Prothrombotic responses to exercise are little influenced by clopidogrel treatment

AIMS: Adenosine diphosphate (ADP) is involved in shear-induced platelet activation, which may be important for platelet responses to stress. We therefore tested the hypothesis that ADP receptor antagonism by clopidogrel treatment would attenuate exercise-induced platelet activation. METHODS AND RESULTS: Fifteen healthy volunteers performed exhaustive exercise without and with clopidogrel pretreatment (75 mg/day; 7 days) in a randomised crossover study. Filtragometry readings (reflecting platelet aggregability in vivo) and 11-dehydro-thromboxane B(2) (TxM) in plasma were determined before and after exercise. Platelet and leukocyte activity, platelet-platelet (PPA), and platelet-leukocyte aggregates (PLAs) in vivo and their responsiveness to agonist stimulation in vitro were assessed by flow cytometry. Clopidogrel treatment inhibited ADP-induced platelet P-selectin expression by 72% (54-85%). Exercise increased platelet aggregation (filtragometry and PPAs), elevated plasma TxM, increased single platelet P-selectin expression, elevated circulating PLAs, and enhanced ADP and thrombin-stimulated P-selectin expression. Clopidogrel prolonged filtragometry readings and attenuated agonist stimulated P-selectin expression at rest, but did not influence TxM in plasma or urine or attenuate platelet or leukocyte responses to exercise. Clopidogrel treatment did not influence plasma CD40L (ligand) at rest or after exercise. CONCLUSION: Clopidogrel treatment attenuates platelet activity in vivo at rest, but exercise counteracts the platelet stabilizing effects of clopidogrel. The hypothesis that ADP is involved in stress-induced platelet activation was not supported.     

The additive antiplatelet action of clopidogrel in patients with coronary artery disease treated with aspirin

We searched for additional anti-platelet effects of clopidogrel in coronary artery disease (CAD) patients treated with aspirin. Response to clopidogrel was also stratified according to aspirin resistance. Out of 76 screened aspirin-treated CAD male patients, five were aspirin-resistant based on arachidonic acid (AA) and ADP aggregometry. These five patients and 15 aspirin-sensitive patients entered the proper study. Platelet function was assessed at baseline and after one week of additional clopidogrel treatment using aggregometry, flow cytometry (ADP, TRAP-6) and platelet reactivity index (PRI) based on VASP (vasodilatorstimulated phosphoprotein) expression. We evaluated the same markers in 15 healthy men after aspirin treatment. In healthy subjects aspirin did not affect resting or ADP-induced activated GPIIb/IIIa and P-selectin expression. The P-selectin expression on ADP-activated platelets was increased (p < 0.01) in aspirin treated ASA-resistant CAD patients as compared to ASA-sensitive group or aspirin-treated healthy subjects. Clopidogrel significantly decreased ADP and AA-induced platelet aggregation and overcame aspirin resistance in four of five patients. Expression of ADP-induced activation markers was significantly lowered after clopidogrel in all patients. Out of 20 patients, five did not respond to clopidogrel (<10% inhibition of ADP aggregation), and this group showed no change in expression of ADP-induced activation markers after clopidogrel. Clopidogrel treatment significantly reduced PRI only in the clopidogrel-sensitive group. In conclusion, the addition of clopidogrel to aspirin provides greater inhibition of platelets and can overcome aspirin resistance. Flow cytometric analysis of platelets is useful for monitoring of clopidogrel therapy.     

Inhibition of ADP-induced P-selectin expression and platelet-leukocyte conjugate formation by clopidogrel and the P2Y12 receptor antagonist AR-C69931MX but not aspirin

Platelet-leukocyte interactions are recognised to have pro-inflammatory effects, which may be important in the pathophysiology of ischaemic heart disease. Clopidogrel and the novel intravenous antithrombotic agent AR-C69931MX act at the level of the platelet P2Y12 receptor, which is known to amplify platelet activation, aggregation and other responses induced by numerous platelet agonists. We studied the effects of clopidogrel and aspirin on ADP-induced platelet-leukocyte conjugate formation and P-selectin expression in healthy volunteers. The effects of clopidogrel and AR-C69931MX administered to patients with ischaemic heart disease were also assessed. AR-C69931MX and aspirin were also studied in vitro. Clopidogrel and AR-C69931MX suppressed ADP-induced platelet aggregation, P-selectin expression and platelet-leukocyte conjugate formation whereas aspirin had no inhibitory effect. These effects of clopidogrel and AR-C69931MX may confer therapeutic benefits in the management of acute coronary syndromes.     

