PB0881 - Thrombin generation after initial cessation of bleeding is required for hemostatic plug stability via PAR-mediated platelet activation.

Background: Coagulation disorders and anticoagulant therapeutics are known to impair hemostatic plug stability resulting in rebleeding. We and others have shown that thrombin is not uniformly distributed in time and space during hemostasis, however, the importance of thrombin spatiotemporal distribution for maintaining hemostasis after the initial cessation of bleeding is unknown.

Aims: We sought to determine whether continued thrombin generation after initial hemostasis is necessary for maintenance of hemostatic plug stability in vivo. The contribution of thrombin-mediated platelet activation during the post-hemostatic period was also explored.

Methods: The hemostatic response was assessed using a mouse jugular puncture model coupled with 2-photon fluorescence imaging to assess platelet accumulation, activation and fibrin deposition. The factor Xa antagonist rivaroxaban was administered either pre-injury or after bleeding had stopped to evaluate the role of thrombin generation during initial hemostasis vs. the post-hemostatic period (up to 30 minutes post-injury). Par4-deficient mice were used to examine the role of thrombin-mediated platelet activation.

Results: Rivaroxaban administered pre-injury nearly eliminated fibrin deposition (p < 0.01), and impaired platelet accumulation specifically within the intraluminal component of hemostatic plugs (p < 0.001). These changes in hemostatic plug structure did not extend the initial time to hemostasis, but resulted in impaired hemostatic plug stability with approximately 50% rebleeding within 30 minutes (p < 0.0001). Rivaroxaban administered after initial cessation of bleeding had no effect on overall fibrin formation, suggesting that the bulk of fibrin is deposited during initial hemostasis. However, post-hemostasis rivaroxaban attenuated intraluminal platelet accumulation (p < 0.05) and significantly impaired hemostatic plug stability (p < 0.0001). Par4-deficiency also impaired intraluminal platelet accumulation (p < 0.01) and increased rebleeding events (p < 0.001) despite normal fibrin formation. These findings suggest that thrombin-mediated platelet activation contributes to maintenance of hemostatic plug stability.

Conclusion(s): Thrombin generation beyond the initial cessation of bleeding is necessary for continued PAR-mediated platelet activation to maintain hemostatic plug stability.