(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin

BACKGROUND: Activation of two receptors for adenosine diphosphate (ADP), P2Y(1) and P2Y(12), is necessary for ADP-induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y(12), such as clopidogrel, are uniquely mediated by inhibition of P2Y(12)-dependent PA. However, as P2Y(12) is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI(2)), which inhibit PA by stimulating AC. OBJECTIVES: To test whether inhibition of P2Y(12) potentiates the antiplatelet effects of PGI(2). METHODS: We measured the effects of PGI(2) (0.01-10 microm) on PA of washed human platelets induced by thrombin (0.5 U mL(-1)) in the presence or absence of ARC69931MX (anti-P2Y(12)) or MRS2500 (anti-P2Y(1)). Results: PGI(2) inhibited PA in the presence of anti-P2Y(12), but not in the presence of anti-P2Y(1) or in the absence of inhibitors. In contrast, dibutyryl-cyclicAMP inhibited PA both in the presence and absence of anti-P2Y(1) or anti-P2Y(12). PGI(2) increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti-P2Y(12). CONCLUSIONS: PGI(2) did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y(12). Anti-P2Y(12) drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI(2), which may contribute to their antithrombotic effect.     

The P2Y1 receptor plays an essential role in the platelet shape change induced by collagen when TxA2 formation is prevented.

ADP and TxA2 are secondary agonists which play an important role as cofactors when platelets are activated by agonists such as collagen or thrombin. The aim of the present study was to characterize the role of the ADP receptor P2Y(1) in collagen-induced activation of washed platelets. Inhibition of P2Y(1) alone with the selective antagonist MRS2179 prolonged the lag phase preceding aggregation in response to low or high concentrations of fibrillar collagen, without affecting the maximum amplitude of aggregation or secretion. A combination of MRS2179 and aspirin resulted in complete inhibition of platelet shape change at low and high collagen concentrations, together with a profound decrease in aggregation and secretion. Scanning electron microscopy showed that these platelets had conserved the discoid morphology typical of the resting state. A lack of shape change was also observed in aspirin-treated P2Y(1)- and G(alphaq)-deficient mouse platelets and in delta-storage pool-deficient platelets from Fawn Hooded rats. In contrast, when the second ADP receptor P2Y(12) was inhibited with AR-C69931MX, aspirin-treated platelets were still able to change shape and displayed only a moderate decrease in aggregation and secretion. In conclusion, this study provides evidence that collagen requires not only the TxA2 receptor Tpalpha, but also P2Y(1), to induce platelet shape change.     

The platelet P2 receptors as molecular targets for old and new antiplatelet drugs

Platelet activation by ADP and ATP plays a crucial role in haemostasis and thrombosis, and their so-called P2 receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X1 and the 2 G protein-coupled P2Y1 and P2Y12 ADP receptors selectively contribute to platelet aggregation. The P2Y1 receptor is responsible for ADP-induced shape change and weak and transient aggregation, while the P2Y12 receptor is responsible for the completion and amplification of the response to ADP and to all platelet agonists, including thromboxane A2 (TXA2), thrombin, and collagen. The P2X1 receptor is involved in platelet shape change and in activation by collagen under shear conditions. Due to its central role in the formation and stabilization of a thrombus, the P2Y12 receptor is a well-established target of antithrombotic drugs like ticlopidine or clopidogrel, which have proved efficacy in many clinical trials and experimental models of thrombosis. Competitive P2Y12 antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y1 and P2X1 knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs.     

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.     

A clopidogrel-insensitive inducible pool of P2Y12 receptors contributes to thrombus formation: inhibition by elinogrel, a direct-acting, reversible P2Y12 antagonist

It is known that hepatic metabolism limits the antiaggregatory activity of clopidogrel and, as a consequence, its clinical benefits. In this study, we investigated whether other factors exist that could account for clopidogrel's suboptimal antithrombotic activity. Using an in vivo murine FeCl(3) thrombosis model coupled with intravital microscopy, we found that at equivalent, maximal levels of inhibition of ADP-induced platelet aggregation, clopidogrel (50 mg/kg p.o.) failed to reproduce the phenotype associated with P2Y(12) deficiency. However, elinogrel (60 mg/kg p.o.), a direct-acting reversible P2Y(12) antagonist, achieved maximal levels of inhibition in vivo, and its administration (1 mg/kg i.v.) abolished residual thrombosis associated with clopidogrel dosing. Because elinogrel is constantly present in the plasma, whereas the active metabolite of clopidogrel exists for ～2 h, we evaluated whether an intracellular pool of P2Y(12) exists that would be inaccessible to clopidogrel and contribute to its limited antithrombotic activity. Using saturation [(3)H]2-(methylthio)ADP ([(3)H]2MeSADP) binding studies, we first demonstrated that platelet stimulation with thrombin and convulxin (mouse) and thrombin receptor activating peptide (TRAP) (human) significantly increased surface expression of P2Y(12) relative to that of resting platelets. We next found that clopidogrel dose-dependently inhibited ADP-induced aggregation, signaling (cAMP), and surface P2Y(12) on resting mouse platelets, achieving complete inhibition at the highest dose (50 mg/kg), but failed to block this inducible pool. Thus, an inducible pool of P2Y(12) exists on platelets that can be exposed upon platelet activation by strong agonists. This inducible pool is not blocked completely by clopidogrel, contributes to thrombosis in vivo, and can be blocked by elinogrel.     

