A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-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. 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 gamma-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1,500 daltons. The previously described protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-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 gamma-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 gamma-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 gamma-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen.
Koopman
J
J
Gaubius Institute TNO, Leiden, The Netherlands.
Haverkate
F
F
Briët
E
E
Lord
S T
ST
D000328:Adult; D001483:Base Sequence; D002118:Calcium / Q000097:blood*; D002872:Chromosome Deletion; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015241:Fibrinogens, Abnormal / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes
D015241:Fibrinogens, Abnormal; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; C069866:fibrinogen Vlissingen; D005337:Fibrin; D005340:Fibrinogen; D002118:Calcium