A gamma methionine-310 to threonine substitution and consequent N-glycosylation at gamma asparagine-308 identified in a congenital dysfibrinogenemia associated with posttraumatic bleeding, fibrinogen Asahi.

Abstract

In an abnormal fibrinogen with severely impaired polymerization of fibrin monomers, we identified a methionine-to-threonine substitution at position 310 of the 'y chain. Furthermore, asparagine at position 308 was found to be N-glycosylated due to a newly formed consensus sequence, asparagine(308)-glycine(309)-threonine(310). The two structural defects in the mutant oy chain may well perturb the conformation required for fibrin monomer polymerization that is specifically assigned to the D domain of fibrinogen. This alteration also seems to affect the intermolecular y chain cross-linking of fibrin and fibrinogen, although the amine acceptor y glutamine-398 was found to function normally. These functional abnormalities may well be related to posttraumatic hemorrhage as observed in a 33-yr-old man with moderate hemorrhagic diathesis related to injuries since his early adolescence.The structure of the extra carbohydrate moiety attached to asparagine-308 was found to be identical with those derived from the normal B,8 and y chains as evidenced by HPLC. IntroductionThe carboxy-terminal region of the fibrinogen y chain has been shown to participate in various functions of fibrinogen including fibrin polymerization (1-7).To form stable fibrin clots, several reactions take place sequentially or simultaneously. They include proteolytic cleavage of fibrinopeptides A and B by thrombin, polymerization of fibrin monomers to form fibrin gels, and intermolecular cross-linking mediated by activated blood coagulation factor XIII (Factor XIIIa) (4, 5). Although polymerization and gelation of fibrin have been extensively studied (1-3), little is known about the mechanisms involved in the reactions. For this purpose, congenital dysfibrinogens with well-elucidated structures may deserve careful study.

Fibrinogen Lima: a homozygous dysfibrinogen with an A alpha-arginine-141 to serine substitution associated with extra N-glycosylation at A alpha-asparagine-139. Impaired fibrin gel formation but normal fibrin-facilitated plasminogen activation catalyzed by tissue-type plasminogen activator.

“…Several possibilities could be raised to account for the higher molecular weight for the abnormal Ace chain, and linking to an extra oligosaccharide was thought to be a highly proba- ble candidate as has recently been shown in two other abnormal fibrinogens reported from this laboratory (9,20). Indeed, the abnormal Aa chain was found to stain positively for carbohydrate with biotinylated concanavalin A, whereas the normal Aa chain, in which the carbohydrate moieties had been reported to be absent (29), was negative for the staining (Fig.…”

“…The extra glycosylation is attributed to a newly created asparagine-X-serine/threonine-type consensus sequence, asparaginevaline-serine, for the N-glycosylation ofthe asparagine residue. This asparagine-X-serine-type tripeptide is unique for human fibrinogen, because the glycosylation sequences so far shown for normal (35) and three abnormal fibrinogens (9,20,36) are all asparagine-X-threonine types. The extra oligosaccharide in fibrinogen Lima was found to have a biantennary oligosaccharide with a structure ofGal2 *Man3 GlcNAc4 (see the legend to Fig.…”

“…Fibrinogen fractions thus prepared were found to be > 95% pure as examined by SDS-PAGE (7,8). Portions of the fibrinogen fractions were further treated with an anti-factor XIII (subunit A) monoclonal antibody bound to protein A-Sepharose (Pharmacia AB, Uppsala, Sweden) to eliminate factor XIII, a trace contaminant, as described (9).…”

“…Plasmic degradation of fibrinogen and factor XIIIa-mediated cross-linking of fibrin were conducted essentially as described elsewhere (9,19). Plasmic degradation of cross-linked fibrin was also analyzed by utilizing fibrinogen labeled with 1251 by the lactoperoxidase method essentially according to the manual supplied by the manufacturer (Na'251 from Amersham Japan, Tokyo; the specific activities were 1.246 X I03 cpm/mg for the patient's and 2.023 X 103 cpm/ mg for normal fibrinogen).…”

Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole ‘a’ and calcium binding sites

“…Heterozygous N308K, N308I, G309D, and M310T variant fibrinogens [8] near the T305 residue were reported as dysfibrinogenemias and their thrombin-catalyzed fibrin polymerization was impaired, but to a lesser degree than T305A [20,[27][28][29].…”