Enhanced platelet activation by prolactin in patients with ischemic stroke

Prolactin and leptin are newly recognised platelet co-stimulators due to potentiation of ADP-induced platelet aggregation. Elevated leptin levels have recently been found to be a risk factor for ischemic stroke in both men and women, and especially in combination with increased blood pressure for hemorrhagic stroke in men. Until now an association between hyperprolactinemia and ischemic stroke has not been investigated systematically. We determined plasma prolactin and leptin levels as well as platelet P-selectin expression in 36 patients with ischemic stroke or transient ischemic attack and detected a significant correlation between increased prolactin values and enhanced ADP stimulated P-selectin expression on platelets. In contrast, no correlation of leptin values with platelet P-selectin expression was found. Next we determined plasma prolactin and leptin as well as acquired and congenital risk factors of thrombophilia in patients with first-ever non-hemorrhagic stroke with or without atrial fibrillation. Excluding patients with such preexisting risk factors, 21 patients with and 59 patients without atrial fibrillation were identified. Patients without atrial fibrillation revealed significantly higher plasma prolactin levels than patients with atrial fibrillation. Furthermore, the influence of aspirin or clopidogrel on prolactin stimulated P-selectin expression in vitro was tested, showing that aspirin was without effect, whereas clopidogrel significantly inhibited platelet P-selectin expression. In conclusion, hyperprolactinemia might be a novel risk factor for stroke mediating its thrombogenic effect through enhanced platelet reactivity, and this might correspond to a higher efficacy of antiplatelet combination therapy with clopidogrel compared to aspirin therapy alone.     

Clopidogrel-induced qualitative changes in thrombus formation correlate with stent patency in injured pig cervical arteries

Thienopyridines (ticlopidine or clopidogrel) alone or in combination with aspirin are now the reference antiplatelet therapy after stent implantation. To better understand the high efficacy and low risk of bleeding with these agents, we tested clopidogrel alone or with aspirin in an acute ex vivo flow chamber model and in a subacute in vivo arterial thrombosis model. Clopidogrel induced a dose-dependent increase in bleeding time (BT), inhibited ADP-induced platelet aggregation and in the flow chamber reduced thrombus size, and changed thrombus structure to broad-based structure composed of nondegranulated loosely attached platelets contrasting with the tight clumps of degranulated platelets seen without clopidogrel. The in vivo model involved angioplasty and stenting at the site of a preinduced arterial lesion and thrombosis in pig carotid arteries. Clopidogrel alone or with aspirin (but not aspirin alone) decreased the number of stented vessels occluded for more than 24 h and conversely reduced the number of occluding thrombus. At 96 h after stenting, 100% and 90% of the arteries were patent with clopidogrel/aspirin and clopidogrel alone, respectively (vs. 67% and 44% with aspirin and saline, respectively). Clopidogrel destabilizes thrombus without complete abolishment of platelet reactivity.     

Clopidogrel inhibits platelet-leukocyte adhesion and platelet-dependent leukocyte activation

