ORIENTATION OF THE CARBOXY-TERMINAL REGIONS OF FIBRIN GAMMA-CHAIN DIMERS DETERMINED FROM THE CROSS-LINKED PRODUCTS FORMED IN MIXTURES OF FIBRIN, FRAGMENT-D, AND FACTOR-XIIIA

There are two schools of thought regarding the orientation of the inte rmolecular epsilon-amino-(gamma-glutamyl) lysine isopeptide bonds form ed between gamma chains in the D domains of assembled fibrin fibers. S ome investigators believe that these bonds are oriented parallel to th e 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, op posing strands ('DD-transverse'). To distinguish between these two pos sibilities, the structure of crosslinked products formed in mixtures o f fibrin, plasmic fragment D, and factor XIIIa were analyzed, based up on 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-termina l gamma chains in the D:fibrin:D complex became aligned in a DD-long c onfiguration, only crosslinked fragment D dimers ('D-D') will result a nd the fibrin 'template' will not become crosslinked to the associated D fragments, If instead, gamma chain crosslinks form transversely bet ween the D fragments and fibrin, covalently linked D-fibrin complexes will result. SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin a nd fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimer s were also formed at the same levels as were formed in mixtures of fr agment D and factor XIIIa alone. Electron microscopic images of D-fibr in-D complexes prepared under physiological buffer conditions demonstr ated 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 als o positioned transversely in an assembled fibrin polymer.