Abstract

The objectives of this renewal proposal are 1) to determine the intracellular mechanisms by which the component chains of fibrinogen are assembled into a functional biologically active protein and 2) to map domains which play important roles in fibrin polymerization and crosslinking using recombinant engineered fibrinogen. To accomplish these objectives, the Applicant will 1) use a reconstituted ER vesicle system to characterize ER factors involved in fibrinogen assembly and to determine the association of fibrinogen precursors with such factors, 2) express deletion and substitution mutants of each of the fibrinogen chains in COS cells to map the critical domains necessary for assembly and secretion, 3) perform pulse-chase studies in HepG2 cells to define the sequential steps in fibrinogen assembly, 4) mutagenize secretion factors in a yeast expression system to study fibrinogen assembly and secretion, and 5) mutagenize recombinant fibrinogen in putative crosslinking and polymerization sites to define structure-function relationships.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL037457-09
Application #
2218465
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1987-02-01
Project End
1999-05-31
Budget Start
1995-07-01
Budget End
1996-05-31
Support Year
9
Fiscal Year
1995
Total Cost
Indirect Cost

  Institution

Name
New York Blood Center
Department
Type
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Xia, H; Redman, C (2000) Enhanced secretion of ApoB by transfected HepG2 cells overexpressing fibrinogen. Biochem Biophys Res Commun 273:377-84
Xia, H; Redman, C (1999) The degradation of nascent fibrinogen chains is mediated by the ubiquitin proteasome pathway. Biochem Biophys Res Commun 261:590-7
Spraggon, G; Applegate, D; Everse, S J et al. (1998) Crystal structure of a recombinant alphaEC domain from human fibrinogen-420. Proc Natl Acad Sci U S A 95:9099-104
Fu, Y; Zhang, J Z; Redman, C M et al. (1998) Formation of the human fibrinogen subclass fib420: disulfide bonds and glycosylation in its unique (alphaE chain) domains. Blood 92:3302-8
Applegate, D; Haraga, L; Hertzberg, K M et al. (1998) The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets. Blood 92:3669-74
Roy, S; Sun, A; Redman, C (1996) In vitro assembly of the component chains of fibrinogen requires endoplasmic reticulum factors. J Biol Chem 271:24544-50
Zhang, J Z; Redman, C M (1996) Assembly and secretion of fibrinogen. Involvement of amino-terminal domains in dimer formation. J Biol Chem 271:12674-80
Zhang, J Z; Redman, C (1996) Fibrinogen assembly and secretion. Role of intrachain disulfide loops. J Biol Chem 271:30083-8
Roy, S N; Kudryk, B; Redman, C M (1995) Secretion of biologically active recombinant fibrinogen by yeast. J Biol Chem 270:23761-7
Zhang, J Z; Redman, C M (1994) Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen. J Biol Chem 269:652-8

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