DETERMINATION OF THE TOPOLOGY OF FACTOR-XIIIA-INDUCED FIBRIN GAMMA-CHAIN CROSS-LINKS BY ELECTRON-MICROSCOPY OF LIGATED FRAGMENTS

After fibrin polymerizes to form a clot, the transglutaminase Factor X IIIa cross-links the gamma and alpha chains to stabilize the clot. The re has been conflicting evidence on whether the gamma chain isopeptide bonds occur between molecules that are interacting in a longitudinal (end-to-end) manner or transverse (half-staggered) manner between the two strands of the protofibril. Since the topology of the cross-links has important consequences for fibrin structure, as well as for its st ability and susceptibility to and pattern of fibrinolysis, cross-linke d fibrin fragments were examined by electron microscopy to distinguish between these two possibilities for the arrangement of the ligated mo lecules. Cross-linked fibrin clots were produced by prolonged incubati on of fibrinogen with thrombin and Factor XIII, and then digested with plasmin. The resulting soluble cross-linked fibrin complexes were rot ary-shadowed with tungsten and examined by electron microscopy, reveal ing protofibril-like structures consisting of clusters of globular dom ains with a repeat of 22.5 nm. Longer plasmin digestion times yielded increasingly shorter structures. Rotary-shadowed cross-linked fibrin f ragments, produced by dilution of the complexes into 0.125% acetic aci d at pH 3.5 to dissociate all non-covalently linked fragments, showed uniformly single-stranded structures with a characteristic spacing of nodules, consistent with longitudinal cross-linking. Long, thin strand s were seen at short digestion times, while shorter strands appeared w ith longer digestion. The smallest structures observed included two no dules together, and two such nodules with another nodule at a short di stance from one or both ends, compatible with fragments DD, DY, and YY . Longer strands had the appearance of fibrin molecules that were link ed end-to-end, usually with a fragment D or Y at each end. In conclusi on, these results are consistent with previously proposed structures o f these derivatives and clearly demonstrate that the interactions betw een cross-linked gamma chains are longitudinal (end-to-end) and not tr ansverse.