Regular Article THROMBOSIS AND HEMOSTASIS Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network

Platelet accumulation is a hallmark of hemostasis and a contributing factor in heart attacks and strokes. Platelet activation is driven by receptor-mediated signaling in response to stimuli of varying potency, such as collagen, thrombin, adenosine 59-diphosphate (ADP), and thromboxane A2 (TxA2). This has led to a model of the hemostatic response in which redundant elements of the platelet-signaling network work in concert to produce platelet aggregation, thrombin generation, and a hemostatic mass composed of activated platelets interspersed with fibrin. Interestingly, despite the long-recognized ability of multiple platelet agonists to drive platelet activation to completion in vitro, observations performed in vivo show that platelet activation is not uniform throughout a hemostatic plug. Rather, some of the platelets accumulating at a site of injury retain a discoid, or “resting,” morphology, cytosolic calcium mobilization is heterogeneous, and a-granule secretion occurs nonuniformly throughout the growing hemostatic mass. Consistent with these recent observations performed in vivo, variations in the extent of platelet activation during the hemostatic response have been demonstrated by electron microscopy studies dating back to the 1960s that examined thrombi formed in vivo and ex vivo. These observations raise a number of questions. If the hemostatic response normally produces a mixed population of platelets with varying degrees of activation, what are the implications for achieving a stable plug and for avoiding unnecessary vascular occlusion? How can a common signaling network produce distinguishable outcomes among participating platelets and how might different agonists contribute to these outcomes? How does the growing hemostatic structure alter the conditions experienced by individual platelets and what impact does that have on subsequent events? Finally, how might differences in the clinical impact of antiplatelet agents taken to prevent adverse cardiovascular events be understood in the context of the heterogeneous platelet activation observed during the hemostatic response? With these questions in mind, our first goal in the present study was to determine how variations in platelet activation in vivo arise through the integration of distinct elements of the platelet-signaling network. Our second goal was to determine how regional variations in the extent of platelet activation affect the stability of the hemostatic mass and the passage of plasma-borne molecules within the mass. To achieve these goals, we used a combination of highresolution intravital confocal microscopy, genetically engineered