The major objective of this proposal is to characterize the interactions of fibrinogen and fibronectin with specific hepatic and endothelial cell receptors, and to identify the cellular transglutaminase responsible fort he covalent multimerization of the bound glycoproteins. Radiolabeled fibrinogen and fibronectin will be incubated with the cells in suspension, or will be immobilized onto plastic petri dishes and used to support cell attachment. The radiolabeled proteins will be analyzed by SDS-PAGE and autoradiography following interaction with cells. Identification of the cellular receptor(s) mediating ligand-cell interactions will be approached using affinity chromatography with immobilized ligand or with monoclonal antibodies against the ligand-receptor complex. To identify the cellular transglutaminase, these enzymes will be purified from isolated hepatocytes and endothelial cells using a variety of chromatographic techniques coupled with affinity chromatography. The potential role of cytokines, such as interleukin-1 or tumor necrosis factor, as regulatory agents modulating the transglutaminase-mediated cross-linking of fibrinogen and fibronectin by endothelial cells will be investigated. The possible role of tissue transglutaminase in the organization (via covalent bond formation between fibrinogen and fibronectin) of the extracellular matrix will also be evaluated. The characterization of the carbohydrate composition of fibrinogen and prothrombin in liver disease will be continued. Sialic acid will be measured and glycopeptides fractionated by concanavalin A chromatography. Oligosaccharide structure will be analyzed by chromatographic techniques. in addition, the in vivo metabolism of human prothrombin and asialoprothrombin in rabbits and the interaction of these proteins with isolated rabbit hepatocytes will be studied, and the catabolic properties of the asialoderivative correlated with the oligosaccharide structure.

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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Thomas Jefferson University
Schools of Medicine
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