Abstract 41: Integrin Outside-in Signaling Regulates Local Microenvironments Within Hemostatic Plugs

Abstract

Prior studies have demonstrated that platelet activation within a hemostatic plug is heterogeneous. In particular, we recently determined that following vascular injury in vivo, a platelet mass is composed of distinct regions defined by the extent of platelet activation and other properties. At least two regions are apparent: a core of fully activated and degranulated platelets immediately adjacent to the site of injury, which is overlaid by a shell of less activated platelets that have not undergone a-granule secretion. Here, we tested the hypothesis that platelet accumulation and activation are regulated by the interaction of platelet signaling pathways with local microenvironments present within the different regions of a hemostatic plug using novel confocal intravital imaging approaches. We also hypothesized that integrin outside-in signaling plays a role in shaping the core region microenvironment. The results reveal that plasma volume (i.e. porosity) and solute transport velocity are significantly reduced in the interplatelet space of a hemostatic plug as compared to the bulk blood flow following laser-induced injury in mouse cremaster arterioles. Further, porosity and solute transport are significantly reduced in the core region of the hemostatic plug as a result of tight platelet packing, as compared to the outer shell region composed of loosely adherent platelets. To examine the impact of integrin outside-in signaling we utilized mice lacking beta 3 integrin cytoplasmic tail tyrosine phosphorylation (diYF). Prior studies have shown unstable platelet aggregation and defective fibrin clot retraction in diYF mice. We found diYF mice formed smaller hemostatic plugs following vascular injury in vivo. This decrease in total platelet accumulation was associated with reduced formation of the hemostatic plug core region and decreased fibrin generation, indicating decreased thrombin activity within the hemostatic plug. Taken together, these results support the hypothesis that tight platelet packing provides a protected microenvironment within a hemostatic plug in which bioactive molecules may accumulate to support platelet activation. They also identify a novel role for aIIbb3 outside-in signaling in hemostatic plug formation.