No abstract

Following proteolytic conversion of fibrinogen to fibrin, clot assembly commences with formation of double-stranded fibrils that subsequently branch extensively in forming a three-dimensional network. Plasmin digests of fibrin clots that had first been covalently crosslinked by plasma transglutaminase (factor XIIIa) contained multimeric proteolytic fragments composed of crosslinked outer (D) domains of neighboring fibrin molecules. Two of these were larger than the well-known "D dimer" fragment and corresponded to D trimers and D tetramers, respectively. Whereas D dimers originate from crosslinked D domains at bimolecular junctions within two-stranded fibrils, D trimers and D tetramers evidently arise through crosslinking of contiguous D domains at trimolecular and tetramolecular junctions or at fibril branch points, respectively. Measurement of the widths of fibrils comprising trifunctional branches in thin fiber networks revealed tetramolecular branch points, which are formed by bifurcation of two double-stranded fibrils. In addition, another type of trifunctional structure, which we term the trimolecular branch point, was composed of three double-stranded fibrils. Crosslinking of D domains to form trimers may occur at this type ofjunction. These findings add to our understanding of the crosslinking arrangements that stabilize fibrin clot structure and the ways that fibrin molecules polymerize to form branches in the clot matrix.Following proteolytic conversion of fibrinogen to fibrin, polymer assembly commences with formation of doublestranded fibrils in which fibrin molecules, by virtue of noncovalent intermolecular interactions between outer (D) and central (E) domains (1-6), are arranged in a staggered overlapping manner (7-13) (Fig. 1). Subsequently, lateral fibril associations result in increased fiber thickness (11,(16)(17)(18), which is believed to account for interfibril connections and the trifunctional branching structures that comprise the three-dimensional matrix (11,(18)(19)(20)(21). In the presence of plasma transglutaminase (factor XIIIa) and Ca2', fibrin molecules undergo covalent crosslinking by formation of E-(y-glutamyl)lysine [E-('y-Glu)Lys] isopeptide bonds (22,23). Intermolecular crosslinking between D domains forms dimers (24), which occur as reciprocal bridges between a lysine at position 406 of one 'y chain and a glutamine at position 398 or 399 of another (25)(26)(27)(28). In addition, slower intermolecular crosslinking among a chains creates oligomers and larger a-chain polymers (29-31).Plasmin digestion of crosslinked fibrin results in early release of crosslink-containing a-chain segments from core structures (32-36). Thus, their existence in fibrin does not contribute significantly to the structure of major intermediate or terminal plasmin core fragments. In contrast, the intermolecular e-(y-Glu)Lys y-chain bonds result in degradation products unique to crosslinked fibrin, of which the bimolecular fragment, "D dimer," is the most abundant and best characterized (32, 37-39...

The molecular defect in the abnormal fibrinogen Dusart (Paris V) that is associated with thrombophilia was determined by sequence analysis of genomic DNA that had been amplified using the polymerase chain reaction. The propositus was heterozygous for a single base change (C -> T) in the Aa-chain gene, resulting in the amino acid substitution Aa 554 Arg Cys. Restriction analysis of the amplified DNA derived from the family members showed that his father and his two sons were also heterozygous. Electron microscopic studies on fibrin formed from purified fibrinogen Dusart demonstrated fibers that were much thinner than in normal fibrin. In contrast to the previously observed defective binding of plasminogen, the binding of thrombospondin to immobilized fibrinogen Dusart was similar to that of normal fibrinogen. Immunoblot analysis of plasma fibrinogen demonstrated that a substantial part of the fibrinogen Dusart molecules were disulfide-linked to albumin. The plasma of the affected family members also contained fibrinogen-albumin complexes. Furthermore, small amounts of high molecular weight complexes containing fibrinogen were detected in all the heterozygous individuals. These data indicate that the molecular abnormality in fibrinogen Dusart (Aa 554 Arg -n Cys) results in defective lateral association of the fibrin fibers and disulfide-linked complex formation with albumin, and is associated with a family history of recurrent thrombosis in the affected individuals. (J. Clin. Invest. 1993.

IntroductionIntermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in Invest. 1995Invest. . 96:1053Invest. -1058

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.