Leveraging Human Genetics to Estimate Clinical Risk Reductions Achievable by Inhibiting Factor XI.

Background and Purpose- Coagulation factor XI (FXI) is a novel target for antithrombotic therapy addressed by various therapeutic modalities currently in clinical development. The expected magnitude of thrombotic event reduction mediated by targeting FXI is unclear. Methods- We analyzed the association of 2 common genetic variants, which alter levels of FXI, with a range of human phenotypes. We combined variants into a genetic score standardized to a 30% increase in relative activated partial thromboplastin time, equivalent to what can be achieved with pharmacological FXI reduction. Using data from 371 695 participants in the United Kingdom Biobank and 2 large-scale genome-wide association studies, we examined the effect of this FXI score on thrombotic and bleeding end points. Results- Genetic disposition to lower FXI levels was associated with reduced risks of venous thrombosis (odds ratio, 95% CI; P value; odds ratio=0.1, 0.07-0.14; P=3×10-43) and ischemic stroke (odds ratio=0.47, 0.36-0.61; P=2×10-8) but not with major bleeding (odds ratio=0.7, 0.45-1.04; P=0.0739). The observed relative risk reductions were consistent within a range of subgroups that were at high risk for thrombosis. Consistently, we observed higher absolute risk reductions conferred by genetically lower FXI levels in high-risk subgroups, such as patients with atrial fibrillation. Conclusions- Human genetic data suggest that pharmacological inhibition of FXI may achieve considerable reductions in ischemic stroke risk without clear evidence for an associated risk of major bleeding. The quantitative framework developed can be used to support the estimation of achievable risk reductions with pharmacological modulation of FXI.

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication.

In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y12 inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, 16 patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and 30 days later. Platelet function in response to ADP was normalized between five and 30 days after treatment cessation and in vitro addition of the reversible P2Y12 receptor antagonist ticagrelor fully suppressed the regained activation response. Discontinuation of prasugrel resulted in the formation of an emerging subpopulation of ADP-responsive platelets, exhibiting high expression of active integrin αIIbß3. Two different mRNA probes, thiazole orange and the novel 5'Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due to the increased activity of juvenile platelets.

Optimization of ketone-based P2Y(12) receptor antagonists as antithrombotic agents: pharmacodynamics and receptor kinetics considerations.

Modification of a series of P2Y12 receptor antagonists by replacement of the ester functionality was aimed at minimizing the risk of in vivo metabolic instability and pharmacokinetic variability. The resulting ketones were then optimized for their P2Y12 antagonistic and anticoagulation effects in combination with their physicochemical and absorption profiles. The most promising compound showed very potent antiplatelet action in vivo. However, pharmacodynamic-pharmacokinetic analysis did not reveal a significant separation between its anti-platelet and bleeding effects. The relevance of receptor binding kinetics to the in vivo profile is described.

Characterization of the adenosine pharmacology of ticagrelor reveals therapeutically relevant inhibition of equilibrative nucleoside transporter 1.

INTRODUCTION: Studies have shown that ticagrelor has a further adenosine-mediated mechanism of action in addition to its potent inhibition of the P2Y12 receptor, which may explain some of ticagrelor's clinical characteristics. This study aimed to further characterize the adenosine pharmacology of ticagrelor, its major metabolites, and other P2Y12 receptor antagonists. METHODS: Inhibition of nucleoside transporter-mediated [(3)H]adenosine uptake by ticagrelor, its major metabolites, and alternative P2Y12 antagonists was examined in recombinant Madin-Darby canine kidney (MDCK) cells. The pharmacology of ticagrelor and its major metabolites at adenosine A1, A2A, A2B, and A3 receptor subtypes was examined using in vitro radioligand binding and functional assays and ex vivo C-fiber experiments in rat and guinea pig vagus nerves. RESULTS: Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [(3)H]adenosine uptake in equilibrative nucleoside transporter (ENT) 1-expressing MDCK cells, whereas cangrelor and the active metabolites of prasugrel or clopidogrel had no effect. No significant inhibitory activity was observed in MDCK cells expressing ENT2 or concentrative nucleoside transporters 2/3. Ticagrelor demonstrated high affinity (inhibition constant [Ki] = 41 nmol/L) for ENT1. In adenosine receptor-binding experiments, ticagrelor and its major circulating metabolite, AR-C124910XX, had low affinity (Ki > 6 µmol/L) for each of the adenosine A1, A2A, and A2B receptors, whereas ticagrelor had a submicromolar (Ki = 190 nmol/L) affinity for the adenosine A3 receptor. However, in functional assays, at high concentrations (10 µmol/L) ticagrelor only partially inhibited 3 mmol/L adenosine-induced depolarizations in the guinea pig and rat vagus nerve preparations (by 35% and 49%, respectively). CONCLUSIONS: Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.

