Roles of Low Specificity and Cofactor Interaction Sites on Thrombin during Factor XIII Activation

Abstract

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.Following initiation of coagulation, a series of carefully regulated serine proteinase reactions take place resulting in the generation of thrombin and the formation of an insoluble fibrin clot. Thrombin is a serine proteinase responsible for proteolytic cleavage of multiple substrates involved in the coagulation pathway (1, 2). One of the main roles of thrombin is the conversion of fibrinogen to an insoluble fibrin clot. This process is initiated by proteolytic cleavage of two pairs of fibrinopeptides, FPA 1 and FPB, from the A␣-and B␤-chains of fibrinogen, respectively. FPA is cleaved first, leading to spontaneous polymerization of the fibrin monomers. This is shortly followed by proteolysis of the B␤-chain, which is associated with lateral aggregation of the fibrin protofibrils to produce thicker fiber bundles (3, 4). To ensure a more stable clot structure, activated factor XIII (factor XIIIa), a transglutaminase, covalently crosslinks specific glutamine and lysine side chains of the protofibrils, resulting in increased resistance of the clot to chemical, physical, and proteolytic insults (5, 6).Factor XIII is a heterologous tetramer with a molecular mass of 324,000 Daltons. It consists of two A-subunits that contain the active site of the transglutaminase and two B-subunits that serve a carrier function for the hydrophobic A-subunit in the aqueous environment of human plasma (5, 6). Thrombin activates factor XIII by cleavage of a 37 amino acid activation peptide from the factor XIII A-subunits (7). Consequently, ...

Proteolytic inactivation of ADAMTS13 by thrombin and plasmin

“…Competition between cofactors for exosite binding have been proposed as a mechanism for directing the activities of thrombin. 37 In binding TM, the specificity of thrombin toward fibrinogen, protein C, and PAR molecules is dramatically altered. 38 This serves to restrict its procoagulant activities to the site of vascular injury.…”

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…21,35,36 So although on one hand the ␥Ј chain is responsible for antithrombin I activity and reduction of intrinsic activation, FpA release on the other hand is normal and thrombin is afforded protection against serpin inactivation in the presence of glycosaminoglycans. It appears that the ␥Ј chain provides another example where exosite binding is involved in directing thrombin's activity, 19,37 possibly by inhibiting thrombin generation in the solution phase and localizing thrombin to the fibrin surface.…”

Thrombomodulin Changes the Molecular Surface of Interaction and the Rate of Complex Formation between Thrombin and Protein C

“…We dissected the epitopes of thrombin recognizing protein C in the absence and presence of thrombomodulin using a panel of 77 Ala mutants. Ala-scanning mutagenesis is the methodology of choice to identify functional epitopes (20 -22) and has been successfully applied to thrombin (15,(23)(24)(25)(26)(27)(28)(29). Previous studies have identified thrombin residues potentially important for protein C recognition but have been limited to exosite regions and explored only marginally the 60-loop region and the Na ϩ binding site comprising loops 220 and 186 (24).…”