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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL053280-01
Application #
2231116
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1995-01-15
Project End
1999-12-31
Budget Start
1995-01-15
Budget End
1995-12-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Washington University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Melman, Lora; Geuze, Hans J; Li, Yonghe et al. (2002) Proteasome regulates the delivery of LDL receptor-related protein into the degradation pathway. Mol Biol Cell 13:3325-35
Obermoeller-McCormick, L M; Li, Y; Osaka, H et al. (2001) Dissection of receptor folding and ligand-binding property with functional minireceptors of LDL receptor-related protein. J Cell Sci 114:899-908
Liu, C X; Li, Y; Obermoeller-McCormick, L M et al. (2001) The putative tumor suppressor LRP1B, a novel member of the low density lipoprotein (LDL) receptor family, exhibits both overlapping and distinct properties with the LDL receptor-related protein. J Biol Chem 276:28889-96
Narita, M; Rudolph, A E; Miletich, J P et al. (1998) The low-density lipoprotein receptor-related protein (LRP) mediates clearance of coagulation factor Xa in vivo. Blood 91:555-60
Mast, A E; Higuchi, D A; Huang, Z F et al. (1997) Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor. Biochem J 327 ( Pt 2):577-83
Warshawsky, I; Herz, J; Broze Jr, G J et al. (1996) The low density lipoprotein receptor-related protein can function independently from heparan sulfate proteoglycans in tissue factor pathway inhibitor endocytosis. J Biol Chem 271:25873-9
Warshawsky, I; Schwartz, A L (1996) The 39-kDa protein regulates LRP activity in cultured endothelial and smooth muscle cells. Eur J Cell Biol 69:156-65
Warshawsky, I; Bu, G; Mast, A et al. (1995) The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo. J Clin Invest 95:1773-81
Narita, M; Bu, G; Olins, G M et al. (1995) Two receptor systems are involved in the plasma clearance of tissue factor pathway inhibitor in vivo. J Biol Chem 270:24800-4
Bu, G; Geuze, H J; Strous, G J et al. (1995) 39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein. EMBO J 14:2269-80

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