A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the γ-chain gene, causing defective calcium binding and impaired fibrin polymerization

A congenitally abnormal fibrinogen (Vlissingen) was isolated from the blood of a young woman suffering from massive pulmonary embolism. Fibrinogen Vlissingen showed an abnormal clotting time with both thrombin and Reptilase(TM). The release of the fibrino-peptides A and B by thrombin was normal, but fibrin polymerization was impaired both in the presence and absence of Ca²⁺ ions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed according to Laemmli the γ-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1500 daltons. The previously described protective effect of Ca²⁺ ions on plasmin degradation of the carboxyl terminus of the γ-chain of normal fibrinogen was only partially detectable in fibrinogen Vlissingen. In addition the binding of Ca²⁺ ions was decreased. Fibrinogen Vlissingen bound 2.4 Ca²⁺ ions per fibrinogen molecule at pH 7.4, whereas normal fibrinogen bound 3.1 Ca²⁺ ions. At pH 5.8 fibrinogen Vlissingen bound 1.1 Ca²⁺ ions, whereas normal fibrinogen bound 2.0 Ca²⁺ ions per molecule fibrinogen in the D-domains, again indicating a structural change in the carboxyl terminus of fibrinogen. The structural defect was determined by sequence analysis of DNA amplified by use of the polymerase chain reaction. Exons VIII, IX, and X of the γ-chain gene were amplified and the DNA sequence of the amplified fragments was determined. A 6-base deletion was found in 50% of the fragments corresponding to exon VIII, indicating that the patient was heterozygous for the mutation. This deletion codes for amino acids Asn-319 and Asp-320 in the normal fibrinogen γ-chain. The data indicate that Asn-319 and Asp-320 are crucial for maintaining the integrity of the carboxyl-terminal polymerization sites, the protective effect of Ca²⁺ ions on plasmin degradation of the carboxyl terminus of the γ-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen. Chemicals/CAS: Calcium, 7440-70-2; Fibrin, 9001-31-4; fibrinogen Vlissingen; Fibrinogen, 9001-32-5; Fibrinogens, Abnormal; Macromolecular Systems; Oligonucleotide Probes