Coordination of platelet agonist signaling during the hemostatic response in vivo

Abstract

Key Points• Coordinated thromboxane A 2 and ADP/P2Y 12 signaling is required for platelet accumulation in the outer shell region of hemostatic plugs.• Platelet activation within the hemostatic plug core region is predominantly mediated by thrombin.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 59-diphosphate (ADP)/P2Y 12 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 A 2 (TxA 2 ) 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 TxA 2 signaling is critical and nonredundant with ADP/P2Y 12 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 P2Y 12 antagonist. Finally, dual P2Y 12 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, P2Y 12 , and TxA 2 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.

GRK6 Regulates the Hemostatic Response to Injury through Its Rate-Limiting Effects on GPCR Signaling in Platelets

“…As noted previously, thrombin is generated close to the site of injury and can induce maximal platelet activation independent of TxA 2 and P2Y 12 signaling. 18,35 This is consistent with our observation that the initial hemostatic plug forms much faster in GRK6 2/2 mice than in WT control, with an increase in the rate of the platelet accumulation. Furthermore, the increased size of the core region is driven by the gradient of thrombin concentration emanating from the site of the injury.…”

“…31,32 Recently, a model of the hemostatic response has been established in which the development of gradients of platelet-soluble agonists presents within the evolving platelet plug, resulting in a gradient of platelet activation emanating from the injury site. 18,19,[33][34][35] In the core region of the thrombus, thrombin mediates platelet activation with minimal requirement of ADP and TxA 2 . In contrast, ADP and TxA 2 signaling are critical for outer shell region formation.…”

Platelet Shp2 negatively regulates thrombus stability under high shear stress

“…This indicates that Shp2 mainly regulates the stability of the shell region of the thrombus. Coinciding with the findings that the shell region of the thrombus is influenced by ADP or TXA2 [18] and the core is under the regulation of thrombin [41,42], Shp2 selectively modulates TXA2-stimulated platelet dense granule secretion, but not platelet responses evoked by thrombin. Moreover, Shp2 responds to TXA2 stimulation by regulating outside-in signaling, consistent with the previous report that TXA2 relies on integrin outside-in signaling to regulate platelet dense granule secretion [32].…”

Activated αIIbβ3 on platelets mediates flow-dependent NETosis via SLC44A2

“…For example, platelets in the core of a hemostatic plug/thrombus are exposed to higher concentrations of agonists and are more highly activated (i.e. P-selectin-positive procoagulant platelets) than those in the surrounding shell (P-selectin-negative) (de Witt et al, 2014;Shen et al, 2017;Stalker et al, 2013;Welsh et al, 2014). Thus, platelets exhibit a 'tunable' activation response determined by agonist availability.…”