This proposal is based on the hypothesis that prothrombin and factor X (factor Xa) contain discrete functional domains which are involved in discrete portions of the expression of the biological activity of these complex proteins. It is proposed to identify these regions by the combined use of specific chemical modification and limited proteolysis. Prothrombin will be modified with 2-hydroxy-5-nitrobenzyl bromide or with tetranitromethane. The modified protein(s) will be analyzed with respect to the extent of modification and changes in functional properties. The two reagents selected can identify functional tryptophanyl and tyrosyl residues respectively and, in addition, provide the basis for the introduction of structural probes whose spectral and fluorescence properties can measure changes in the microenvironment around the modified residues. Limited proteolysis of the native prothrombin in the presence and absence of ligands such as divalent cations, phospholipid and other components of the prothrombinase complex will provide derivatives for comparison with native prothrombin. Similar studies are proposed for the elucidation of functional domains in factor X and factor Xa. The primary effort will be directed toward the study of factor Xa since our emphasis is directed toward the study of the prothrombinase complex. The proposed experiments will provide a molecular understanding of prothrombinase complex. Such understanding is critical to the elucidation of the mechanism of normal blood coagulation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL034032-02
Application #
3346563
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Dentistry/Oral Hygn
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Manly, D T; Featherstone, G L; Jenzano, J W et al. (1988) Influence of divalent and monovalent cations on some active site properties of human factor Xa. Thromb Res 49:343-51
Lundblad, R L (1988) A hydrophobic site in human prothrombin present in a calcium-stabilized conformer. Biochem Biophys Res Commun 157:295-300
Lundblad, R L; Jenzano, J W; Roberts, H R (1987) Interaction of polylysine with bovine factor Xa: effect of divalent cations. Thromb Res 48:395-402
Tarvers, R C; Roberts, H R; Straight, D L et al. (1987) Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions. Arch Biochem Biophys 257:439-43
Tarvers, R C; Noyes, C M; Tarvers, J K et al. (1986) Mechanism of the calcium-dependent self-association of bovine prothrombin. Use of a covalent cross-linking reagent to study the reaction. J Biol Chem 261:4855-9
Tarvers, R C (1985) Calcium-dependent changes in properties of human prothrombin: a study using high-performance size-exclusion chromatography and gel-permeation chromatography. Arch Biochem Biophys 241:639-48
Tarvers, R C; Church, F C (1985) Use of high-performance size-exclusion chromatography to measure protein molecular weight and hydrodynamic radius. An investigation of the properties of the TSK 3000 SW column. Int J Pept Protein Res 26:539-49
Tarvers, R C (1985) Purification of human prothrombin fragment 1 using hydrophobic interaction chromatography on phenyl-sepharose. Thromb Res 40:235-41