Lead optimization of ethyl 6-aminonicotinate acyl sulfonamides as antagonists of the P2Y12 receptor. separation of the antithrombotic effect and bleeding for candidate drug AZD1283.

Synthesis and structure-activity relationships of ethyl 6-aminonicotinate acyl sulfonamides, which are potent antagonists of the P2Y12 receptor, are presented. Shifting from 5-chlorothienyl to benzyl sulfonamides significantly increased the potency in the residual platelet count assay. Evaluation of PK parameters in vivo in dog for six compounds showed a 10-fold higher clearance for the azetidines than for the matched-pair piperidines. In a modified Folts model in dog, both piperidine 3 and azetidine 13 dose-dependently induced increases in blood flow and inhibition of ADP-induced platelet aggregation with antithrombotic ED50 values of 3.0 and 10 µg/kg/min, respectively. The doses that induced a larger than 3-fold increase in bleeding time were 33 and 100 µg/kg/min for 3 and 13, respectively. Thus, the therapeutic index (TI) was ≥ 10 for both compounds. On the basis of these data, compound 3 was progressed into human clinical trials as candidate drug AZD1283.

Synthesis, structure-property relationships and pharmacokinetic evaluation of ethyl 6-aminonicotinate sulfonylureas as antagonists of the P2Y12 receptor.

The present paper describes the development of a new series of P2Y12 receptor antagonists based on our previously reported piperazinyl urea series 1 (IC50 binding affinity = 0.33 µM, aq solubility <0.1 µM, microsomal CLint (HLM) ≥300 µM/min/mg). By replacement of the urea functionality with a sulfonylurea group we observed increased affinity along with improved stability and solubility as exemplified by 47 (IC50 binding affinity = 0.042 µM, aq solubility = 90 µM, microsomal CLint (HLM) = 70 µM/min/mg). Further improvements in affinity and metabolic stability were achieved by replacing the central piperazine ring with a 3-aminoazetidine as exemplified by 3 (IC50 binding affinity = 0.0062 µM, aq solubility = 83 µM, microsomal CLint (HLM) = 28 µM/min/mg). The improved affinity observed in the in vitro binding assay also translated to the potency observed in the WPA aggregation assay (47: 19 nM and 3: 9.5 nM) and the observed in vitro ADME properties translates to the in vivo PK properties observed in rat. In addition, we found that the chemical stability of the sulfonylureas during prolonged storage in solution was related to the sulfonyl urea linker and depended on the type of solvent and the substitution pattern of the sulfonyl urea functionality.

Ticagrelor enhances adenosine-induced coronary vasodilatory responses in humans.

OBJECTIVES: This study was undertaken to determine if ticagrelor augments adenosine-induced coronary blood flow and the sensation of dyspnea in human subjects. BACKGROUND: Ticagrelor is a P2Y(12) receptor antagonist that showed superior clinical benefit versus clopidogrel in a phase III trial (PLATO [Platelet Inhibition and Patient Outcomes]). Ticagrelor has been shown to inhibit cell uptake of adenosine and enhance adenosine-mediated hyperemia responses in a dog model. METHODS: In this double-blind, placebo-controlled study, 40 healthy male subjects were randomized to receive a single dose of ticagrelor (180 mg) or placebo in a crossover fashion. Coronary blood flow velocity (CBFV) was measured by using transthoracic Doppler echocardiography at rest after multiple stepwise adenosine infusions given before and after study drug, and again after the infusion of theophylline. RESULTS: Ticagrelor significantly increased the area under the curve of CBFV versus the adenosine dose compared with placebo (p = 0.008). There was a significant correlation between ticagrelor plasma concentrations and increases in the area under the curve (p < 0.001). In both treatment groups, the adenosine-induced increase in CBFV was significantly attenuated by theophylline, with no significant differences between subjects receiving ticagrelor or placebo (p = 0.39). Furthermore, ticagrelor significantly enhanced the sensation of dyspnea during adenosine infusion, and the effects were diminished by theophylline. CONCLUSIONS: Ticagrelor enhanced adenosine-induced CBFV and the sensation of dyspnea in these healthy male subjects via an adenosine-mediated mechanism. (Study to Assess the Effect of Ticagrelor on Coronary Blood Flow in Healthy Male Subjects; NCT01226602).

