Summary. A high-affinity thrombin-binding site in an alternately processed fibrinogen variant, the gA/g 0 isoform, is characterized in this report. The binding site has been shown to be situated between g 0 414 and 427, and Tyr418 and 422 in this part of the g 0 chain are known to be sulfated. A synthetic peptide corresponding to the g 0 chain carboxyl terminus is shown to bind thrombin with abinding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the g 0 peptide via exosite II. Thus, thrombin binding to the g 0 chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin-bound thrombin can retain enzymatic activity, and why fibrin-bound thrombin is heparin-resistant.

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, ␥A/␥A fibrin, or a minor variant form, ␥A/␥ fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, ␥A/␥ fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin ␥A and ␥ chains and a significantly more rapid cross-linking of fibrin ␣ chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, ␥A/␥ fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.Plasma coagulation factor XIII (EC 2.3.2.13) is the zymogen precursor to the active transglutaminase factor XIIIa. Plasma factor XIII consists of two a subunits (M r ϳ83,000) and two b subunits (M r ϳ80,000) with the stoichiometry a 2 b 2 , whereas a form of factor XIII stored in platelets lacks the b subunits (1-4). In the final stages of blood coagulation, thrombin activates plasma factor XIII by cleaving an activation peptide (M r ϳ4000) from the amino terminus of each a subunit (3). In the presence of calcium, active factor XIII (factor XIIIa) then catalyzes the formation of isopeptide bonds in polymerized fibrin strands between adjacent glutamine/lysine side chains (5). Cross-linking occurs in the ␣ and ␥ chains of fibrin but not in the ␤ chains, and it results in the formation of ␥-␥ dimers, ␥ chain multimers, ␣ chain multimers, and complexes between ␣ and ␥ chains (6). Cross-linked fibrin shows increased stability (7) and increased proteolytic resistance to fibrinolytic enzymes (8).Factor XIII activation by thrombin is a multistep process that is modulated by calcium and by the presence of fibrinogen. An explicit model of factor XIII activation has been proposed (9) in which the initial activation step is thrombin cleavage of the activation peptide from the a subunit, which is then referred to as the aЈ subunit. However, the a 2 Јb 2 complex is not catalytically active. In the presence of high concentrations of calcium, i.e. Ͼ10 mM, the a 2 Јb 2 complex dissociates into the a 2 Ј and b 2 subunits. Fibrin facilitates this dissociation step such that it occurs at physiologic calcium concentrations (ϳ1.5 mM), thereby increasing the rate of factor XIII activation under physiologic conditions. Finally, a conformati...

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.