Development of a Stable Thrombotic Core with Limited Access to Plasma Proteins During Thrombus Formation In Vivo

Abstract 2013 Several studies examining thrombus formation in vivo have described the formation of a thrombotic core composed of stably adherent, apparently activated platelets covered by several layers of minimally activated, loosely adherent platelets. Using confocal fluorescence intravital microscopy in mouse cremaster muscle arterioles, we sought to 1) determine whether the stable thrombus core could be defined by the presence of platelet activation markers (e.g. P-selectin); 2) define the spatial and temporal characteristics of the stable thrombus core as it develops; and 3) determine whether the accessibility of plasma proteins to the inner regions of a stable thrombus is limited. We found that P-selectin platelet surface expression is associated with stable platelet incorporation into a growing thrombus following either laser- or wall puncture-induced vascular injury in vivo. The P-selectin positive core originated at the site of vascular injury and subsequently expanded outward into the central region of the developing thrombus with kinetics that were distinct from total platelet accumulation. Further, using two related approaches, we determined that the accessibility of plasma components to the stable thrombus core is limited. In the first approach, fluorescently labeled anti-P-selectin antibody was infused after a stable thrombus had formed (25 minutes after injury), and was found to bind to a monolayer of platelets on the luminal surface of the stable thrombus core, but was unable to penetrate this layer of platelets and bind to platelets in the central region of the thrombus. In the second approach, we directly measured the porosity of thrombi as they evolved by infusing fluorescently labeled dextran to illuminate the plasma in the gaps between platelets. We found that the porosity of the stable thrombus core was significantly decreased as compared to the porosity of the growing platelet mass before the core developed. Taken together, these results demonstrate that a stable core composed of degranulated platelets develops during thrombus formation in vivo with spatial localization and kinetics that are distinct from total platelet accumulation, and that development of this core limits the accessibility of plasma components to the central region of a stable thrombus. Disclosures: No relevant conflicts of interest to declare.