Evaluation of ticagrelor pharmacodynamic interactions with reversibly binding or non-reversibly binding P2Y(12) antagonists in an ex-vivo canine model.

INTRODUCTION: As ticagrelor, clopidogrel and cangrelor therapies may be used in the same clinical setting, their potential pharmacodynamic interactions are of interest. Hence, we investigated possible interactions between these agents in dogs using a variety of switching protocols. METHODS: Six male dogs all received 7 different dosing regimens separated by 1-5week washout periods: cangrelor (1µg/kg/min, intravenous infusion); ticagrelor (0.8mg/kg, oral); clopidogrel (3mg/kg, intravenous injection); cangrelor together with ticagrelor initiated 10minutes after cangrelor infusion start or clopidogrel given 30minutes after cangrelor infusion start; ticagrelor followed by clopidogrel given 3 or 7hours after ticagrelor dosing. ADP-induced whole blood platelet aggregation was measured by impedance aggregometry. RESULTS: Mean maximum inhibition of platelet aggregation (IPA) was 81-87% at 6minutes (cangrelor), 3hours (ticagrelor) and 4hours (clopidogrel) postdosing and platelet function recovered after 1.5hours, 12hours, and 9days, respectively. IPA at 2hours post clopidogrel was reduced to 39% when clopidogrel was given during cangrelor infusion versus 69% for clopidogrel alone. With clopidogrel dosed 3hours after ticagrelor, IPA was reduced after washout of ticagrelor to 38% at 24hrs vs. 68% for clopidogrel alone, but an interaction was not seen when clopidogrel was dosed 7hours after ticagrelor. No pharmacodynamic interaction occurred between ticagrelor and cangrelor. CONCLUSIONS: The extent of the pharmacodynamic drug-drug interactions observed between clopidogrel and cangrelor or ticagrelor apparently depends on the level of receptor occupancy when clopidogrel is administered. Importantly, no significant pharmacodynamic interaction occurred between ticagrelor/clopidogrel when clopidogrel was given at clinical trough IPA levels with ticagrelor. No significant pharmacodynamic interaction occurred with cangrelor and ticagrelor.

Ticagrelor inhibits adenosine uptake in vitro and enhances adenosine-mediated hyperemia responses in a canine model.

AIMS: A routine secondary pharmacology screen indicated that reversibly binding oral P2Y(12) receptor antagonist ticagrelor could inhibit adenosine uptake in human erythrocytes, suggesting that ticagrelor may potentiate adenosine-mediated responses in vivo. The aim of this study was to further characterize the adenosine uptake inhibition in vitro and study possible physiological consequences of adenosine uptake inhibition by ticagrelor in an anesthetized dog model of coronary blood flow compared to dipyridamole. METHODS AND RESULTS: We measured [2-3H]adenosine uptake in purified human erythrocytes and several cell lines in the presence of ticagrelor or the known uptake inhibitor dipyridamole as a comparator. Using an open-chest dog model (beagles), we measured the left anterior descending (LAD) coronary artery blood flow during reactive hyperemia after 1 minute occlusion or intracoronary infusion of adenosine before and after administration of vehicle, ticagrelor, or dipyridamole (each n = 8). Ticagrelor concentration-dependently inhibited adenosine uptake in human erythrocytes and in cell lines of rat, canine, or human origin. In the dog model, ticagrelor and dipyridamole dose-dependently augmented reactive hyperemia after LAD occlusion, as assessed by percentage repayment of flow debt relative to control (both Ps < .05). Ticagrelor and dipyridamole also dose-dependently augmented intracoronary adenosine-induced increases in LAD blood flow relative to control (both Ps < .05). CONCLUSION: Ticagrelor inhibits adenosine uptake in vitro and subsequently augments cardiac blood flow in a canine model of reactive hypoxia- or adenosine-induced blood flow increases. These findings suggest that ticagrelor may have additional benefits in patients with acute coronary syndrome beyond inhibition of platelet aggregation.

