P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries

Abstract

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40–50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12–/– mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12–/– platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12–/– mice on collagen were less dense and less activated than their WT counterparts. P2Y12–/– platelet activation was also reduced in response to ADP or a PAR-4–activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population

Central role of the P2Y12 receptor in platelet activation

“…The tools available for the analysis of P2Y 12 receptor function have facilitated characterization of the implications of P2Y 12 antagonism, though the studies performed thus far also raise more questions. The differences in embolization between homozygous and heterozygous mice observed by Andre et al (16) raise several important questions. Would complete blockade of the P2Y 12 receptor be a better therapeutic goal, and would it be more beneficial than clopidogrel because of abolished receptor function?…”

“…Andre et al (16) used several approaches to explore the effects of P2Y 12 receptor deficiency on thrombus formation. FeCl 3 -induced vessel wall injury of the mouse mesenteric artery resulted in occlusion of the wild-type mouse vessel; however, the P2Y 12 -deficient mouse vessel remained unoccluded in eight out of nine mice (16). The appearance of the first thrombus was delayed and only small "unstable" thrombi formed in P2Y 12 -/-mice (16).…”

Central role of the P2Y12 receptor in platelet activation

“…Consistent with observations in patients deficient in the P2Y 12 receptor, mice lacking the P2Y 12 receptor have increased tail bleeding times (19,36,40,41). However, heterozygous mice show little or no change in bleeding times (16). It is currently unclear why a 50% decrease in the population of functional P2Y 12 receptors, either following clopidogrel treatment or in humans heterozygous for the P2Y 12 receptor, prolongs bleeding time, while heterozygous mice show little change in bleeding time ( Table 1).…”

“…Treatment with AR-C69931MX also decreased the reocclusion rate and improved myocardial tissue perfusion in a canine model of coronary electrolytic injury (53). Andre et al (16) used several approaches to explore the effects of P2Y 12 receptor deficiency on thrombus formation. FeCl 3 -induced vessel wall injury of the mouse mesenteric artery resulted in occlusion of the wild-type mouse vessel; however, the P2Y 12 -deficient mouse vessel remained unoccluded in eight out of nine mice (16).…”

Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2

“…A second receptor for ADP on platelets, P2Y12, is coupled primarily to the G i family member, G i2 (4,33). Studies showing that platelets lacking G i2 or the P2Y12 receptor have reduced responses to thrombin (2,4,34) and that blockade of P2Y12 destabilized platelet aggregates (35) raised the possibility that thrombin-mediated Akt phosphorylation might be mediated by ADP release. Our studies of Akt phosphorylation in Gα i2 -versus Gα q -null platelets showed that thrombin can stimulate Akt phosphorylation under conditions where ADP cannot, suggesting that platelets stimulated with thrombin (or U46619) do not require secreted ADP to activate Akt, although ADP can amplify Akt activation by stimulating G i2 -coupled P2Y12 receptors.…”