The long-term goal is to increase knowledge of mechanisms that regulate blood coagulation so that new antithrombotic therapies and preventative measures can be developed. Defects in coagulation regulation lead to increased risk of vascular disease, a major cause of death. Studies will focus on a novel coagulation regulator, protein Z-dependent protease inhibitor (ZPI) that rapidly inactivates Factor Xa (FXa) in the presence of protein Z, Ca 2+ and phospholipids (PL). Our studies show that ZPI/protein Z also inhibits FIXa. Low protein Z levels are associated with stroke, suggesting physiologic relevance. We hypothesize that: A) ZPI and protein Z are significant down-regulators of both FXa and FIXa; B) Mechanisms of ZPI inhibition differ from those of most serpins; D) ZPI inhibits FIXa and FXa by different mechanisms; and C) Defining ZPI mechanisms and sites of protein-protein interaction may lead to new antithrombotic therapies.
Specific aims are: 1) Assess whether ZPI and protein Z are significant inhibitors of FXa and FIXa. We will determine if inhibition of both FXa and FIXa occurs at plasma levels of ZPI/protein Z, prothrombin and FX. We will assess the importance of ZPI inhibition of FXa vs. FlXa and determine if combined inhibitory effects are additive. We will find if FXa-ZPI or FlXa-ZPI are major complexes formed when FIXa or FXa are added to plasma. 2) Define mechanisms for ZPI/protein Z inhibition of FXa. Most serpins do not require a protein cofactor, PL or Ca 2+, and serpin-protease complexes are usually covalent and irreversible. Inhibition of FXa by ZPI seems to differ in these respects. We will elucidate ZPI's unusual mechanisms. We will find if cleavage of ZPI by FXa occurs and if ternary complexes of ZPI-protein Z-FXa can form. 3) Determine how mechanisms of ZPI inhibition of FIXa and FXa differ. FXa inhibition by ZPI strictly requires protein Z and FXa is partly protected from ZPI by FVa. We will extend findings suggesting that ZPI inhibition of FIXa does not require protein Z and that FVIIla enhances ZPI inhibition of FIXa. We will find the reasons for the differences and discover if ZPI inhibition of FIXa differs from that of FXa with respect to binary/ternary complexes, covalency of complexes, or reversibility. 4) Elucidate structure-function relationships for ZPI and protein Z that lead to FIXa or FXa inhibition. We will define some of the regions crucial for interaction between these proteins by performing functional studies with modified proteins, selected mutants of rZPI and peptides representing candidate sequences for interactions. We will find whether a ZPI polymorphism we identified affects function.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Link, Rebecca P
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Scripps Research Institute
La Jolla
United States
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Fernandes, N; Mosnier, L O; Tonnu, L et al. (2010) Znýý(+) -containing protein S inhibits extrinsic factor X-activating complex independently of tissue factor pathway inhibitor. J Thromb Haemost 8:1976-85
Fernandez, Jose A; Heeb, Mary J; Xu, Xiao et al. (2009) Species-specific anticoagulant and mitogenic activities of murine protein S. Haematologica 94:1721-31
Heeb, Mary J; Prashun, Duane; Griffin, John H et al. (2009) Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor. FASEB J 23:2244-53
Heeb, M J; Gandrille, S; Fernandez, J A et al. (2008) Late onset thrombosis in a case of severe protein S deficiency due to compound heterozygosity for PROS1 mutations. J Thromb Haemost 6:1235-7
Heeb, Mary J (2008) Role of the PROS1 gene in thrombosis: lessons and controversies. Expert Rev Hematol 1:9-12
Heeb, Mary J; Fisher, Mark; Paganini-Hill, Annlia (2007) Association of low protein Z levels with ischemic stroke in young women. Thromb Haemost 97:495-6
Fernandez, Jose A; Heeb, Mary Jo (2007) Role of protein C inhibitor and tissue factor in fertilization. Semin Thromb Hemost 33:13-20
Heeb, M J; Radtke, K-P; Fernandez, J A et al. (2006) Plasma contains protein S monomers and multimers with similar direct anticoagulant activity. J Thromb Haemost 4:2215-22
Heeb, M J; Schuck, P; Xu, X (2006) Protein S multimers and monomers each have direct anticoagulant activity. J Thromb Haemost 4:385-91
Hall, Michael O; Obin, Martin S; Heeb, Mary J et al. (2005) Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells. Exp Eye Res 81:581-91

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