Contact with the N Termini in the Central E Domain Enhances the Reactivities of the Distal D Domains of Fibrin to Factor XIIIa

Abstract

The reaction of Factor XIII a with fibrin is the last enzyme-catalyzed step on the coagulation cascade, leading to the formation of a normal blood clot. The finding that fibrin is preferred by the cross-linking enzyme about 10-fold over the circulating fibrinogen suggests the operation of a unique substrate-level control for the orderly functioning of the physiological process in the forward direction. An important task is to elucidate the molecular mechanism for the transmission of the signal generated by the thrombin-catalyzed cleavage in the central E domain of fibrin to the distant Factor XIII areactive glutamine residues. By focusing on the substrate sites present in ␥ chain remnants of D type domains of fibrinogen and by employing the approach of fragment complementation with the regulatory E domain, which represents the thrombin-modified portion of fibrin, we have now succeeded in reconstructing in solution the phenomenon of kinetic enhancement for the reaction with Factor XIII a .Two D type preparations (truncated fibrinogen, ϳ250 kDa and D, ϳ105 kDa) were obtained by digestion of human fibrinogen with endo Lys-C. Neither product could be cross-linked by Factor XIII a , but as shown by the incorporation of dansylcadaverine, both were acceptor substrates for the enzyme. The plasmin-derived D (ϳ105-kDa) product, however, could be cross-linked into DD dimers. In all cases, the admixture of E fragments exerted a remarkable boosting effect on the reactions with Factor XIII a . Even with native fibrinogen as substrate, cross-linking of ␥ chains was enhanced in the presence of E. Nondenaturing electrophoresis was used to demonstrate the complex forming potential of E fragments with fibrinogen, truncated fibrinogen, D, or D. The GPRP tetrapeptide mimic of the GPRV N-terminal sequence of the ␣ chains in the E fragments, abolished both complex formation and the kinetic boosting effect of E on the reactions of substrates with Factor XIII a . Thus, the N-terminal ␣ chain sequences seem to act as organizing templates for spatially orienting the D domains, probably during the protofibrillar assembly of the fibrin units, for favorable reaction with Factor XIII a .

The complementary aggregation sites of fibrin investigated through examination of polymers of fibrinogen with fragment E

“…Kabi, Stockholm, Sweden) and prepared as described (21). Recombinant human fibrinogen, prepared as described (22,23), was a gift from Oleg V. Gorkun and Susan T. Lord (University of North Carolina, Chapel Hill).…”

“…Recombinant human fibrinogen, prepared as described (22,23), was a gift from Oleg V. Gorkun and Susan T. Lord (University of North Carolina, Chapel Hill). Fragment E was prepared by methods similar to those used previously (21). Factor XIII was purified as described (24,25).…”

Sol Sherry Lecture in Thrombosis

“…In addition, it has become evident that the fibrin substrate itself acts to coordinate the orderly sequence of reactions during the late stages of coagulation; as a feed-forward regulator, 1) it accelerates the cleavage of factor XIII by thrombin, 63 2) it enables the subunits of the thrombinmodified zymogen to dissociate at the 1.5 mmol/L concentration of Ca 2ϩ in plasma, 64 and 3) the noncovalent assembly of fibrin units speeds up the end-to-end fusion of the ␥ chains in the D domains of the protein. 65,66 These unique controls ( Figure 5) must have evolved to ensure that in the physiological sequence of events, only fibrin, and not the parent fibrinogen molecule, should be the target for cross-linking by factor XIIIa.…”

Cross‐Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase

“…For removal of free dansyl-c-aca-QQIV, pellets were serially extracted (8 >< 1 ml) with N,N-dimethylformamide containing 1% N-methylmorpholine and 5% H20 (Samokhin and Lorand, 1995). Each extraction proceeded at 55°Cfor 30 mm and was followed by centrifugation (10 mm X 14,000 g).…”

“…Reactions were terminated by addition of 50~sl of 100 mM EDTA. The stoichiometry of DC and dansyl-e-aca-QQIV incorporation was estimated from auquots of stoppedreactions (20~tgof tau protein) as described previously (Samokhin and Lorand, 1995).…”