Abstract 530: Hemostasis Revisited: a Combined Light and Electron Microscopy Analysis of Hemostatic Plug Formation in vivo

Abstract

We have previously shown in the microcirculation that gradients of platelet agonists develop during the hemostatic response as a consequence of the solute transport properties within the evolving platelet mass. These agonist gradients result in the formation of a gradient of platelet activation extending from the site of vascular injury. Here we utilized a combined light and electron microscopy approach to examine the organization of hemostatic plugs formed in response to larger injuries that result in significant bleeding in the mouse jugular vein. The results show that the hemostatic response to vessel wall puncture in a large vein is similarly characterized by a gradient of platelet activation. Specifically, we found that 1) platelets are the primary cellular component of hemostatic plugs in large veins; 2) platelet activation is heterogeneous, with fully activated and degranulated platelets present at the interface of the damaged vessel wall and extending to the extravascular side of hemostatic plugs, while platelets aggregated on the luminal side of plugs were minimally activated; 3) fibrin, and by extension thrombin activity, is localized primarily in the extravascular space; 4) P2Y 12 antagonism impairs platelet aggregation, resulting in a prolonged bleeding time when the hole created by the puncture injury does not completely fill with platelets; and 5) the importance of P2Y 12 signaling in establishing a hemostatically competent plug is related to the size of the hole through which blood escapes the vasculature. Taken together, these results provide novel insights into the mechanisms of hemostatic plug formation in vivo, demonstrate in detail the mechanism by which P2Y 12 antagonists result in bleeding, and highlight differences in the composition and morphology of hemostatic plugs versus pathologic thrombi.