Abstract 363: Platelet Mass Retraction is a Negative Feedback Regulator of Thrombin Generation and Activity

Abstract

Endogenous anticoagulants, including antithrombin III, tissue factor pathway inhibitor, and activated protein C (aPC) provide a biochemical means to inhibit thrombin generation and/or activity. In addition, we have previously shown that platelet mass retraction prevents the escape and exchange of solutes, thereby providing a biophysical mechanism to limit thrombin generation and activity. The relative contribution of each of these biochemical and biophysical mechanisms to the termination of thrombin activity during the hemostatic response in vivo remains poorly understood. Here, using computational simulations coupled with in vivo experimental models, we tested the hypotheses that 1) platelet mass retraction itself is physiologically relevant to inhibit thrombin generation; and 2) that given the spatial localization of protein C activation on the endothelial cell surface, aPC mediated thrombin inhibition has negligible effects on the hemostatic response. Our computational model has several innovative aspects, including 3D representations of the vessel, extravascular space, and injury, blood flow, and a simplified but anatomically correct model of coagulation. Our simulations show that tissue factor localization combined with flow exiting the injury limit thrombin generation/activity to the extravascular space. Our experiments using a mouse jugular puncture injury model show rich deposition of fibrin in the extravascular space and little to no fibrin in the luminal side. Additionally, our simulations illustrate how platelet mass retraction inhibits thrombin generation by decreasing the delivery of substrates, such as prothrombin and factor X, and at the same time it limits thrombin activity by constraining its movement. These effects are mediated by physical forces only and are independent of the biochemical inhibitory pathways. Our in vivo experiments using aPC inhibitory antibodies show that lack of aPC mediated thrombin inhibition has negligible effects on the hemostatic response. In conclusion, this study suggests that platelet mass retraction is an underappreciated mechanism that negatively regulates thrombin generation, while aPC mediated inhibition of thrombin generation plays a minor role in hemostasis.