Role of Platelet Transfusion in the Reversal of Anti-Platelet Therapy

Antiplatelet therapy is extensively used in the primary and secondary prophylaxis of arterial thrombotic disorders. Aspirin, the most commonly used antiplatelet agent, is a cyclooxygenase−1 inhibitor and considered a mild to moderate inhibitor of platelet function. Therefore, often a second antiplatelet agent is necessary in certain clinical conditions requiring greater inhibition of platelet function. An adenosine diphosphate (ADP) receptor, P2Y12, is an important target for this purpose; several agents inhibit this receptor providing potent antiplatelet effect. One of the side effects of these agents is bleeding, which in some patients may require reversal of antiplatelet effect. Similarly, patients undergoing emergent surgeries may benefit from reversal of antiplatelet effect to avoid excessive surgical bleeding. This article reviews current literature on this topic.     

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Summary.  In order to investigate the role of the platelet P2Y₁ receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [³³P]2MeSADP in the presence or absence of the selective P2Y₁ antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y₁ receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y₁ receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y₁ expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y₁ receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.     

Inhibition of localized thrombosis in P2Y1-deficient mice and rodents treated with MRS2179, a P2Y1 receptor antagonist

Summary.  Previous studies in experimental models revealed a role for the P2Y₁ platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y₁ receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl₃, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y₁-deficient mice and mice treated with the P2Y₁ antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y₁ deficiency with P2Y₁₂ inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y₁ receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs.     

Thienopyridines, but not elinogrel, result in off-target effects at the vessel wall that contribute to bleeding

Clinical studies with clopidogrel or prasugrel show that although increased inhibition of P2Y(12) and platelet function improves efficacy, bleeding is also increased. Other preclinical and clinical studies have suggested a greater therapeutic index (TI) with reversible inhibitors and disproportionate effects of thienopyridines on bleeding at high doses. We used multiple in vivo (FeCl(3)-induced arterial thrombosis in mesenteric arteries, blood loss after tail transsection, and platelet deposition and wound closure time in a micropuncture model in mesenteric veins) and ex vivo (light transmittance aggregometry, prothrombin time, and activated partial thromboplastin time) mouse models to 1) compare the TI of clopidogrel, prasugrel, and elinogrel, a reversible, competitive antagonist, with that in P2Y(12)(-/-) mice and 2) determine whether the bleeding consequences of the thienopyridines are attributed only to the inhibition of P2Y(12). Data indicated greater (elinogrel) and decreased (thienopyridines) TI compared with that in P2Y(12)(-/-) mice. The impaired TI associated with the thienopyridines was not attributed to non-P2Y(12) activities on platelet function or coagulation but was related to a direct effect at the vessel wall (inhibition of vascular tone). Further analysis showed that the prasugrel off-target effect was dose- and time-dependent and of a reversible nature. In conclusion, the TI of thienopyridines in the mouse may be decreased by P2Y(12)-independent off-target effects at the vessel wall, whereas that of elinogrel may be enhanced by the reversible, competitive nature of the antiplatelet agent.     

Platelet activation by ADP: the role of ADP antagonists

ADP plays a key role in haemostasis and thrombosis. Despite its early identification in 1961 as the first known aggregating agent, the molecular basis of ADP-induced platelet activation is only beginning to be understood. Two purinergic receptors contribute separately to the complex process of ADP-induced platelet aggregation: the P2Y1 metabotropic receptor responsible for mobilization of ionized calcium from internal stores, which initiates aggregation, and P2Y receptor coupled to adenylyl cyclase inhibition, which is essential for the full aggregation response to ADP and stabilization of platelet aggregates. The latter is the molecular target of the ADP-selective antiaggregating drugs ticlopidine and clopidogrel and the ATP analogues of the AR-C series. In addition, it is probably defective in patients with a bleeding diathesis characterized by selective impairment of platelet responses to ADP. Finally, the P2X1 ionotropic receptor is also present in platelets, but its role is not yet known. Studies with P2Y1 knock-out mice as well as the use of selective P2Y1 antagonists have shown that, in addition to the P2Y receptor, which is the target of clopidogrel, the P2Y1 receptor is an important potential target for new antithrombotic drugs.     

