Development of a Stable Thrombotic Core with Limited Access to Plasma Proteins During Thrombus Formation In Vivo
Timothy J. Stalker, PhD*,1,
Elizabeth A. Traxler, MS*,1,
Scott L. Diamond, PhD2 and
Lawrence F. Brass, MD, PhD3
1 Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA, 2 Institute for Medicine and Biology, University of Pennsylvania, Philadelphia, PA, USA, 3 University of Pennsylvania, Philadelphia, PA, USA
Several studies examining thrombus formation in vivo have describedthe formation of a thrombotic core composed of stably adherent,apparently activated platelets covered by several layers ofminimally activated, loosely adherent platelets. Using confocalfluorescence intravital microscopy in mouse cremaster musclearterioles, we sought to 1) determine whether the stable thrombuscore could be defined by the presence of platelet activationmarkers (e.g. P-selectin); 2) define the spatial and temporalcharacteristics of the stable thrombus core as it develops;and 3) determine whether the accessibility of plasma proteinsto the inner regions of a stable thrombus is limited. We foundthat P-selectin platelet surface expression is associated withstable platelet incorporation into a growing thrombus followingeither laser- or wall puncture-induced vascular injury in vivo.The P-selectin positive core originated at the site of vascularinjury and subsequently expanded outward into the central regionof the developing thrombus with kinetics that were distinctfrom total platelet accumulation. Further, using two relatedapproaches, we determined that the accessibility of plasma componentsto the stable thrombus core is limited. In the first approach,fluorescently labeled anti-P-selectin antibody was infused aftera stable thrombus had formed (25 minutes after injury), andwas found to bind to a monolayer of platelets on the luminalsurface of the stable thrombus core, but was unable to penetratethis layer of platelets and bind to platelets in the centralregion of the thrombus. In the second approach, we directlymeasured the porosity of thrombi as they evolved by infusingfluorescently labeled dextran to illuminate the plasma in thegaps between platelets. We found that the porosity of the stablethrombus core was significantly decreased as compared to theporosity of the growing platelet mass before the core developed.Taken together, these results demonstrate that a stable corecomposed of degranulated platelets develops during thrombusformation in vivo with spatial localization and kinetics thatare distinct from total platelet accumulation, and that developmentof this core limits the accessibility of plasma components tothe central region of a stable thrombus.
Disclosures: No relevant conflicts of interest to declare.
* Asterisk with author names denotes non-ASH members.