Abstract 525: Integration of Platelet Agonist Signaling During the Hemostatic Response in vivo

Abstract

The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation emanating from the site of injury. While thrombin and ADP mediated platelet activation are clearly critical for establishment of this gradient, it remains unclear to what extent there is overlap of these and other platelet signaling pathways in time and space. In other words, does a single agonist dominate the platelet activation state within specific regions of a hemostatic plug, or do multiple agonist pathways need to be integrated to produce the gradient of platelet activation observed? Further, how do the relationships among different agonists change in the face of anti-platelet or anti-coagulant therapy and what are the consequences for hemostatic plug organization? Here, we used a combination of genetic and pharmacologic approaches coupled with real-time intravital imaging to examine how thrombin, thromboxane A 2 , P2Y 12 and epinephrine signaling are coordinated in time and space to regulate the development of platelet activation gradients at a site of vascular injury in vivo. We found that both thromboxane A 2 /TP signaling and ADP/P2Y 12 signaling are required for accumulation of minimally activated platelets in the outer shell region of hemostatic plugs. Interestingly, dual inhibition of both thromboxane and P2Y 12 signaling did not have an additive effect, but rather was similar to inhibition of either pathway alone. Epinephrine, which activates a Gi signaling pathway similar to ADP/P2Y 12 , was completely dispensable for hemostatic plug formation, even in the absence of P2Y 12 signaling. Finally, inhibition of P2Y 12 in the setting of sub-maximal thrombin activity revealed no role for P2Y 12 signaling in the development of robust platelet activation within the hemostatic plug core region. Taken together, these data shed new light on the way multiple platelet signaling pathways are integrated during the hemostatic response in vivo, and predict the outcome of therapeutically targeting specific platelet signaling pathways alone and in combination on hemostatic plug organization.