Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin.

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 location of the carboxy-terminal region of γ chains in fibrinogen and fibrin D domains

“…This ␥ chain flexibility would also help to explain the formation of interfibrillar crosslinked ␥ trimers and ␥ tetramers, occurring in a setting of previously formed ␥ dimers (24) and is consistent with the fact that neither Yee et al (27) nor Spraggon et al (21) visualized this segment of the ␥ chain in their crystal structures. The idea that this region is flexible also is strongly suggested by our present observation that C-terminal ␥ chains are situated in several locations in the D domain ranging from distal to proximal.…”

“…Recently, the ''end-to-end'' crosslinking idea received somewhat revised support from studies of crystals of crosslinked D dimer fragments (21), in which crosslinked C-terminal ␥ chains, though not situated at the extreme end of the molecule as originally envisioned, were inferred nevertheless to traverse the abutting D domains in an end-to-end alignment. However, several independent lines of evidence, beginning with reports by Selmayr et al (22,23), have indicated that the crosslinked ␥ chain segments in fibrin do not straddle the ends of adjacent D domains but rather tend to extend transversely between each fibril strand (1,(24)(25)(26). Evidence in favor of the transverse bond arrangement includes among the above cited articles, the electron microscopic demonstration of double-stranded crosslinked fibrinogen fibrils, visualization of bridging filaments representing transversely positioned ␥ chains within crosslinked fibrinogen fibrils (1) and in crosslinked D-fibrin-D complexes (26), and evidence indicating that the abutting ends of fibrin D domains where end-to-end intermolecular contacts are now recognized to occur (the so-called D:D site) (1,21,25), do not as first proposed (20), contain the C-terminal ␥ chain segment.…”

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

“…These findings indicate that the Tokyo II Fibrin polymerization begins after cleavage and release of two amino-terminal A fibrinopeptides from the central 'E' domain of fibrinogen molecules, exposing each of the 'A' polymerization sites they mask in precursor fibrinogen molecules ( 1-5 ). Each exposed 'A' site then combines noncovalently with a constitutive complementary 'a' site in the outer 'D' domain of a neighboring fibrin molecule, a necessary interaction ('D:E') for producing the half-staggered, end-to-middle domain pairing that results in twisting (6-10), double-stranded fibrils (6)(7)(8)(9)(10)(11)(12)(13)(14) (Fig. 1).…”

Vibrio vulnificus Secretes a Broad-Specificity Metalloprotease Capable of Interfering with Blood Homeostasis through Prothrombin Activation and Fibrinolysis

“…Factor XIIIa then introduces intermolecular covalent [ε-(␥-Glu)Lys] isopeptide bonds into these polymers, creating ␥-dimers. This is followed by the formation of crosslinks between complementary sites on ␣ chains and among ␥-dimers, completing the mature network structure (31). During fibrinolysis, the fibrin polymer is converted into soluble product by the action of plasmin, which is generated from plasminogen by urokinase and tissue plasminogen activator (tPA).…”