Clopidogrel is considered to be an important therapeutic advance in anti-platelet therapy. We investigated whether inhibition by clopidogrel results in a reduced capacity of platelets to adhere and stimulate pro-atherothrombotic and inflammatory functions in polymorphonuclear leukocytes (PMN) and in monocytes (MN). An eventual effect on these processes could further substantiate anti-atherothrombotic properties of this drug. The effects of clopidogrel or of its active metabolite were investigated on ADP or thrombin receptor-induced platelet activation and on platelet-leukocyte interactions ex vivo in the mouse or in vitro in isolated human cells or whole blood, respectively. Clopidogrel inhibited platelet aggregation, expression of P-selectin, platelet-PMN adhesion and platelet-dependent ROS production in mouse PMN. Similarly pretreatment of human platelets with the active metabolite of clopidogrel in vitro resulted in a profound inhibition of platelet P-selectin expression, platelet-PMN adhesion and production of ROS by PMN. Pretreatment with the active metabolite of clopidogrel significantly impaired the ability of platelets to up-regulate the expression of TF procoagulant activity in MN, in a washed cell system. Moreover, the active metabolite of clopidogrel inhibited rapidTF exposure on platelet as well as on leukocyte surfaces in whole blood. By reducing platelet-dependent up-regulation of inflammatory and pro-atherothrombotic functions in leukocytes, clopidogrel may reduce inflammation that underlies the chronic process of atherosclerosis and its acute complications.     

Effect of clopidogrel on thrombin generation in platelet-rich plasma in the rat.

Clopidogrel (25 mg/kg, p.o.), a potent and selective inhibitor of ADP-induced platelet aggregation, significantly inhibited, in the presence of platelets, ex vivo thrombin generation triggered by low concentrations of tissue factor. Clopidogrel reduced the area under the curve (23%, p <0.05) and the thrombin peak concentration (35%, p <0.05) but did not affect the lag phase of thrombin generation. Under the same experimental conditions, heparin (100 microg/ml) inhibited thrombin generation mostly by delaying and by reducing the burst of thrombin. In a stasis-induced venous thrombosis model in rats under low thrombogenic challenge, clopidogrel inhibited thrombus formation (ED50 = 7.9+/-1.5 mg/kg, p.o. - n = 10), confirming the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation and venous thrombosis.     

Genetic polymorphisms of the platelet receptors P2Y(12), P2Y(1) and GP IIIa and response to aspirin and clopidogrel

INTRODUCTION: There is wide variability in the responses of individual patients to aspirin and clopidogrel. Polymorphisms of several platelet receptors have been related to increased platelet aggregation. We therefore aimed to evaluate whether these polymorphisms are related to altered response to aspirin or clopidogrel. MATERIALS AND METHODS: Patients (n=120) undergoing percutaneous coronary intervention who received aspirin for > or =1 week but not clopidogrel were included. Blood samples were drawn at baseline and 20-24h after a 300-mg clopidogrel dose. Aspirin insensitivity was defined as 5 microM ADP-induced aggregation > or =70% and 0.5 mg/mL arachidonic acid-induced aggregation > or =20%. Clopidogrel insensitivity was defined as baseline minus post-treatment aggregation < or =10% in response to 5 and 20 microM ADP. PlA polymorphism of glycoprotein IIIa, T744C polymorphism of the P2Y(12) gene and the 1622A>G polymorphism of the P2Y(1) gene were genotyped by polymerase chain reaction. RESULTS: There were no differences in polymorphism frequencies between drug-insensitive vs. drug-sensitive patients. There were also no significant differences in response to aspirin (assessed by arachidonic acid-induced aggregation) or to clopidogrel (assessed by ADP-induced aggregation or activation markers) when patients were grouped according to genotype. The only trend observed was lower reduction in PAC-1 binding following clopidogrel in PlA(2) carriers (P=0.065). CONCLUSIONS: We did not find an association between polymorphisms in the platelet receptors GP IIIa, P2Y(12) or P2Y(1) and response to aspirin or clopidogrel in cardiac patients. These findings suggest that the variability in response to anti-platelet drugs is multi-factorial and is not caused only by single gene mutations.     