Prevention of arterial thrombosis by intravenously administered platelet P2T receptor antagonist AR-C69931MX in a canine model

P2Y1, P2X1, and P2T receptors mediate ADP-induced platelet aggregation. The antithrombotic effects of AR-C69931MX (N6-[2-methylthio)ethyl]-2-[3,3,3-trifluoropropylthio]-5'-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid), a selective P2T platelet receptor antagonist, was assessed in a canine model of arterial thrombosis. Placebo or AR-C69931MX (4.0 microg/kg/min for 6 h) pretreatment was administered as an intravenous infusion beginning 15 min before inducing vessel wall injury. A 300-microA anodal current was applied to the intima of the carotid artery for 180 min or discontinued 30 min after cessation of blood flow due to thrombus formation. Each of five control animals developed occlusive thrombi within 3 h after induction of vessel wall injury. In contrast, carotid artery blood flow in five of six AR-C69931MX-treated animals was maintained for the duration of the protocol. Ex vivo platelet aggregation in response to adenosine diphosphate was inhibited at the first measurement time point of 75 min after the start of drug infusion and remained inhibited during drug administration. Bleeding time values were increased in the drug-treated group. Values for both the ex vivo platelet aggregation and the bleeding times returned to control values shortly after discontinuation of AR-C69931MX. The results indicate that AR-C69931MX antagonizes the ex vivo and in vivo aggregatory actions of ADP, and displays a rapid onset and offset of action with the ability to prevent occlusive arterial thrombus formation. AR-C69931MX may be suitable for the management of patients who require short-term modulation of platelet function.     

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrel''s active metabolite

BACKGROUND AND METHODS: Prasugrel is a novel orally active thienopyridine prodrug with potent and long-lasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y(12) receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated. RESULTS: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5'-diphosphate (ADP) (10 microm) with an IC(50) value of 1.8 microm. Clopidogrel AM similarly inhibited platelet aggregation with an IC(50) value of 2.4 microm. Similar results were also observed for ADP-induced (10 microm) decreases in prostaglandin E(1)-stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities. CONCLUSIONS: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.     

P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.     

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.     

Impaired activation of murine platelets lacking G alpha(i2)

The intracellular signaling pathways by which G protein-coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G(i) pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet G alpha(i) subtype, G alpha(i2), has been disrupted. In intact platelets from G alpha(i2)-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin alpha IIb beta 3 (GPIIb-IIIa) were strongly reduced in platelets from G alpha(i2)-deficient mice. In addition, G alpha(i2)-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase--coupled platelet ADP receptor (P2Y(12)) on wild-type platelets with a selective antagonist. These observations suggest that G alpha(i2) is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the G alpha(i2)-linked ADP receptor.     

The future of platelet function testing to guide therapy in clopidogrel low and enhanced responders

Dual oral antiplatelet therapy with aspirin and clopidogrel is the therapy of choice in patients with acute coronary syndromes and in patients undergoing coronary stent placement to lower the risk of thrombotic events. Responsiveness to aspirin and especially to clopidogrel is not uniform and is subject to considerable interindividual variability. Furthermore, there is a broad consensus that clopidogrel low response or so-called high on-treatment platelet reactivity is linked to the occurrence of ischemic events. On the other hand, evidence is accumulating that enhanced clopidogrel responders are at increased risk of bleeding. Newer antiplatelet drugs, such as prasugrel and ticagrelor, are more potent and produce more consistent inhibition of platelet aggregation via the P2Y(12) ADP platelet receptor. A variety of methods of platelet function testing are available for evaluating platelet inhibition in percutaneous coronary intervention-treated patients in order to help determine the individual risk for ischemic and bleeding complications. Although not yet routinely undertaken, platelet function testing offers the potential to tailor antiplatelet therapy for individual patients. Whether alteration of therapy based on platelet function testing improves patients' outcomes remains unclear and is currently under investigation. This article reviews the impact of antiplatelet drug responsiveness on clinical outcomes with a focus on P2Y(12) receptor inhibition as well as on current and future concepts for personalized antiplatelet strategies.     

Variable effect of P2Y12 inhibition on platelet thrombus volume in flowing blood

Summary. Background and objectives: Patients treated with percutaneous coronary intervention receive aspirin and P2Y12 ADP receptor inhibitors to reduce thrombotic complications. The choice of methodology for monitoring the effects of treatment and assessing its efficacy is still a topic of debate. We evaluated how decreased P2Y12 function influences platelet aggregate (thrombus) size measured ex vivo. Methods and results: We used confocal videomicroscopy to measure in real time the volume of platelet thrombi forming upon blood perfusion over fibrillar collagen type I at a wall shear rate of 1500 s^?1. The average volume was significantly smaller in 31 patients receiving aspirin and clopidogrel (19) or ticlopidine (12) than in 21 controls, but individual values were above the lower limit of the normal distribution, albeit mostly within the lower quartile, in 61.3% of cases. Disaggregation of platelet thrombi at later perfusion times occurred frequently in the patients. Vasodilator‐stimulated phosphoprotein phosphorylation, reflecting P2Y12 inhibition, was also decreased in the patient group, and only 22.6% of individual values were above the lower normal limit. We found no correlation between volume of thrombus formed on collagen fibrils and level of P2Y12 inhibition, suggesting that additional and individually variable factors can influence the inhibitory effect of treatment on platelet function. Conclusions: Measurements of platelet thrombus formation in flowing blood reflects the consequences of antiplatelet therapy in a manner that is not proportional to P2Y12 inhibition. Combining the results of the two assays may improve the assessment of thrombotic risk.