The abnormal adherence of sickle red blood cells (SS RBC) to the vessel wall is generally held to be of central importance to the pathogenesis of vaso-occlusive events in sickle cell disease (SCD). There is extensive clinical evidence indicating that increased thrombin levels are generated in patients with SCD, yet the relevance of thrombin to the pathogenesis of sickle vaso-occlusion in the microcirculation remains to be established. Thrombin is the central bioregulatory enzyme in hemostasis. It converts coagulation factors V, VIII and XIII to their active forms, cleaves fibrinogen promoting the formation of fibrin, and potently activates platelets. Thrombin also exerts direct effects on vascular endothelial cells (EC) producing wide ranging alterations in phenotype. The overall hypothesis of this proposal is that increased thrombin generating in SCD promotes SS RBC adhesion and consequent vaso-occlusive events. We will employ in vitro and intravital microscopic assays of SS RBC adhesion employing cultured vascular EC and in murine models to address this hypothesis. The proposed studies have been designed to determine the contributions of thrombin-activated EC and platelets to sickle vaso- occlusive phenomena. We will examine the direct effects of thrombin on vascular EC including von Willebrand factor (vWF) secretion. We will assess the contribution of platelets and platelet activation to SS RBC/EC adhesion. The proposed intravital studies will utilize several mouse strains, singly and in combination, that are either currently available or which will be developed in the context of this application. These include mice that bear a SCD phenotype (SAD), or in which there is variable thrombomodulin (TM) activity (and thus altered thrombin generation), absence of thrombospondin-1 (TSP) expression, or increased expression of ATP diphosphohydrolase (ATPDase; CD 39). We will also examine further the interaction of vWF and TSP as these pertain to SS RBC binding to EC and to the subendothelial matrix. We will test the hypothesis that the molecular context of vWF is crucial to its ability to support SS RBC adhesion. The results of these investigations are likely to provide a basis for novel therapeutic approaches to the treatments of patients with SCD.
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