Murine Thrombus Organization Limits Access to High Platelet Activation States While Supporting Platelet Recruitment

- Exposure of high activation platelet surfaces to intravascular factors peaks post bleeding cessation. - Modulating exposure of highly activated platelet surfaces to circulating coagulation factors is a potential target for limiting thrombosis. Platelet aggregation at sites of vascular injury is essential for hemostasis. However, the mechanisms that prevent excessive clot growth are not fully understood. In the prevailing Core and Shell model, based largely on small vessel injury studies, a central Core of highly activated platelets is surrounded by a limited signal intensity Shell of less activated, minimally degranulated platelets. Recent reports, especially in mouse models of profuse bleeding, suggest thrombus architecture and platelet activation states are more heterogeneous than the binary Core and Shell model proposes. Here, we performed high-resolution morphometric mapping of individual platelet activation states in mouse jugular vein and femoral artery puncture wound thrombi, using serial block face scanning electron microscopy and wide-area transmission electron microscopy. Manually annotated images were analyzed at multiple time points, revealing initial,1-min, near complete intermixing of platelet activation states with no distinct core of highly activated platelets. At 5 min, highly activated, degranulated platelets became concentrated along the interior surfaces of vaulted thrombus structures. At 20 min, platelet numbers decreased and distinct clustering of degranulated, highly activated platelets was observed within central portions of the intravascular platelet-rich crown, limiting their access to the circulation. Deletion of the α-granule v-SNARE, VAMP8, increased both the frequency and clustering of highly activated platelets. Similar patterns were observed in femoral artery wounds. We conclude that thrombus organization is more complex than previously recognized and provide evidence that progressive structural changes help limit procoagulant surface exposure and thrombus growth during hemostasis following puncture wounding.