Characterization of an abnormal fibrinogen Osaka V with the replacement of gamma-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.

Prolonged thrombin time was completely corrected by the addition of millimolar concentrations of calcium in a new abnormal fibrinogen, Osaka V. Analysis of lysyl endopeptidase digests of A alpha-, B beta-, or gamma-chains by high performance liquid chromatography, and the following amino acid sequence analysis of relevant peptides revealed that about 50% of the gamma-chain has a replacement of gamma-arginine 375 by glycine. When fibrinogen was digested with plasmin in the presence of millimolar concentration of calcium, the amount of fragment D1 was about 50% of the normal control, and the rest was further cleaved to fragment D2, D3, or D62 with an apparent Mr of 62,000. Plasmic digestion of cross-linked fibrin in the presence of calcium resulted in the appearance of an abnormal fragment with an apparent Mr of 123,000 as well as fragments D2, D3, and D62, concomitant with the decrease of D dimer. The gamma-remnant of the abnormal fragment proved to be a cross-linked complex of the normal D1 gamma-remnant and residues 374-406/411 of the abnormal gamma-chain. The number of high affinity Ca(2+)-binding sites for the normal fibrinogen and fibrinogen Osaka V obtained by equilibrium dialysis was 2.88 (about 3) and 1.85, respectively, and that for the abnormal molecules was calculated as 0.9 (about 1) from their relative amounts in the samples, suggesting the lack of two Ca(2+)-binding sites in the D-domains. These data suggest that the normal structure of the COOH-terminal portion of the gamma-chain including residue 375 is required for the full expression of high affinity calcium binding to D-domains, the ability to be protected by calcium against plasmic digestion, and fibrin polymerization. During these studies, we found that the NH2-terminal amino acid of the gamma-remnant in fragments D or D dimer which were obtained after prolonged digestion with plasmin is gamma-Met89.