Orientation of the Carboxy-terminal Regions of Fibrin γ Chain Dimers Determined from the Crosslinked Products Formed in Mixtures of Fibrin, Fragment D, and Factor XIIIa

Abstract

There are two schools of thought regarding the orientation of the intermolecular epsilon-amino-(gamma-glutamyl) lysine isopeptide bonds formed between gamma chains in the D domains of assembled fibrin fibers. Some investigators believe that these bonds are oriented parallel to the direction of fiber growth (longitudinally) at the contacting ends of fibrin D domains ('DD-long'), whereas others believe that these bonds are oriented across the two-stranded fibril, between D domains in opposing strands ('DD-transverse'). To distinguish between these two possibilities, the structure of crosslinked products formed in mixtures of fibrin, plasmic fragment D, and factor XIIIa were analyzed, based upon this rationale: Complex formation between D fragments and a fibrin template depends upon the non-covalent 'D:E' interaction between each fibrin E domain and two D fragments ('D:fibrin:D'). If carboxy-terminal gamma chains in the D:fibrin:D complex become aligned in a DD-long configuration, only crosslinked fragment D dimers ('D-D') will result and the fibrin 'template' will not become crosslinked to the associated D fragments. If instead, gamma chain crosslinks form transversely between the D fragments and fibrin, covalently linked D-fibrin complexes will result. SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin and fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimers were also formed at the same levels as were formed in mixtures of fragment D and factor XIIIa alone. Electron microscopic images of D-fibrin-D complexes prepared under physiological buffer conditions demonstrated that the D fragments were associated with the central E domain of the fibrin molecule, but that they could be dissociated from this non-covalent association in 2% acetic acid. These findings indicate that gamma chain crosslinks occur transversely in D:fibrin:D complexes and permit the extrapolated conclusion that gamma chain crosslinks are also positioned transversely in an assembled fibrin polymer.