Both the intrinsic and extrinsic coagulation pathways terminate with the activation of the blood protein Factor X. Activated Factor X in turn forms a supramolecular complex, termed prothrombinase, consisting of an enzyme (Factor X alpha), a protein cofactor (Factor V alpha), calcium ion, and a phospholipid surface. This assembled complex catalyzes the proteolytic activation of prothrombin to the protease thrombin. The expedient formation of thrombin at wound sites thus requires the precise recognition and binding of the enzyme to a cofactor, a surface, and a substrate. Each of these interactions is mediated by distinct regions on the surface of Factor X. The approach outlined in this proposal will define those peptide sites on Factor X involved in recognition and binding to prothrombin, Factor V alpha, and phospholipid. The approach is to use isolated peptide fragments of Factor X, synthetic peptides from the known sequence of Factor X, and antibodies to specific topographic sites on Factor X in well- defined functional assays for Factor X. Peptides that will be synthesized include the variable region sequences of Factor X and sequences from the Gla domain. Several available monoclonal antibodies will be examined for their ability to inhibit Factor X activity and the antigenic determinant recognized by the inhibitory antibody alpha Beta F chi-2 beta will be localized. Antibodies produced to the synthetic peptides will also be examined for their ability to bind Factor X and to inhibit prothrombin activation, prothrombinase complex assembly or Factor X binding to phospholipid vesicles. This combined approach on the structure/function of Factor X using peptide fragments, synthetic peptides, and site- specific antibodies will provide more information about the relevant surface features of the Factor X molecule important for its function than would each approach used separately. Such topographic information will be extremely valuable in understanding the assembly, function, and regulation of prothrombinase, and at the same time, assist the design and modification of genetically engineered blood coagulation proteins.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL040467-02
Application #
3471960
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1989-07-01
Project End
1994-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Church, W R; Messier, T L; Ouellette, L A et al. (1994) A kringle-specific monoclonal antibody. Hybridoma 13:423-9
Bovill, E G; Soll, R F; Lynch, M et al. (1993) Vitamin K1 metabolism and the production of des-carboxy prothrombin and protein C in the term and premature neonate. Blood 81:77-83
Ouellette, L A; Messier, T L; Church, W R (1992) Neutralization of factor X activity by factor X-specific monoclonal antibodies. Blood Coagul Fibrinolysis 3:563-74
Church, W R; Messier, T L (1991) Inhibition of plasminogen activation by monoclonal antibodies to the kringle 5-B chain segment of human plasminogen. Hybridoma 10:659-72
Messier, T L; Pittman, D D; Long, G L et al. (1991) Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X. Gene 99:291-4
Church, W R; Ouellette, L A; Messier, T L (1991) Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2. J Biol Chem 266:8384-91
Huang, R S; Sorisky, A; Church, W R et al. (1991) ""Thrombin"" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides. J Biol Chem 266:18435-8
Mann, K G; Nesheim, M E; Church, W R et al. (1990) Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 76:1-16