PB0891 - Evolution of the Platelet Plug and Leukocyte Recruitment After Initial Hemostasis In Vivo

Background: Platelets express the leukocyte recruiting adhesion molecule P-selectin upon activation and subsequent alpha-granule release. Leukocyte migration through the endothelium and interactions with platelets via platelet-leukocyte aggregates are well described; however, the ability of leukocytes to infiltrate and migrate through a hemostatic plug via platelet interaction after vascular injury remains poorly understood.

Aims: This study aims to elucidate changes in platelet plug architecture and the ability of leukocytes to migrate through the plug for up to 60 minutes after hemostatic injury.

Methods: A mouse jugular puncture injury model was used in conjunction with quantitative multi-photon microscopy to measure platelet accumulation, alpha granule secretion, fibrin deposition, and leukocyte recruitment over one hour post-injury.

Results: Bleeding cessation was achieved within 47±2.22 seconds following puncture injury of the mouse jugular vein. We found that platelet recruitment continued beyond initial hemostasis, as total platelet accumulation was significantly increased 30 minutes post-injury as compared to 5 min post-injury (p < 0.05). Fibrin deposition on the extravascular side of the injury site also continued to increase following initial hemostasis (p < 0.05, 5 vs. 30 min post-injury). In association with these changes in plug architecture, we observed a time dependent increase in leukocyte recruitment to the platelet plug within the first hour post-injury, particularly on the intraluminal side of the vessel (p < 0.0001, 5 vs. 60 min post-injury).

Conclusion(s): Taken together, our results describe changes in hemostatic plug structure and function that occur after hemostasis is achieved. Continued fibrin deposition suggests ongoing localized thrombin activity over an extended period at the injury site. Concurrently, leukocytes are recruited and infiltrate the interior of the platelet plug. These findings suggest continued evolution of hemostatic plug structure and function after bleeding cessation occurs to facilitate plug stabilization and wound healing.