The overall goal of these studies is to define the in vivo role of tissue factor (TF) in hemostasis as in pertains to hemophilia. The information gained from the proposed series of experiments in this Project will be directly relevant to the development of new therapies, namely Factor VIIa (FVIIa) mutants, for patients with hemophilia and inhibitors. FVIIa in high doses has recently been approved by t he FDA for the treatment of bleeding experienced by patients who have an inhibitor to FVIII or FIX Modification of the membrane contact site in FVIII-as proposed in Project 2- may lead to the development of FVIIa products that are superior to wild type FVIIa with respect to treatment of bleeding episodes at a lower dose (and therefore cost). However, it is conceivable that unwanted thrombogenicity will also be enhanced by manipulation of the membrane-binding site of the Gla domain. Therefore, in order to define the mode of action, efficacy, and safety of these FVII mutants, an understanding of the in vivo expression of their essential ligand-TF-is essential. To this end, this project will study t he intravascular availability and state of activation of TF in humans, and in an animal model of hemophilia. First, we will define the biochemical basis of the phenomenon of TF 'encryption' (defined as the ability to bind FVII(a) while failing to express full pro-coagulant activity (PCA)). In particular, the roles of membrane phosphatidylserine (PS) asymmetry and TF quaternary structure in modulating the expression of TF PCA will be examined. The results of these experiments will guide the design of inhibitory molecules that specifically target either the active or encrypted forms of circulating TF. These inhibitors will be used as probes of the functional availability of intravascular TF, which will be quantified in two candidate 'pools' (cell-associated and microparticle-associated) in normals, in patients with hemophilia, and in a rabbit model of acquired hemophilia. Finally, in experiments that will dovetail closely with Projects 2 and 4, the rabbit model will be used to examine the role played by TF in physiological hemostasis, and in pharmacological hemostasis induced by wild-type and mutant forms of FVIIa.
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