title: Orientation of the carboxy-terminal regions of fibrin gamma 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.
authors.affiliation: University of Wisconsin Medical School, Sinai Samaritan Medical Center, Milwaukee 53233, USA.
authors.forename: K R
authors.initials: KR
authors.lastname: Siebenlist
authors.forename: D A
authors.initials: DA
authors.lastname: Meh
authors.forename: J S
authors.initials: JS
authors.lastname: Wall
authors.forename: J F
authors.initials: JF
authors.lastname: Hainfeld
authors.forename: M W
authors.initials: MW
authors.lastname: Mosesson
mesh_terms: D005337:Fibrin / Q000378:metabolism* / Q000648:ultrastructure; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D005338:Fibrin Fibrinogen Degradation Products; C015214:fibrinogen D fragment; D005337:Fibrin; D011503:Transglutaminases