Role of newly formed platelets in thrombus formation in rat after clopidogrel treatment: comparison to the reversible binding P2Y12 antagonist ticagrelor.

Platelet P2Y12 receptors play an important role in arterial thrombosis by stimulating thrombus growth. Both irreversibly (clopidogrel) and reversibly binding (ticagrelor, AZD6140) P2Y12 antagonists are clinically used for restricted periods, but possible differences in platelet function recovery after drug cessation have not been investigated. We treated WKY rats with a single, high dose of 200 mg/kg clopidogrel or 40 mg/kg ticagrelor. Blood was collected at different time points after treatment. Flow cytometry confirmed full platelet protection against ADP-induced αIIbß3 activation shortly after clopidogrel or ticagrelor treatment. At later time points after clopidogrel treatment, a subpopulation of juvenile platelets appeared that was fully responsive to ADP. Addition of ticagrelor to clopidogrel-treated blood reduced αIIbß3 activation of the unprotected platelets. In contrast, at later time points after ticagrelor treatment, all platelets gradually lost their protection against ADP activation. Perfusion experiments showed abolishment of thrombus formation shortly after clopidogrel or ticagrelor treatment. Thrombus formation on collagen was determined under high shear flow conditions. At later time points, large thrombi formed in the clopidogrel but not in the ticagrelor group, and unprotected, juvenile platelets preferentially incorporated into the formed thrombi. However, platelets from both groups were still similarly reduced in assays of whole blood aggregation. Conclusively, recovery of rat platelet function after ticagrelor differs mechanistically from that after clopidogrel. This difference is masked by conventional platelet aggregation methods, but is revealed by thrombus formation measurement under flow. Juvenile platelets formed at later time points after clopidogrel treatment promoted thrombus formation.

A novel series of piperazinyl-pyridine ureas as antagonists of the purinergic P2Y12 receptor.

A novel series of P2Y(12) antagonists for development of drugs within the antiplatelet area is presented. The synthesis of the piperazinyl-pyridine urea derivatives and their structure-activity relationships (SAR) are described. Several compounds showed P2Y(12) antagonistic activities in the sub-micromolar range.

Adjunctive treatment with ticagrelor, but not clopidogrel, added to tPA enables sustained coronary artery recanalisation with recovery of myocardium perfusion in a canine coronary thrombosis model.

Reperfusion therapy for myocardial infarction is limited by significant re-occlusion rates and less-than-optimal myocardial tissue perfusion. It was the objective of this study to assess and compare the effect of ticagrelor, the first reversibly binding oral P2Y12 receptor antagonist, with that of clopidogrel, in conjunction with thrombolytic therapy, on platelet aggregation, thrombus formation, and myocardial perfusion in a canine model. Thrombus formation was induced by electrolytic injury and blood flow was measured with a Doppler ultrasonic flowmeter. All animals received tissue plasminogen activator (tPA) (1 mg/kg over 20 min); 10 animals received clopidogrel (10 mg/kg IV bolus over 5 min), 10 animals received ticagrelor initiated with a 1-min bolus (75 microg/kg/min), followed by continuous infusion (10 microg/kg/min) for 2 h, and 10 animals received IV saline. Re-occlusion rate and cyclic flow variation decreased with ticagrelor compared to saline groups (p<0.05). Adenosine phosphate (ADP)-induced platelet aggregation decreased with ticagrelor (1.9% +/- 2.67) and clopidogrel (1.11% +/- 2.0) vs. saline (26.3% +/- 23.5, p<0.05) at the end of adjunctive therapy. Bleeding time increased in the clopidogrel compared to the ticagrelor group (p=0.01). Infarct size was reduced with ticagrelor compared to the clopidogrel and saline groups (p<0.05). Blood flow remained significantly below baseline values at 20 min after tPA administration in the saline and clopidogrel groups but not in the ticagrelor group. In conclusion, in a dog coronary thrombosis model, ticagrelor blocks ADP-induced platelet activation and aggregation; prevents platelet-mediated thrombosis; prolongs reperfusion time and reduces re-occlusion and cyclic flow variation; and significantly decreases infarct size and rapidly restores myocardial tissue perfusion.

