Thrombin binding to fibrinogen at its substrate recognition sites in the central E domain leads to proteolytic conversion of fibrinogen to fibrin. Fibrin itself has an appreciable non-substrate thrombin binding potential termed 'anti-thrombin I', that is defined by two classes of thrombin-binding sites, one of low affinity in the E domain, and the other of high affinity in certain D domains. The low affinity sites represent a residual aspect of fibrinogen substrate binding, whereas the high affinity thrombin binding site in human fibrin(ogen) is situated exclusively in a gamma chain variant termed gamma', which contains a unique C-terminal sequence from residue 408 to 427 (y' l-427L) that also binds plasma factor XIII. The thrombin binding site on y' 1-427L encompasses residues 414 to 427, and Tyr sulfation at 418 and 422 as well as the ultimate tripeptide sequence (DDL), are important for thrombin binding. We believe that the most important physiological aspect of anti-thrombin I activity in blood is that non-substrate thrombin binding to fibrin sequesters thrombin within the forming clot, removing it from the thrombin-generating environment, thereby reducing thrombin feedback clotting activation as well as other direct thrombin effects. The goal of this proposal is to evaluate that hypothesis by investigating the role of non-substrate thrombin binding sites in murine fibrin that are representative of human anti-thrombin I activity. To accomplish this we will prepare genetically-altered mice in which (1) the non-thrombin-binding murine y' chain sequence has been replaced by the thrombin-binding human y' sequence (hu-y' fibrinogen), or (2) the low-affinity thrombin-binding site has been mutated at BB 68 to create fibrinogen with both defective thrombin substrate-recognition and low-affinity non-substrate thrombin-binding [Naples I (BB A68T)].
In Aim 1 we will characterize the biochemical properties of hu-y' fibrin(ogen), determine the in vivo consequences of introducing hu-y' on thrombosis, factor XIII-mediated crosslinking, and fibrinolysis, and carry out an interaction screen in thrombomodulin-deficient mice (TM pro, TM flox) to assess whether the hu-y' phenotype ameliorates or exacerbates the prethrombotic or flagrantly thrombotic state. We will also evaluate the content and in vivo metabolic conversion of intact y' 1-427L chains to non-thrombin-binding (des-EDDL) y' 1-423P chains, using ELISA methodology that has been developed for measuring human y' chains.
In Aim 2 we will characterize the biochemical properties of murine Naples I fibrin(ogen), evaluate the in vivo consequences of introducing the Naples I mutation on thrombosis, and carry out an interaction screen in prethrombotic TM pro mice to determine whether the Naples I phenotype exacerbates the hypercoagulable state. We expect to resolve any controversy that now exists as to whether human y' chains have beneficial or deleterious in vivo effects on thrombosis or fibrinolysis. By comparing analyses on the hu-y' and Naples I phenotypes, we will be able to define the specific roles of the high- and low-affinity components of anti-thrombin I in the prevention and initiation of thrombosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL070627-02
Application #
6612843
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
2002-01-01
Project End
2005-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
2
Fiscal Year
2003
Total Cost
$342,137
Indirect Cost
Name
Bloodcenter of Wisconsin, Inc.
Department
Type
DUNS #
057163172
City
Milwaukee
State
WI
Country
United States
Zip Code
53233
Guo, Yi-He; Hernandez, Irene; Isermann, Berend et al. (2009) Caveolin-1-dependent apoptosis induced by fibrin degradation products. Blood 113:4431-9
Mosesson, M W; Cooley, B C; Hernandez, I et al. (2009) Thrombosis risk modification in transgenic mice containing the human fibrinogen thrombin-binding gamma' chain sequence. J Thromb Haemost 7:102-10
Mosesson, M W; Siebenlist, K R; Hernandez, I et al. (2008) Evidence that alpha2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation. J Thromb Haemost 6:1565-70
Davis, Ryan L; Mosesson, Michael W; Kerlin, Bryce A et al. (2007) Fibrinogen Columbus: a novel gamma Gly200Val mutation causing hypofibrinogenemia in a family with associated thrombophilia. Haematologica 92:1151-2
Brennan, Stephen O; Davis, Ryan L; Mosesson, Michael W et al. (2007) Congenital hypodysfibrinogenaemia (Fibrinogen Des Moines) due to a gamma320Asp deletion at the Ca2+ binding site. Thromb Haemost 98:467-9
Pineda, Agustin O; Chen, Zhi-Wei; Marino, Francesca et al. (2007) Crystal structure of thrombin in complex with fibrinogen gamma'peptide. Biophys Chem 125:556-9
Mosesson, M W; Hernandez, I; Raife, T J et al. (2007) Plasma fibrinogen gamma'chain content in the thrombotic microangiopathy syndrome. J Thromb Haemost 5:62-9
Mosesson, M W (2005) Fibrinogen and fibrin structure and functions. J Thromb Haemost 3:1894-904
Siebenlist, Kevin R; Mosesson, Michael W; Hernandez, Irene et al. (2005) Studies on the basis for the properties of fibrin produced from fibrinogen-containing gamma' chains. Blood 106:2730-6
Pechik, Igor; Madrazo, Joel; Mosesson, Michael W et al. (2004) Crystal structure of the complex between thrombin and the central ""E"" region of fibrin. Proc Natl Acad Sci U S A 101:2718-23

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