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.

Fragment D prepared from human fibrinogen was labeled specifically by photoactivation of the peptide ['4C]Gly-Pro-Arg-N-(4-azido-2-nitrophenyl)Lys amide. The preparation was freed of excess labeling reagents and then reduced and alkylated. The component a, (3, and y chains were purified by chromatography on carboxymethylcellulose and the radioactivity was found to be restricted to the y chain. Isolated y chains were digested with various endopeptidases, both alone and in tandem, and the products were fractionated by gradient HPLC. The amino acid compositions of all labeled peptides led to the conclusion that the modification occurs exclusively on V-chain Tyr-363.The identification of those parts of the fibrinogen molecule that interact during the initial polymerization event leading to formation of the fibrin clot has been a subject of interest for almost 50 years (for a review of early efforts, see ref. 1). The finding that simple peptides patterned on the amino-terminal sequences of fibrin can block polymerization provided convincing evidence that these newly exposed groups formed one part of the interaction (2). Binding studies with these same peptides also implicated the carboxyl-terminal portions of fibrinogen y chains as the sites of the complementary interaction (3,4).Localizing these latter sites has proved difficult and controversial (5-8). In the preceding article (9), photoaffinity labeling was used to demonstrate that the binding site for peptides with the sequence Gly-Pro-Arg must be close to an amino acid in the segment comprising y-chain residues 337-379. In this article we show that the photolabeled amino acid is Tyr-363 on the y chain. MATERIALS AND METHODSOur initial work on photoaffinity labeling of fibrinogen and its fragments (9) had several technical limitations that hindered localization of the labeled residues more precisely. Accordingly, a number of changes were incorporated into the present study that greatly improved the sensitivity of our analyses. These changes included preparing peptides of significantly greater specific activity, photolabeling under mild conditions aimed at preventing oxidation, using specific proteases in place of CnBr, and using a more sensitive amino acid analyzer. Only those methods and materials that differ significantly from those used in the earlier stages of the study are described here.Enzymes. Lysyl endopeptidase was obtained from Wako Pure Chemical, Osaka. Digestions were carried out in a 0.05 M Tris buffer (pH 9.0) containing 3 M urea, for 24 hr at 370C. The ratio of enzyme to substrate was 1:20 (wt/wt). Asparaginyl endopeptidase was obtained from Takara Shuzo, Kyoto. Digestions were conducted in a 0.02 M sodium acetate buffer (pH 5.0) containing 0.01 M dithiothreitol and 0.001 M EDTA, for 16 hr at 370C. The protease from Staphylococcus aureus, strain V8 (Staphylococcus V8 protease) was purchased from ICN and used in 0.1 M ammonium bicarbonate (pH 8.0) for 12 hr at 370C.Peptide Synthesis. Peptides were synthesized in the same manner as reported ...

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An Aa-arginine-141 to serine substitution has been identified in a homozygous dysfibrinogen, fibrinogen Lima, associated with impaired fibrin polymerization. The point mutation created an asparagine-X-serine-type glycosylation sequence, and indeed, extra, mainly disialylated biantennary oligosaccharides have been isolated from Aa asparagine-139 of the patient's fibrinogen. This type of glycosylation sequence is unique for human fibrinogen, because the sequences shown for normal and abnormal fibrinogens are all asparagine-X-threonine types. The terminal sialic acids of the extra oligosaccharides seem to have largely contributed to the impaired fibrin gel formation, as evidenced by its correction to a near normal level by desialylation. Nevertheless, the polymerizing fibrin facilitated tissuetype plasminogen activator-catalyzed plasmin formation in a normal fashion, indicating that the initial two-stranded fibrin protofibrils had been constructed normally. Thus the impaired fibrin gel formation could be attributed to the delay in their subsequent lateral association, most probably because of the repulsive forces generated by the negative electric charge of the extra sialic acids. The substitution of a basic residue arginine to a noncharged residue serine may also have contributed to the impaired function in a similar manner or by steric hindrance in association with bulky extra oligosaccharide chains. (J. Clin. Invest. 1992. 90:67-76.) Key words: amino acid substitutioncongenital homozygous dysfibrinogen * extra asparagine-linked oligosaccharide * fibrin-facilitated plasminogen activation * lateral association of fibrin protofibrils