Stabilizing role of platelet P2Y(12) receptors in shear-dependent thrombus formation on ruptured plaques.

BACKGROUND: In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y(12) receptors. However, such models do not predict involvement of P2Y(12) in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y(12) in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis. METHODOLOGY: Plaques in the carotid arteries from Apoe(-/-) mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y(12) blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated. PRINCIPAL FINDINGS: Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y(12) blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y(12) reduced the thrombi and increased embolization events. These P2Y(12) effects were present at shear rates >500 s(-1), and they persisted in the presence of coagulation. P2Y(12)-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding. CONCLUSIONS/SIGNIFICANCE: Platelet P2Y(12) receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y(12) function is restricted to high shear flow conditions, and is preserved in the presence of coagulation.

Comparison of ticagrelor and thienopyridine P2Y(12) binding characteristics and antithrombotic and bleeding effects in rat and dog models of thrombosis/hemostasis.

Ticagrelor (AZD6140), the first reversibly binding oral P2Y(12) receptor antagonist, blocks adenosine diphosphate (ADP)-induced platelet aggregation via a mode of action distinct from that of thienopyridine antiplatelet agents. The latter must be metabolically activated and binds irreversibly to P2Y(12) for the life of the platelet, precluding restoration of hemostatic function without the generation of new platelets. In in vitro studies comparing binding characteristics of ticagrelor and compound 105, a chemical compound indistinguishable from the active metabolite of prasugrel, ticagrelor exhibited 1) an approximately 100-fold higher affinity for P2Y(12) and rapid achievement of equilibrium (vs no equilibrium reached with compound 105) as assessed by radioligand displacement in a receptor filtration binding assay, 2) 48-fold greater potency in a functional receptor assay using recombinant human P2Y(12), and 3) 63-fold greater potency in inhibiting ADP-induced aggregation in washed human platelets. In rat and dog models of thrombosis/hemostasis, there was greater separation between doses that provided antithrombotic effect and those that increased bleeding for ticagrelor compared with clopidogrel and compound 072, a chemical compound indistinguishable from the prasugrel parent compound. The ratio of dose resulting in 3-fold increase in bleeding time to dose resulting in 50% restoration of blood flow in rats was 9.7 for ticagrelor compared with 2.0 for clopidogrel and 1.4 for compound 072. Similar results were observed in dogs. Our findings suggest that reversibility of P2Y(12) binding with ticagrelor may account for the greater separation between antithrombotic effects and increased bleeding compared with the irreversible binding of clopidogrel and prasugrel.

Ticagrelor: the first reversibly binding oral P2Y12 receptor antagonist.

Ticagrelor (AZD6140) is the first reversibly binding oral P2Y(12) receptor antagonist that blocks ADP-induced platelet aggregation. Unlike thienopyridines, which irreversibly bind to the P2Y(12) receptor for the lifetime of the platelet, ticagrelor binds reversibly to the receptor and exhibits rapid onset and offset of effect, which closely follow drug exposure levels. Animal models indicate greater separation between antithrombotic effects and bleeding effects with ticagrelor than with thienopyridines. Unlike the thienopyridines, ticagrelor does not require metabolic activation. It is quickly absorbed and exhibits a rapid antiplatelet effect, with higher and more consistent levels of inhibition of platelet aggregation (IPA) being maintained across the dosing interval than with clopidogrel. IPA levels decline with plasma drug levels after discontinuation of dosing. In the phase II DISPERSE-2 trial of 990 patients with non-ST-elevation acute coronary syndromes (ACS), ticagrelor treatment with 90 mg and 180 mg twice daily showed comparable rates of major and minor bleeding compared with clopidogrel 75 mg while there were numerically fewer myocardial infarctions. Ticagrelor resulted in greater IPA in clopidogrel-naïve patients and produced substantial additional reductions in platelet aggregation activity in patients pretreated with clopidogrel. Ticagrelor treatment was well tolerated in DISPERSE-2, and discontinuation rates were comparable to those observed for clopidogrel. An increased risk of mild to moderate dyspnea and mostly asymptomatic ventricular pauses were observed in phase II studies. The mechanisms for these effects are currently being investigated. The efficacy and safety of ticagrelor are being further evaluated in the phase III PLATO trial, involving approximately 18,000 patients with ACS, including both ST-elevation and non-ST-elevation ACS.

