunt CRP increase in ACS and PCI
• Reverse endothelial dysfunction induced by PCI
• Reduce leukocyte-platelet aggregation in ACS
Furman MI et al. J Thromb Haemost. 2005;3:312-20.
Giugliano RP, Braunwald E. J Am Coll Cardiol. 2005;46:906-19.
Gp IIb/IIIa ANTAGONISTS
• Platelet Gp IIb/IIIa receptors play a pivotal
role in platelet-mediated thrombus
formation, binding to binds to fibrinogen
• IIb/IIIa antagonists differ in receptor affinity,
reversibility, and specificity
Efficacy of GP IIb/IIIa inhibition
on death or MI in PCI or ACS
Death or MI at 30 days
P < 10–9
*Does not include 345 patients In the tirofiban only
group, which was stopped prematurely
Odds ratio (95% CI)
Antman EM et al. Am Heart J. 2003;146:S18-S22.
Altered platelet functions in
↓ Membrane fluidity
↓ Prostacyclin production
Altered Ca+2 and Mg+2
↓ NO production
↑ Arachidonic acid
↑ Thromboxane A2
(eg, GP IIb/IIIa, P-selectin)
These changes contribute to increased platelet
aggregability and adhesiveness in diabetes
Colwell JA, Nesto RW. Diabetes Care. 2003;26:2181-8.
PURSUIT: Outcomes in diabetic
vs nondiabetic US patients
30-day death or MI
Odds ratio (95% CI)
Lincoff AM et al. Circulation. 2000;102:1093-100.
Will any drug ever prevent thrombosis without causing