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IntroductionPlatelet aggregation at sites of vascular injury is essential for the formation of the primary hemostatic plug and also for the development of pathological thrombi at sites of atherosclerotic plaque rupture. The initial contact of platelets with the injured vessel wall (platelet adhesion) is a complex process involving multiple adhesive substrates (von Willebrand factor [vWf], collagen) and receptors on the platelet surface (GPIb/V/IX, integrins α IIb β 3 and α 2 β 1 ) (1). The interaction between matrix-bound vWf and GPIbα on the platelet surface serves primarily to tether platelets to the area of vascular injury (2, 3), particularly under conditions of high shear stress, as a prerequisite step for integrin-mediated cell arrest (4). Whereas the molecular events underlying platelet adhesion under different shear conditions have been well delineated, the mechanism(s) by which platelets in freeflowing blood subsequently adhere to the initial layer of adherent platelets (platelet cohesion or aggregation) under flow have been less clearly defined.The traditional model of platelet aggregation, in which integrin α IIb β 3 was thought to have an exclusive role in mediating platelet-platelet adhesion contacts, has been largely determined from studies using a platelet aggregometer (5). With this method, the addition of a soluble agonist to a stirred platelet suspension induces activation of integrin α IIb β 3, converting it from a low-to a high-affinity receptor capable of binding soluble fibrinogen. The dimeric nature of fibrinogen enables it to cross-link adjacent activated platelets leading to stable platelet aggregation. Studies of platelet aggregation under high shear conditions, using a coneplate viscometer, have demonstrated that plasma vWf becomes the relevant ligand responsible for platelet activation (6). Shear-induced binding of soluble vWf to GPIbα initiates platelet activation independent of the addition of exogenous stimuli. Whereas the vWf-GPIbα interaction is indispensable for the initiation of platelet-platelet adhesion contacts under high shear, irreversible platelet aggregation requires a second adhesive interaction between vWf and integrin α IIb β 3 (7).The molecular events governing the formation of stable adhesion contacts between platelets in suspension have been well delineated; however, the mechanism by In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin α IIb β 3 demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin α IIb β 3 mediated cell ...

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin ␣ IIb ␤ 3 , involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin ␣ IIb ␤ 3 engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin ␣ IIb ␤ 3 , in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin ␣ IIb ␤ 3 -dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin ␣ IIb ␤ 3 that serves to regulate the dynamics of platelet adhesion under flow.

Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcR␥-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/ FcR␥-chain complex (FcR␥ ؊/؊ ). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcR␥ is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcR␥ was associated with a 30% reduction in thrombus growth. Analysis of FcR␥ ؊/؊ platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcR␥ deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcR␥ compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcR␥ ؊/؊ mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcR␥ deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents. (Blood. 2006;107:4346-4353)