THE LOCATION OF THE CARBOXY-TERMINAL REGION OF GAMMA-CHAINS IN FIBRINOGEN AND FIBRIN D-DOMAINS

Elongated fibrinogen molecules are comprised of two outer ''D'' domain s, each connected through a ''coiled-coil'' region to the central ''E' ' domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations t hat result in double-stranded fibrils. Factor XIIIa mediates crosslink ing of the C-terminal regions of gamma chains in each D domain (the ga mma(XL) site) by incorporating intermolecular epsilon-(gamma-glutamyl) lysine bonds between amine donor gamma 406 lysine of one gamma chain a nd a glutamine acceptor at gamma 398 or gamma 399 of another. Several lines of evidence show that crosslinked gamma chains extend ''transver sely'' between the strands of each fibril, but other data suggest inst ead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gamma(XL) site in fibrinogen and assembled fibrin fib rils, we incorporated an amine donor, thioacetyl cadaverine, into glut amine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron d ense gold cluster compound, undecagold monoaminopropyl maleimide (Au-1 1). Fibrinogen was examined by scanning transmission electron microsco py to locate Au-11-cadaverine-labeled gamma 398/399 D domain sites. Se venty-nine percent of D domain Au-11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the re maining Au-11 clusters in a distal position. In fibrin fibrils, D doma in Au-11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibri n are oriented toward the central domain and indicate that gamma(XL) s ites in fibrils are situated predominantly between strands, suitably a ligned for transverse crosslinking.