Prevalence of clopidogrel non-responders among patients with stable angina pectoris scheduled for elective coronary stent placement

Dual antiplatelet therapy with aspirin and clopidogrel decreases the rate of stent thrombosis in patients undergoing percutaneous coronary intervention (PCI). However, despite intensified antiplatelet treatment, up to 4.7% of the patients undergoing coronary stenting develop thrombotic stent occlusion, suggesting incomplete platelet inhibition due to clopidogrel resistance. We evaluated the percentage of clopidogrel non-responders among 105 patients with coronary artery disease (CAD) undergoing elective PCI. All patients were treated regularly with aspirin 100 mg/d and received a loading dose of 600 mg clopidogrel followed by a maintenance dose of 75 mg/d before PCI. Clopidogrel non-responders were defined by an inhibition of ADP (5 and 20 Mol/L) induced platelet aggregation that was less than 10% when compared to baseline values 4 h after clopidogrel intake. Semi-responders were identified by an inhibition of 10 to 29%. Patients with an inhibition over 30% were regarded as responders. We found that 5 (ADP 5 Mol/L) to 11% (ADP 20 Mol/L) of the patients were non-responders and 9 to 26% were semi-responders. Among the group of non-responders there were two incidents of subacute stent thrombosis after PCI. We conclude that a subgroup of patients undergoing PCI does not adequately respond to clopidogrel, which may correspond to the occurrence of thromboischemic complications. Point-of-care testing may help to identify these patients who may then benefit from an alternative antiplatelet therapy.     

Monitoring platelet inhibition after clopidogrel with the VerifyNow-P2Y12(R) rapid analyzer: the VERIfy Thrombosis risk ASsessment (VERITAS) study

INTRODUCTION: Clopidogrel inhibits platelet P2Y12 ADP receptors, while ADP, as an inductor of aggregation, stimulates both P2Y12 and P2Y1 platelet receptors. Despite a clinical loading dose routine with clopidogrel, some patients still experience coronary stent thrombosis suggesting persistent platelet activation. The VerifyNow-P2Y12 is a rapid assay that test platelet activity over 3 min and uses of the combination of ADP and prostaglandin E1 (PGE1) to directly measure the effects of clopidogrel on the P2Y12 receptor. ADP is used to maximally activate the platelets by binding to the P2Y1 and P2Y12 platelet receptors, while PGE1 is used to suppress the ADP-induced P2Y1-mediated increase in intracellular calcium levels. OBJECTIVE: The VERIfy Thrombosis risk ASsessment (VERITAS) was a prospective study designed to measure platelet response to clopidogrel therapy in subjects with multiple risk factors or history of vascular disease using this novel point-of-care assay. METHODS: 166 participants were enrolled in 4 participating sites. Data from 147 participants were analyzed after exclusion of 19 patients due to protocol violations. Platelets were assessed twice at baseline (before clopidogrel) and at 24 h post-loading 450 mg (110 participants) or 7 days after chronic clopidogrel treatment (75 mg/day) (37 patients). All participants received aspirin 81-325 mg for at least 2 days before the study enrollment. Results from the VerifyNow-P2Y12 assay are reported in P2Y12 reaction units (PRU). RESULTS: Clopidogrel therapy resulted in a mean 64.0+/-25.3% PRU reduction. No participant reached PRU inhibition below 10% of baseline. Distribution of PRU values for the VerifyNow-P2Y12 assay shows a separation from baseline to post-clopidogrel assay values with some overlap due to high inter-individual variations in response. CONCLUSIONS: VerifyNow-P2Y12 is a reliable, fast and sensitive device suitable for monitoring of platelet inhibition during clopidogrel therapy.     

Lack of association between the 807 C/T polymorphism of glycoprotein Ia gene and post-treatment platelet reactivity after aspirin and clopidogrel in patients with acute coronary syndrome

