Redundancy and interaction of thrombin- and collagen-mediated platelet activation in tail bleeding and carotid thrombosis in mice.

OBJECTIVE: Current antiplatelet strategies to prevent myocardial infarction and stroke are limited by bleeding risk. A better understanding of the roles of distinct platelet-activating pathways is needed. We determined whether platelet activation by 2 key primary activators, thrombin and collagen, plays distinct, redundant, or interacting roles in tail bleeding and carotid thrombosis in mice. APPROACH AND RESULTS: Platelets from mice deficient for the thrombin receptor protease-activated receptor-4 (Par4) and the collagen receptor glycoprotein VI protein (GPVI) lack responses to thrombin and collagen, respectively. We examined tail bleeding and FeCl3-induced carotid artery occlusion in mice lacking Par4, GPVI, or both. We also examined a series of Par mutants with increasing impairment of thrombin signaling in platelets. Ablation of thrombin signaling alone by Par4 deficiency increased blood loss in the tail bleeding assay and impaired occlusive thrombus formation in the carotid occlusion assay. GPVI deficiency alone had no effect. Superimposing GPVI deficiency on Par4 deficiency markedly increased effect size in both assays. In contrast to complete ablation of thrombin signaling, 9- and 19-fold increases in EC50 for thrombin-induced platelet activation had only modest effects. CONCLUSIONS: The observation that loss of Par4 uncovered large effects of GPVI deficiency implies that Par4 and GPVI made independent, partially redundant contributions to occlusive thrombus formation in the carotid and to hemostatic clot formation in the tail under the experimental conditions examined. At face value, these results suggest that thrombin- and collagen-induced platelet activation can play partially redundant roles, despite important differences in how these agonists are made available to platelets.

Roles and interactions among protease-activated receptors and P2ry12 in hemostasis and thrombosis.

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. Par3(-/-) and Par4(+/-) mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. P2ry12(+/-) mouse platelets showed partially decreased responses to ADP, resembling those in clopidogrel-treated human platelets. Par3(-/-) mice showed nearly complete protection against carotid artery thrombosis caused by low FeCl(3) injury. Par4(+/-) and P2ry12(+/-) mice showed partial protection. Increasing FeCl(3) injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in Par3(-/-):P2ry12(+/-) mice, restored it. Par4(-/-) mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that (i) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; (ii) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and (iii) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies.