Studies on the Basis for the Properties of Fibrin Produced from Fibrinogen-Containing γ′ Chains

Human fibrinogen 1 is homodimeric with respect to its γ chains (`γA-γA\u27), whereas fibrinogen 2 molecules each contain one γA (γA1-411V) and one γ\u27 chain, which differ by containing a unique C-terminal sequence from γ\u27408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the γ\u27 chains. Second, cross-linking of fibrin γ chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to γ\u27 chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to γ\u27 chains

Studies on the Ultrastructure of Fibrin Lacking Fibrinopeptide B (β-Fibrin)

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a “coarse” matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, “fine” fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles

STUDIES ON KINETICS OF INHIBITION AND BINDING OF XIIIa BY A CROSS-REACTING ANTIFIBRINOGEN ANTIBODY*

Coagulation factor XIIIa, plasma transglutaminase(endo-g-glutamine:e-lysine transferase EC 2.3.2.13) cata-lyzes isopeptide bond formation between glutamine andlysine residues and rapidly cross-links fibrin clots. Amonoclonal antibody (5A2) directed to a fibrinogen Aa-chain segment 529 –539 was previously observed fromanalysis of end-stage plasma clots to block fibrina-chaincross-linking. This prompted the study of its effect onnonfibrinogen substrates, with the prospect that 5A2was inhibiting XIIIa directly. It inhibited XIIIa-cata-lyzed incorporation of the amine donor substrate dan-sylcadaverine into the glutamine acceptor dimethylca-sein in an uncompetitive manner with respect todimethylcasein utilization and competitively with re-spect to dansylcadaverine. Uncompetitive inhibitionwas also observed with the synthetic glutamine sub-strate, LGPGQSKVIG. Theoretically, uncompetitive in-hibition arises from preferential interaction of the in-hibitor with the enzyme-substrate complex but is alsofound to inhibitg-chain cross-linking. The conjunctionof the uncompetitive and competitive modes of inhibi-tion indicates in theory that this bireactant system in-volves an ordered reaction in which docking of the glu-tamine substrate precedes the amine exchange. Thepresence of substrate enhanced binding of 5A2 to XIIIa,an interaction deemed to occur through a C-terminalsegment of the XIIIa A-chain (643– 658,GSDMTVTVQFT-NPLKE), 55% of which comprises sequences occurring inthe fibrinogen epitope Aa-(529 –540) (GSESGIFTNTKE).Removal of the C-terminal domain from XIIIa abolishesthe inhibitory effect of 5A2 on activity. Crystallographicstudies on recombinant XIIIa place the segment 643– 658in the region of the groove through which glutaminesubstrates access the active site and have predicted thatfor catalysis, a conformational change may accompanyglutamine-substrate binding. The uncompetitive inhibi-tion and the substrate-dependent binding of 5A2 pro-vide evidence for the conformational change