Our long range goal is to understand each of the biological functions of fibrinogen and fibrin.
Our specific aims i n this proposal are to define and characterize thrombin-fibrin and thrombin-fibrinogen binding interactions. Thrombin binding to fibrin occurs through its exosite at 'non-substrate' sites, and also in fibrinogen at a 'substrate recognition' site. We will investigate the non-substrate thrombin-binding sites on fibrin, one of which is of low affinity, located near to or contiguous with the fibrinogen substrate recognition site in the E domain (Aim 1). The other site, of high affinity, is located in the carboxyl-terminal region of gamma' chains, which are normal minor (approximately 10%) gamma chain variants. Using a fibrin clot system to measure thrombin binding, we will quantify the interactions at each type of thrombin binding site in fibrin, characterize their structures, and determine the exact gamma' sequences accounting for thrombin binding. We will also determine the crystal structure of thrombin-gamma' peptide complexes to further characterize the thrombin-gamma' chain interaction. Several functional aspects of the thrombin-fibrin binding interaction will be investigated, including: 1) the effect of thrombin binding to fibrin(ogen) on thrombin receptor-mediated platelet aggregation, and on thrombin receptor cleavage in a cell system expressing the thrombin receptor; 2) the mechanism of non-enzymatic thrombin-mediated acceleration of fibrin polymerization; 3) the effect of the gamma ' sequence on fibrinogen to fibrin conversion, fibrin assembly, and fibrin polymer structure; 4) the mechanism of suppressed fibrinolysis in gamma '-containing fibrin. We will also examine thrombin-fibrinogen binding interactions at the substrate recognition site (Aim 2) using active-site inhibited thrombins (e.g., D419N thrombin) to characterize and quantify the thrombin-fibrinogen binding interaction. Using native fibrinogen or proteolytically modified fibrinogen lacking portions of the substrate recognition site, and peptides that are homologous with structures in the central E domain of fibrin(ogen), we will map the substrate recognition site, and homologous with structures in the central E domain of fibrin(ogen), we will map the substrate recognition site, and compare its constituent structures with those comprising the low affinity non-substrate site in fibrin. In summary, our investigations will elucidate the structural and functional roles of the gamma' sequence and the non-substrate thrombin binding site in the fibrin E domain, delineate the constituent structures comprising the fibrinogen substrate recognition site, and characterize and quantify the mechanics of binding with thrombin. These studies will contribute to our understanding of the roles) of thrombin-fibrin(ogen) interactions in mediating fibrin clot formation, clinical thrombosis, fibrinolysis, and wound healing.
|Mosesson, M W; DiOrio, J P; Hernandez, I et al. (2004) The ultrastructure of fibrinogen-420 and the fibrin-420 clot. Biophys Chem 112:209-14|
|Mosesson, Michael W (2003) Antithrombin I. Inhibition of thrombin generation in plasma by fibrin formation. Thromb Haemost 89:9-12|
|Mosesson, M W (2003) Fibrinogen gamma chain functions. J Thromb Haemost 1:231-8|
|Mosesson, M W; Siebenlist, K R; Meh, D A (2001) The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci 936:11-30|
|Siebenlist, K R; Meh, D A; Mosesson, M W (2001) Protransglutaminase (factor XIII) mediated crosslinking of fibrinogen and fibrin. Thromb Haemost 86:1221-8|
|Meh, D A; Siebenlist, K R; Brennan, S O et al. (2001) The amino acid sequence in fibrin responsible for high affinity thrombin binding. Thromb Haemost 85:470-4|
|Meh, D A; Mosesson, M W; DiOrio, J P et al. (2001) Disintegration and reorganization of fibrin networks during tissue-type plasminogen activator-induced clot lysis. Blood Coagul Fibrinolysis 12:627-37|
|Meh, D A; Mosesson, M W; Siebenlist, K R et al. (2001) Fibrinogen naples I (B beta A68T) nonsubstrate thrombin-binding capacities. Thromb Res 103:63-73|
|Siebenlist, K R; Mosesson, M W; Meh, D A et al. (2000) Coexisting dysfibrinogenemia (gammaR275C) and factor V Leiden deficiency associated with thromboembolic disease (fibrinogen Cedar Rapids). Blood Coagul Fibrinolysis 11:293-304|
|Siebenlist, K R; Meh, D A; Mosesson, M W (2000) Position of gamma-chain carboxy-terminal regions in fibrinogen/fibrin cross-linking mixtures. Biochemistry 39:14171-5|