We propose to use recombinant variant fibrinogens in structural and biochemical studies to identify and characterize the residues and domains that are critical for the conversion of soluble fibrinogen to an insoluble fibrin clot. Fibrinogen is converted to fibrin monomers by the serine protease thrombin. Fibrin monomers polymerize to form a fibrin clot. During the past grant period biochemical studies demonstrated that """"""""B:b"""""""" interactions have an unanticipated role in polymerization. To correlate the biochemical data with changes in structure, we determined X-ray crystal structures of variants that examined the """"""""B:b"""""""" interactions. These structure studies not only supported our biochemical findings, but also identified calcium-binding sites that likely modulate polymerization. We now propose to examine the role of """"""""B:b"""""""" interactions and calcium modulation in polymerization. We will test the hypotheses that: 1) both """"""""A:a"""""""" and """"""""B:b"""""""" interactions contribute to protofibril formation and 2) the beta1 calcium binding site modulates polymerization. Fibrin clots are stabilized and strengthened by the FXIIIa-catalyzed formation of isopeptide bonds between monomers. We propose to examine the interactions that are critical for normal crosslink formation and determine the interactions that mediate the strength of fibers and clots. We will test the hypotheses that: 3) the fibrin-enhanced thrombin-catalyzed activation of FXIII reflects the juxtaposition of fibrin-bound thrombin with fibrin-bound FXIII upon association of the D:D/E interface and 4) FXIIIa-catalyzed crosslinks are the molecular interactions that determine the mechanical properties of both fibrin fibers and fibrin clots. Our experiments are designed to provide a molecular analysis of the events that control fibrin clot structure and mechanical properties. Data obtained from these in vitro studies will lead to a more complete understanding of the events that are critical for effective clot formation and clot lysis in vivo.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
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Link, Rebecca P
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Huang, Lihong; Hsiao, Joe Ping-Lin; Powierza, Camilla et al. (2014) Does topology drive fiber polymerization? Biochemistry 53:7824-34
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
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
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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