Coordination of platelet agonist signaling during the hemostatic response in vivo

The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation. We previously showed that thrombin activity at a site of vascular injury is spatially restricted, resulting in robust activation of a subpopulation of platelets in the hemostatic plug core. In contrast, adenosine 5'-diphosphate (ADP)/P2Y₁₂ signaling contributes to the accumulation of partially activated, loosely packed platelets in a shell overlying the core. The contribution of the additional platelet agonists thromboxane A₂ (TxA₂) and epinephrine to this hierarchical organization was not previously shown. Using a combination of genetic and pharmacologic approaches coupled with real-time intravital imaging, we show that TxA₂ signaling is critical and nonredundant with ADP/P2Y₁₂ for platelet accumulation in the shell region but not required for full platelet activation in the hemostatic plug core, where thrombin activity is highest. In contrast, epinephrine signaling is dispensable even in the presence of a P2Y₁₂ antagonist. Finally, dual P2Y₁₂ and thrombin inhibition does not substantially inhibit hemostatic plug core formation any more than thrombin inhibition alone, providing further evidence that thrombin is the primary driver of platelet activation in this region. Taken together, these studies show for the first time how thrombin, P2Y₁₂, and TxA₂ signaling are coordinated during development of a hierarchical organization of hemostatic plugs in vivo and provide novel insights into the impact of dual antiplatelet therapy on hemostasis and thrombosis.