The endogenous mechanisms for the regulation of blood coagulation include the inhibition of coagulation enzymes, the inactivation of coagulation co-factors and clearance mechanisms for the enzymes and co- factors. One key regulatory inhibitor of coagulation is tissue factor pathway inhibitor (TFPI), which produces feedback inhibition of the factor VIIa/tissue factor complex and directly binds and inhibits factor Xa. TFPI is rapidly cleared in vivo via the endocytosis receptor, LRP (low density lipoprotein receptor-related protein). LPR governs the endocytosis of a wide variety of ligands which share an important role in regulation of blood coagulation/fibrinolysis. In addition, a 39 kDa protein serves both as a ligand for LRP and as an inhibitor of LRP function. In addition to LRP, TFPI interacts with cell surface heparan sulfate proteoglycans (HSPGs) which function as a second class of clearance receptors. Recently, we have described a new clearance receptor system, also involving cell surface HSPGs, which recognizes TFPI in the context of factor Xa Thus, our specific aims focus on elucidation of the molecular mechanisms responsible for the rapid clearance of TFPI both in vitro and in vivo. We have available a variety of cell lines which mediate TFPI uptake-degradation as antibodies to each protein, HSPG mutant cells as well as mutant (knockout and over-expression) mice. We shall (1) define the structural features of TFPI which mediate LRP recognition using TFPI mutants, LRP mini-receptors, and sensitive binding/competition assays; (2) identify the HSPG species which serves as the TFPI cell surface receptor in vitro using cell lines, homozygous deficient or over- expressing each of the six glypicans and in vivo using the various glypican (-/-) mice; (3) identify the TFPI-Xa receptor using a series of cell biological approaches including affinity chromatography, expression cloning, expression cloning. These studies will thus provide a basis for elucidating the molecular mechanisms by which blood coagulation proteins are endogenously regulated.
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