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Details of the crystal structure of human thrombin in complex with the central part of human fibrin are revealed and results are presented of the molecular modeling of the fibrinopeptides A and B (fpA- and fpB-) containing portions of the Aalpha and Bbeta chains, in both fibrinogen and protofibrils. Analysis provides a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils at the second stage.

Results demonstrate that transverse cross-linking prevails in fibrinogen/fibrin complexes, which confirms transverse arrangement of the cross-linked gamma chains in an assembled fibrin polymer. No evidence is seen for the longitudinal gamma chain arrangement.

Elongated fibrinogen molecules are comprised of two outer 'D' domains, each connected through a 'coiled-coli' region to the central 'E' domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of [Gamma] chains in each D domain (the [[Gamma].sub.XL] site) by incorporating intermolecular [Epsilon]-([Gamma]-glutamyl)lysine bunds between amine donor [Gamma]406 lysine of one [Gamma] chain and a glutamine acceptor at [Gamma]398 or [Gamma]399 of another. Several lines of evidence show that crosslinked [Gamma] chains extend 'transversely' between the strands of each fibril, but other data suggest instead that crosslinked [Gamma] chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the [[Gamma].sub.XL] site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold mouoaminopropyl maleimide ([Au.sub.11]). Fibrinogen was examined by scanning transmission electron microscopy to locate [Au.sub.11]cadaverine-labeled [Gamma]398/399 D domain sites. Seventy-nine percent of D domain [Au.sub.11] clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining [Au.sub.11] clusters in a distal position. In fibrin fibrils, D domain [Au.sub.11] clusters were located in middle to proximal positions. These findings show that most C-terminal [Gamma] chains in fibrinogen or fibrin are oriented toward the central domain and indicate that [[Gamma].sub.XL] sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.

Gel sieving and ion-exchange chromatography is used to study the binding properties of plasma factor XIII in fibrinogens containing alpha chains. Results indicate that factor XIII binds to fibrinogen molecules via the B subunit of the former. This finding suggests that fibrinogen alpha chains have a physiologically important affinity for the B subunits of plasma factor XIII.

Evidence of the cross-linking of A(alpha).(gamma) chain heterodimers in plasma fibrinogen is provided. The evidence suggests the existence of a subpopulation of native fibrinogen molecules that have intramolecular cross-links between gamma and A(alpha) chains. Furthermore the evidence indicates that these intramolecular cross-links are probably mediated in vivo by transglutaminase.

The factors affecting the formation of gamma-chain multimers in cross-linked fibrin were determined by measuring the D-trimer and D-tetramer content in plasmic digests of fibrin. The results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process. This process can be augmented by low ionic strength, increasing factor VIII concentration, increasing CaCl2 concentration or slow fibrin polymerization resulting in increased fiber thickness or tight fibril packing.