The goal of this proposal is to determine the molecular details of the fibrinogen A alpha chain which contribute to its role as a substrate for thrombin and a participant in fibrin polymerization. Directed mutagenesis will be used to explore the functional contribution of each relevant amino acid residute. Two vectors which express human A alpha polypeptides in E. coli have already been constructed. One expresses the complete A alpha chain while the other expresses the first 50 residues linked by a segment of collagen to the bacterial enzyme, beta-galactosidase. Both peptides are detected by protein blot analysis, with crossreactive bands at the predicted mobilities, and both are cleaved by thrombin. Initial experiments will use the beta-galactosidase fusion product because many of the functionally important residues lie within amino acids 1-50. In addition the fusion peptide can be readily purified based on its beta-galactosidase activity. The A alpha peptide can then be prepared in high purity by digesting away the beta-galactosidase with collagenase. The native A alpha peptide will be assayed as a substrate for thrombin and as an inhibitor of fibrin monomer polymerization. Then mutations will be construted from assembled oligonucleotides, and the activities of the expressed mutant peptides compared to the native activities. It has been proposed that cleavage by thrombin is dependent on the presence of a beta-turn structure at or near amino acids 13 and 14. Molecular modelling will be employed to suggest mutations which will disturb or support such a structure, and the predicted changes tested experimentally. The detailed structure of the polymerization domain will be explored by changing the charges on side chains and making polar/apolar substitutions. Once critical residues have been identified, more subtle changes will be explored and a model developed for the polymerization domain. Similar mutations will be studied in the complete A alpha chain in order to identify long range interactions. The results from the experiments will provide information which can serve as the basis for designing inhibitors of thrombin activity and fibrin polymerization. Such inhibitors could be clinically useful in treating or preventing pathological clot formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031048-07
Application #
3342073
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Huang, Lihong; Hsiao, Joe Ping-Lin; Powierza, Camilla et al. (2014) Does topology drive fiber polymerization? Biochemistry 53:7824-34
Huang, Lihong; Lord, Susan T (2013) The isolation of fibrinogen monomer dramatically influences fibrin polymerization. Thromb Res 131:e258-63
Park, Rojin; Ping, Lifang; Song, Jaewoo et al. (2013) An engineered fibrinogen variant A?Q328,366P does not polymerise normally, but retains the ability to form ? cross-links. Thromb Haemost 109:199-206
Smith, E L; Cardinali, B; Ping, L et al. (2013) Elimination of coagulation factor XIII from fibrinogen preparations. J Thromb Haemost 11:993-5
Hudson, Nathan E; Ding, Feng; Bucay, Igal et al. (2013) Submillisecond elastic recoil reveals molecular origins of fibrin fiber mechanics. Biophys J 104:2671-80
Raynal, Bertrand; Cardinali, Barbara; Grimbergen, Jos et al. (2013) Hydrodynamic characterization of recombinant human fibrinogen species. Thromb Res 132:e48-53
Lord, Susan T (2011) Molecular mechanisms affecting fibrin structure and stability. Arterioscler Thromb Vasc Biol 31:494-9
Ping, Lifang; Huang, Lihong; Cardinali, Barbara et al. (2011) Substitution of the human ?C region with the analogous chicken domain generates a fibrinogen with severely impaired lateral aggregation: fibrin monomers assemble into protofibrils but protofibrils do not assemble into fibers. Biochemistry 50:9066-75
Hantgan, Roy R; Stahle, Mary C; Lord, Susan T (2010) Dynamic regulation of fibrinogen: integrin ?IIb?3 binding. Biochemistry 49:9217-25
Hudson, Nathan E; Houser, John R; O'Brien 3rd, E Timothy et al. (2010) Stiffening of individual fibrin fibers equitably distributes strain and strengthens networks. Biophys J 98:1632-40

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