Vascular occlusion makes a major contribution to the morbidity and mortality of sickle cell disease (SCD). The molecular basis of vascular occlusion in SCD is controversial and the nature of the defect remains poorly defined. This grant will study the role of the thrombin-dependent transglutaminase (TG) Factor XIIIa in the pathogenesis of vascular occlusion in SCD. The relationship between plasma prothrombin F1.2 levels and the vascular occlusive events in SCD will be studied. We will determine whether activated platelets, platelet microparticles or sickle cell membrane vesicles play a role in formation of thrombin in SCD. Plasma levels of cross-linked alpha-2-antiplasmin-fibrinogen complexes will be measured using an ELISA assay to analyze the activity of plasma Factor XIIIa in SCD. In addition, an assay to directly measure Factor XIIIa will be developed using a specific monoclonal antibody. Recently, the thrombin-independent tissue TG was cloned and discovered to be homologous to erythrocyte band 4.2 and the plasma fibrin stabilizing factor called, Factor XIIIa. This raises the intriguing possibility that there could be an important set of interactions between membrane proteins, membrane skeleton proteins and TGs in SCD. Direct binding studies of tissue TG with SCD erythrocytes will be performed since SCD erythrocytes have an increased number of cross-link sites on their surface. The interactions between the fibrin stabilizing enzymes, SCD membrane proteins, and the adhesive glycoproteins (vitronectin, fibronectin, fibrinogen and von Willebrand factor) will be examined in vitro. We will use flow cytometry to determine whether cross-linking between these proteins and SCD erythrocytes occur in vivo. The effect of adhesive glycoproteins and TGs on the stable attachment of SCD erythrocytes to endothelium, subendothelium and fibrin will be tested. We will develop monoclonal antibodies and peptide inhibitors to interfere with TG cross-linking at cell surfaces. The direct binding of the purified TG to Band 3 will be studied in vitro using purified proteins and erythrocyte ghosts depleted of specific membrane components. In addition, the association of the TG with purified membranes from normal and SCD erythrocytes will be quantitated. The effect of TG derived to disrupt TG cross-linking of adhesive glycoproteins will also be studied. This grant attempts to bridge gaps in our understanding of the interactions between coagulation, transglutaminases and erythrocyte membrane function in an attempt to develop better methods for the diagnosis and treatment of vascular occlusive events in SCD.
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