Variability in platelet response to antiplatelet therapy and its clinical relevance have been well described. However, the underlying mechanisms remain unclear. It was the aim of the present study to assess whether the response to aspirin and clopidogrel may be influenced by the 807 C/T polymorphism of the glycoprotein Ia (GpIa) gene in patients with non-ST elevation acute coronary syndrome (NSTE ACS). Six hundred one NSTE ACS patients were included in our study and were divided into three groups: CC homozygotes, CT heterozygotes ad TT homozygotes. All patients received loading doses of 600 mg clopidogrel and 250 mg aspirin at least 12 hours before blood samples were drawn. Post-treatment platelet reactivity was assessed by post treatment ADP 10 microM-induced platelet aggregation (ADP-Ag), VASP phosphorylation (PRI VASP) and P-selectin expression. Non-response to dual antiplatelet therapy was defined by high post-treatment platelet reactivity (HPPR=ADP-Ag > 70%). Significant variability in the distribution of platelet parameters was observed in the overall study population. No significant difference in platelet parameters profiles was observed within patients having the same genotype, for ADP-Ag (p=0.33), PRIVASP (p=0.72) and P-selectin expression (p=0.37). The genotype frequencies of the 807 C/T polymorphism of the GpIa gene were similar in responders and non-responders defined by persistent HPPR (p=0.104). In conclusion, our study did not show any influence of 807 C/T polymorphism of GpIa gene on post-treatment platelet reactivity assessed by ADP-Ag, PRI VASP or P-selectin expression in 601 NSTE ACS patients.     

Clopidogrel resistance?

Clopidogrel is an effective inhibitor of platelet activation and aggregation due to its selective and irreversible blockade of the P2Y(12) receptor. Combination antiplatelet therapy with clopidogrel and aspirin is an important strategy for patients with acute coronary syndromes and those undergoing percutaneous interventions. Despite significant benefits demonstrated with combination antiplatelet treatment in large clinical trials, the occurrence of adverse ischemic events, including stent thrombosis, remains a serious clinical problem. Recent studies have demonstrated distinct response variability and nonresponsiveness to clopidogrel therapy based on ex vivo platelet function measurements. Small scale investigations have suggested that nonresponsiveness may be associated with a heightened risk for adverse clinical events. The above findings have stimulated a close examination of clopidogrel metabolism.     

Anti-thrombotic action of clopidogrel and P1(A1/A2) polymorphism of beta3 integrin in patients with coronary artery disease not being treated with aspirin

Individual variability in response to clopidogrel is known but its mechanism is poorly understood. We examined the relationship between glycoprotein IIIa polymorphism P1(A1/A2) and anti-thrombotic actions of clopidogrel. Clopidogrel (75 mg/d; 2 weeks) was administered to 48 normolipemic patients with coronary artery disease. Bleeding time, thrombin generation at the site of microvascular injury, platelet function under high shear, using PFA-100 with ADP cartridge, and platelet surface activation markers (P-selectin and fibrinogen binding sites on GPIIb/IIIa complex detected by PAC-1 antibody), were studied both before and after clopidogrel treatment. Both unstimulated and low-dose (0.02 microM and 1 microM) in vitro ADP-stimulated platelets were examined. GP IIIa polymorphism was assessed by polymerase chain reaction and restriction fragment length polymorphism analysis. We identified 32 P1(A1/A1) homozygotes, 15 P1(A1/A2 heterozygotes and one P1(A2/A2) homozygote. Clopidogrel significantly prolonged bleeding time in all subjects, but this effect was greater in P1(A2 carriers (p < 0.01). Furthermore, clopidogrel only depressed thrombin generation at the site of microvascular injury (p < 0.01) in P1(A2) patients and prolonged closure time measured in vitro by PFA-100 (p < 0.05).At baseline spontaneous expression of PAC-1 and P-selectin was higher in P1(A2) subjects as compared to P1(A1) homozygotes (p < 0.05 for both antigens). Clopidogrel lowered the expression of both markers affecting more P1(A2) carriers, so that the difference in binding PAC-1 antibody between platelets from P1(A1) and P1(A2) carriers disappeared, while the difference in P-selectin expression slightly diminished. Anti-thrombotic effects of clopidogrel are more pronounced in CAD patients carrying the P1(A2) allele than in P1(A1) homozygotes.     

