Coagulation Factor IX is essential for normal hemostasis. Recent studies have indicated that Factors IX and IXa bind specifically to a shared site on endothelium; cell-bound Factor IX can be activated and cell-bound Factor IXa promotes Factor X activation in the presence of Factor VIII. Preliminary data suggests this cellular binding site may be composed, at least in part, of a protein and this protein is distinct from Factor VIII. We propose to isolate this protein and characterize its role in determining the coagulant properties of cell-bound Factors IX and IXa on quiescent and endotoxin-perturbed endothelial cells. Once the binding protein is isolated, monoclonal and polyclonal antisera will be prepared. Using these immunologic reagents to assess the role of the Factor IX/IXa binding protein, kinetic studies of Factor IX activation and Factor IXa-VIII catalyzed Factor X activation, and radioligand binding studies will be carried out on endothelial cells. The functional characteristics of a recently observed specific Factor IXa endothelial binding site induced by the presence of Factors VIII and X will be studied and its relationship to the shared Factor IX/IXa site assessed. The importance of the Factor IX/IXa binding protein for expression of Factor VIII-bypassing activity by endotoxin-treated endothelial cells which promote direct Factor IXa activation of Factor X will also be examined. Cellular events subsequent to the surface binding of Factors IX and IXa, possible internalization and degradation, will be studied and the role of the Factor IX/IXa binding protein determined. Although Factor IX/IXa binding has been demonstrated on cultered bovine aortic endothelial cells, the presence of this binding in vivo has not been directly demonstrated. Crossed-species animal infusion studies with Factor IX and active site-blocked Factor IXa will be carried out and the level in the plasma of each species' Factor IX antigen measured by specific radioimmunoassays. Since the major vascular surface is comprised by the microvasculature, expression of the Factor IX/IXa binding protein will also be studied on cultured capillary endothelial cells using specific antisera, by radioligand binding and kinetic studies.
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