The endogenous mechanisms for the regulation of blood coagulation include the inhibition of coagulation enzymes, the inactivation of coagulation cofactors and clearance mechanisms for the enzymes and cofactors. One key regulatory inhibitor of coagulation is tissue factor pathway inhibitor (TFPI), which produces feedback inhibition of the factor VIIa/tissue factor complex. TFPI levels are controlled in part via its rapid hepatic clearance. Recent data from our laboratory identify low density lipoprotein receptor-related protein, LRP, as the mediator of the hepatic uptake and degradation of TFPI. LRP also functions as the endocytosis receptor for plasminogen activators and their inhibitors. Thus, our specific aims focus on elucidation of the molecular mechanisms responsible for the rapid hepatic clearance of TFPI both in vitro and in vivo. We have hepatoma cell lines which mediate TFPI uptake/degradation, purified LRP, a 39kDa protein (which inhibits LRP function), TFPI and TFPI mutants as well as antibodies to each. We shall (1) identify the primary binding site for TFPI on hepatocytes using biochemical and cell biological approaching including crosslinking; (2) define the structural features of TFPI which mediate LRP recognition using TFPI mutants and sensitive binding/competition assays; (3) identify domains on the 39kDa protein which inhibit TFPI recognition using 39kDa mutants and binding/competition assays; and (4) define the role of LRP in the clearance of TFPI in vivo using kinetic and tissue distribution studies with TFPI, TFPI mutants, the 39kDa protein and factors VIIa and Xa. These studies will thus provide a basis for elucidating the molecular mechanisms by which blood coagulation proteins are endogenously regulated.
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