Chromatography and electron microscopy of cross-linked fibrin polymers--a new model describing the cross-linking at the DD-trans contact of the fibrin molecules.

Abstract

AbstractPolymerization of fibrin molecules results in the formation of a double‐stranded protofibril. Although convincing data have not been presented, it is classically believed that γ‐chain cross‐linking of fibrin molecules occurs between the longitudinal end‐to‐end contacts (DD‐long contacts) of the molecules within each of the two strands of a protofibril (intrastrand cross‐linking). In this investigation the question addressed was whether γ‐chain cross‐linking takes place across the two strands (interstrand cross‐linking) between the transversal half‐staggered contacts of the molecules. Demonstration of double‐stranded protofibrils in the presence of urea would indicate an interstrand cross‐linking, whereas in the case of intrastrand cross‐linking, the chaotropic agent urea would dissociate the double‐stranded structure to form single‐stranded fibrils. Protofibrils were obtained by generating soluble cross‐linked fibrin polymers (sXLFbP): After incubation of souble fibrin polymers with Factor XIIIa at 37°C, the polymerization and cross‐linking reaction was stopped by the addition of 6M urea and EDTA. Gel filtration of the reaction mixture in the presence of 3M urea was effect in separating sXLFbP from monomeric molecules. The sXLFbP‐containing fractions were adsorbed onto mica in the presence of different concentrations of urea and investigated by electron microscopy after rotary shadowing. In the presence of 3M urea the sXLFbP appeared as double‐stranded protofibrils. In the presence of 4M urea some parts of the double‐stranded structure were found to be unfolded whereas in the presence of 6M urea multiple‐bended single‐stranded fibrils were observed. SDS‐polyacrylamide gel electrophoresis of the sXLFbP demonstrated no γ‐chain cross‐linking within the protofibrils. Ultracentrifugation of the sXLFbP showed that in the presence of 3M urea noncross‐linked fibrin polymers dissociated to monomeric molecules. When sXLFbP was centrifuged into 6M urea on sucrose density gradients, no reduction of the polymer size could be observed. The data indicate that γ‐chain cross‐linking occurs between the transversal contacts of the fibrin molecules within a protofibril, thus generating interstrand cross‐linking. A model of the cross‐linking of polymerized fibrin molecules is developed and the term DD‐trans contact is proposed for this specific alignment of the D‐domains.