Ticagrelor binds to human P2Y(12) independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation.

BACKGROUND: P2Y(12) plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. OBJECTIVE: To characterize the receptor biology of the first reversibly binding oral P2Y(12) antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. METHODS: Ticagrelor displayed apparent non-competitive or insurmountable antagonism of ADP-induced aggregation in human washed platelets. This was investigated using competition binding against [(3)H]ADP, [(33)P]2MeS-ADP and the investigational CPTP compound [(125)I]AZ11931285 at recombinant human P2Y(12). Functional receptor inhibition studies were performed using a GTPgammaS-binding assay, and further binding studies were performed using membranes prepared from washed human platelets. RESULTS: Radioligand-binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y(12) with K(on) and K(off) of (1.1 +/- 0.2) x 10(-4) nm(-1) s(-1) and (8.7 +/- 1.4) x 10(-4) s(-1), respectively. Ticagrelor does not displace [(3)H]ADP from the receptor (K(i) > 10 mum) but binds competitively with [(33)P]2MeS-ADP (K(i) = 4.3 +/- 1.3 nm) and [(125)I]AZ11931285 (K(i) = 0.33 +/- 0.04 nm), and shows apparent non-competitive inhibition of ADP-induced signaling but competitive inhibition of 2MeS-ADP-induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non-competitive binding for ADP and ticagrelor. CONCLUSIONS: These data indicate that P2Y(12) is targeted by ticagrelor via a mechanism that is non-competitive with ADP, suggesting the existence of an independent receptor-binding site for CPTPs.

Evaluation of platelet function, a method comparison.

Platelet function can be studied using many different methods why it is of interest to understand how data from different assays relate to each other. In the present study we compare two methods suitable for screening purposes with two established although laborious methods, impedance aggregometry and platelet-rich plasma (PRP) aggregation. The alternative assays tested were: (i) exposure of active alphaIIbbeta3, in diluted whole blood and (ii) whole blood aggregation assessed by residual platelet counting. The fibrinogen receptor activation assay was found to have the lower variability, higher sensitivity to ADP, and higher signal to noise ratio compared with residual platelet counting. The sensitivity and response profile of the fibrinogen receptor activation assay and residual platelet counting were more similar to PRP aggregation than to impedance aggregometry, whereas impedance aggregometry displayed lower sensitivity to ADP. The two alternative assays correlated well with PRP aggregation as well as with each other. The fibrinogen receptor activation assay displayed the highest potency for AR-C69931MX, possibly due to a lower protein content compared with residual platelet counting. The two studied assays compare well with the more established assays, and are thus both good alternatives for platelet function testing and evaluation of new potential platelet antagonists.

Preclinical and clinical studies with selective reversible direct P2Y12 antagonists.

An important role for adenosine diphosphate (ADP)-induced platelet activation and aggregation was proposed more than 40 years ago. The clinical use of clopidogrel, a prodrug of an irreversible P2Y (12) antagonist, has further proved the relevance of inhibiting signaling via the platelet-specific P2Y (12) ADP receptor in the prevention of cardiovascular events. Pharmacological studies at AstraZeneca R&D Charnwood have identified direct, selective, and competitive P2Y (12) antagonists, including cangrelor (also known as AR-C69931MX), which is suitable for intravenous administration, and AZD6140, which is suitable for oral administration. In preclinical use, these compounds predictably and effectively inhibited platelet aggregation without significant increases in bleeding time. In clinical use, these compounds may have significant advantages over current antiplatelet agents. This article summarizes preclinical and clinical data on cangrelor and AZD6140 and discusses the potential of these compounds as novel antiplatelet therapies.