Vascular injury occurring in response to hemostatic and thrombotic events is a major cause of morbidity and mortality. The initiation of these reactions may involve both soluble and cellular components of the vasculature. Indeed, cellular disruption resulting from trauma and cellular stimulation from the introduction of bacterial products such as endotoxin can initiate the expression of tissue factor, the obligatory cofactor for Factor VII activity, which can contribute to the overall reactions of thrombosis and the Shwartzman reaction leading to intravascular coagulation, respectively. The purpose of this proposal is to investigate the detailed molecular interactions of Factor VII with its cofactor and substrates which should provide a basic understanding of the initiation and regulation of the coagulation system by defining the loci on Factor VII which mediate these molecular associations. Identification of specific regions on Factor VII will primarily involve an immunochemical study using previously generated monoclonal antibodies directed to native Factor VII; some of which neutralize coagulant activity. The possible mechanisms for this neutralization will be investigated. Another aspect of this application will be to identify the specific peptides of Factor VII which are recognized by the monoclonal antibodies. Peptides will be generated from chemical and enzymatic cleavage of Factor VII or the chains of Factor VIIa and bound to immobilized antibodies. The bound peptides will be eluted, identified by amino acid sequence analysis and synthetic peptides made. These epitopes and potential association sites will be investigated in binding and inhibition studies using monoclonal antibodies and the synthetic reagents. New hybridoma antibodies will be generated to novel epitopes to further define the regions on Factor VII mediating these interactions. The second area in the proposal focuses on the isolation and characterization of specific molecular variants of human Factor VII. These studies will include an immunochemical analysis of the epitopes expressed on these abnormal molecules. The binding of normal and variant Factor VII to tissue factors of different species origin will be studied under conditions of limiting cofactor and correlated with biological activity. The ability of the variants to cleave and activate Factors IX and X under these conditions, and to be cleared by Factor Xa will be investigated. Finally, differences in the primary structure will be addressed using peptide mapping and amino acid sequencing approaches.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037770-02
Application #
3353755
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1986-05-01
Project End
1988-04-30
Budget Start
1987-05-01
Budget End
1988-04-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Texas Health Center at Tyler
Department
Type
Hospitals
DUNS #
City
Tyler
State
TX
Country
United States
Zip Code
75708
Shetty, S; Kumar, A; Johnson, A R et al. (1996) Differential expression of the urokinase receptor in fibroblasts from normal and fibrotic human lungs. Am J Respir Cell Mol Biol 15:78-87
Kumar, A; Koenig, K B; Johnson, A R et al. (1995) Inhibition of factor Xa-mediated procoagulant activity of human lung fibroblasts and pleural mesothelial cells. Eur Respir J 8:2038-45
Strange, C; Baumann, M H; Sahn, S A et al. (1995) Effects of intrapleural heparin or urokinase on the extent of tetracycline-induced pleural disease. Am J Respir Crit Care Med 151:508-15
Shetty, S; Kumar, A; Johnson, A et al. (1995) Urokinase receptor in human malignant mesothelioma cells: role in tumor cell mitogenesis and proteolysis. Am J Physiol 268:L972-82
Shetty, S; Kumar, A; Johnson, A R et al. (1995) Regulation of mesothelial cell mitogenesis by antisense oligonucleotides for the urokinase receptor. Antisense Res Dev 5:307-14
Idell, S; Pueblitz, S; Emri, S et al. (1995) Regulation of fibrin deposition by malignant mesothelioma. Am J Pathol 147:1318-29
Kumar, A; Koenig, K B; Johnson, A R et al. (1994) Expression and assembly of procoagulant complexes by human pleural mesothelial cells. Thromb Haemost 71:587-92
Griffith, D E; Johnson, A R; Kumar, A et al. (1994) Growth factors for human pleural mesothelial cells in soluble products from formed clots. Thromb Res 74:207-18
Idell, S; Kumar, A; Zwieb, C et al. (1994) Effects of TGF-beta and TNF-alpha on procoagulant and fibrinolytic pathways of human tracheal epithelial cells. Am J Physiol 267:L693-703
Idell, S; Kumar, A; Koenig, K B et al. (1994) Pathways of fibrin turnover in lavage of premature baboons with hyperoxic lung injury. Am J Respir Crit Care Med 149:767-75

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