Abstract

The major aim of this proposal is to elucidate the origin, composition and the enzymatic mechanisms involved in the generation of fibrinogen complexes. Covalent multimerization products of fibrin(ogen) are present in the circulating plasma, and in the vessel wall of patients with atherosclerosis and possibly with other vascular disorders. One of the major objectives of the proposal is to characterize these fibrinogen complexes which are formed by the cross-linking of the alpha and gamma chains, a reaction which is catalyzed by two different transglutaminases: Factor XIIIa or tissue transglutaminase. Identification of the sub-unit composition of these complexes will be done by separation of the materials, using an original electrophoretic system devised by the applicant, followed by immunostaining with antibodies specific for the alpha and gamma chains of fibrinogen. In some instances, the cross-linked products will be further analyzed using monoclonal antibodies against specific crosslinked regions and by amino acid sequencing.
A second aim of the proposal, consists in the characterization of the mechanisms involved in the removal of fibrin from the circulation. The applicant is proposing to characterize, biochemically and functionally, a fibrin receptor present in macrophages which seem to exhibit some of the biochemical properties of integrins.Isolation of the receptor will be done by affinity chromatography using fibrin or NDSK-fibrin. After isolation, the receptor will be characterized by amino acid sequencing, and with monoclonal antibodies.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL016361-15
Application #
3335195
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1976-04-01
Project End
1996-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
15
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195

  Publications

Shainoff, John R; Smejkal, Gary B; DiBello, Patricia M et al. (2002) Allosteric effects potentiating the release of the second fibrinopeptide A from fibrinogen by thrombin. J Biol Chem 277:19367-73
Mitkevich, O V; Shainoff, J R; DiBello, P M et al. (1998) Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody. J Biol Chem 273:14387-91
Smejkal, G B; Shainoff, J R (1997) Enhanced digital imaging of diaminobenzidene-stained immunoblots. Biotechniques 22:462
Odrljin, T M; Shainoff, J R; Lawrence, S O et al. (1996) Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin beta chain. Blood 88:2050-61
Mitkevich, O V; Sobel, J H; Shainoff, J R et al. (1996) Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases. Blood Coagul Fibrinolysis 7:85-92
Shainoff, J R; Smejkal, G B; Mitkevich, O et al. (1996) Preparative electrophoresis on linear polyacrylamide-agarose composite gels. Electrophoresis 17:179-84
Shainoff, J R; Smejkal, G B; DiBello, P M et al. (1996) Isolation and characterization of the fibrin intermediate arising from cleavage of one fibrinopeptide A from fibrinogen. J Biol Chem 271:24129-37
Shainoff, J R; Estafanous, F G; Yared, J P et al. (1994) Low factor XIIIA levels are associated with increased blood loss after coronary artery bypass grafting. J Thorac Cardiovasc Surg 108:437-45
Shainoff, J R; Urbanic, D A; DiBello, P M et al. (1993) GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood. Blood Coagul Fibrinolysis 4:87-92
Valenzuela, R; Shainoff, J R; DiBello, P M et al. (1992) Immunoelectrophoretic and immunohistochemical characterizations of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudo-intimas. Am J Pathol 141:861-80

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