Variable extent of clopidogrel responsiveness in patients after coronary stenting

Clopidogrel is an effective and specific inhibitor of ADP-induced platelet aggregation. After metabolic activation, the active clopidogrel metabolite irreversibly impairs the human platelet P2Y12 ADP receptor. Gialpha-protein activation and inhibition of vasodilator-stimulated phosphoprotein (VASP) phosphorylation are two key elements of the P2Y12 receptor pathway suitable for quantitation of clopidogrel effects. So far, only limited data exist about a diminished responsiveness to clopidogrel and underlying possible mechanisms. We investigated clopidogrel effects in 57 patients after percutaneous coronary intervention and stent implantation by flow cytometry for the analysis of intracellular VASP phosphorylation. Patients were treated with a 300 mg clopidogrel loading dose, followed by 75 mg/day clopidogrel in combination with 100 mg/day aspirin. Samples were drawn after a median of 5 days of clopidogrel treatment. Considerable differences in the responsiveness to clopidogrel could be observed and it was shown that 17.5% (10/57) of the patients revealed an inadequate responsiveness to clopidogrel despite continuation of clopidogrel intake. Comparable amounts of Gialpha and VASP were found in two clopidogrel low-responding patients as well as in two responding patients. To exclude a molecular defect of P2Y12 ADP receptor, the P2Y12 receptor gene of eight clopidogrel treated patients (seven patients with inadequate responsiveness, one responder) was sequenced. We only found a single silent mutation in exon 2 at position 1828 (GA). We suggest that individual differences in clopidogrel metabolization could cause relevant variations in clopidogrel responsiveness despite the use of a 300 mg clopidogrel loading dose.     

The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin

The novel thienopyridine prodrug prasugrel, a platelet P2Y(12) ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation, there are few data on the direct effects of the prasugrel's active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates, platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent inhibition of these ADP-stimulated thrombo-inflammatory markers. These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC(50) values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y(12)-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y(12). In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y(12)-mediated up-regulation of thrombo-inflammatory markers of platelet activation. This inhibition is enhanced in the presence other blood cells and calcium, but not aspirin.     

Comparison of turbidimetric aggregation and in vitro bleeding time (PFA-100) for monitoring the platelet inhibitory profile of antiplatelet agents in patients undergoing stent implantation

The present study compared classical ADP-induced platelet aggregation vs. PFA-100 closure times using collagen/ADP membrane cartridges to monitor the degree of platelet-inhibiting effect of three drug regimens: ticlopidin, abciximab/ticlopidin and loading dose clopidogrel, each on top of aspirin (acetylsalicylic acid, ASA) during and after elective stent placement (intervention) in a total of 31 patients with acute coronary syndrome. Ticlopidin was started directly after stent implantation, abciximab was started before coronary intervention and given intravenously for 12 h, and a clopidogrel loading dose was given before intervention. The 10 patients treated with ticlopidin (500 mg daily) showed no significant prolongation of PFA closure times and a slight increase of ADP-induced platelet aggregation shortly after intervention. In 11 patients treated with abciximab/ticlopidin, the PFA closure times were significantly prolonged, and ADP-induced platelet aggregation was reduced by more than 80% during the 12-h abciximab infusion after intervention. The 10 patients pretreated with loading dose clopidogrel (450 mg followed by 75 mg daily) showed an intermediate but significant prolongation of PFA closure times and reduction of ADP-induced platelet aggregation at levels between the ticlopidin/aspirin- and the abciximab/ticlopidin/aspirin-treated groups. At 20 h after intervention, a similar degree of PFA closure time prolongation and inhibition of ADP-induced aggregation was observed in the abciximab/ticlopidin/aspirin- and the clopidogrel/aspirin-treated patient groups. Both measurement of PFA-100 closure times and inhibition of ADP-induced platelet aggregation showed a similar degree of platelet inhibition, but had rather broad SD ranges, which limit their precision for the follow-up of individual patients. In conclusion, abciximab on top of ticlopidin/aspirin showed a stronger antiplatelet effect for only less than 20 h, as compared to loading dose clopidogrel/aspirin in acute coronary syndrome patients undergoing stent implantation. Whether such a short-term superiority of abciximab, as compared to loading dose clopidogrel, translates into an overall clinical benefit of thombotic and bleeding complications remains to be established in a randomized clinical trial.     

Future perspectives for optimizing oral antiplatelet therapy

Secondary prevention of stroke and other manifestations of atherothrombosis is essential if the burden of disease associated with these events is to be reduced. Therefore, it is important to identify patients most likely to benefit from antiplatelet therapy. There is a good rationale for combining antiplatelet agents with different modes of action, since different signalling pathways contribute to platelet activation. Based on the promising results obtained with an adenosine diphosphate receptor antagonist-aspirin combination in coronary stenting, several additional trials with clopidogrel plus aspirin are ongoing. They include CURE (Clopidogrel in Unstable angina to prevent Recurrent Events, in unstable angina and non-Q-wave myocardial infarction) and COMMIT (in acute myocardial infarction), which compare clopidogrel with placebo in patients receiving aspirin, and CREDO (Clopidogrel for Reduction of Events During extended Observation), a 1-year treatment follow-up to the clopidogrel arms of the CLASSICS trial (Clopidogrel Aspirin Stent International Cooperative Study). Planned trials with clopidogrel in neurology include SPS3 (Secondary Prevention of Small Subcortical Strokes, in patients with symptomatic lacunar stroke), and MATCH (Management of Atherothrombosis with Clopidogrel in High-risk patients, in patients with stroke or transient ischaemic attack plus one additional risk factor), which will compare the efficacy of clopidogrel plus aspirin versus clopidogrel in reducing important ischaemic events. Combination therapy with an oral glycoprotein (GP) IIb/IIIa receptor antagonist plus aspirin has so far been less promising. Trials of three compounds--orbofiban, xemilofiban and sibrafiban--in combination with aspirin for secondary prevention in cardiac patients have reported increased mortality compared with aspirin alone. A similar effect was seen when sibrafiban monotherapy was compared directly with aspirin alone. Trials of newer oral GP IIb/IIIa inhibitors are under way or are planned. The combination of dipyridamole plus aspirin appears to be superior to aspirin alone for the prevention of stroke in patients with stroke or transient ischaemic attack; the effectiveness of this combination is being further investigated in ESPRIT (European/Australian Stroke Prevention in Reversible Ischaemia Trial).     

Lack of association between the P2Y12 receptor gene polymorphism and platelet response to clopidogrel in patients with coronary artery disease

INTRODUCTION: Clopidogrel inhibits the ADP subtype P2Y(12) receptor. Recently, polymorphisms of this receptor have been associated with different degrees of platelet aggregation in healthy volunteers and have been suggested to modulate clopidogrel response. However, the role of gene sequence variations of the P2Y(12) receptor in patients treated with clopidogrel has not yet been assessed. MATERIALS AND METHODS: The T744C polymorphism of the P2Y(12) receptor gene was assessed in 119 patients: 36 undergoing coronary stenting receiving a 300 mg loading dose (Group A) and 83 on long-term clopidogrel (75 mg/day) treatment (Group B). Patients were divided into 2 subgroups according to the presence or absence of the C allele: carriers (CT heterozygotes and CC homozygotes) and non-carriers (TT homozygotes). Platelet aggregation, assessed by light transmittance aggregometry following ADP, collagen, TRAP and epinephrine stimuli, and platelet activation (GP IIb/IIIa activation and P-selectin expression), assessed by whole blood flow cytometry in ADP and TRAP-stimulated platelets, were performed. Platelet function was assessed at baseline and 4 and 24 h following clopidogrel loading dose in Group A and when patients where on clopidogrel treatment for at least 1 month in Group B. RESULTS: The genotype distribution of Group A was: 22/36 (61.1%) non-carriers and 14/36 (38.9%) carriers of the C allele; Group B: 57/83 (68.7%) non-carriers and 26/83 (31.3%) carriers of the C allele. There were no differences between groups for all the assessed platelet function assays. CONCLUSIONS: The T744C polymorphism of the P2Y(12) receptor gene does not modulate platelet response to clopidogrel either in the early or long-term phases of treatment. This specific gene polymorphism alone is therefore unlikely to be the cause of variability in individual response to antiplatelet therapy.     

Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.