Electrospray ionization mass spectrometry identification of fibrinogen Banks Peninsula (γ280Tyr→Cys): a new variant with defective polymerization

Abstract

Summary. Fibrinogen Banks Peninsula was identified in the mother of a patient referred for investigation following recurrent epistaxis. Coagulation tests revealed prolonged thrombin and reptilase times and a decreased functional fibrinogen level. Thrombin-catalysed release of fibrinopeptides A and B was normal, and no abnormalities were detected by DNA sequencing of the regions encoding the thrombin cleavage sites in the Aa and Bb genes. Reducing SDS-PAGE and reverse-phase HPLC analysis of purified fibrinogen chains were normal, as was electrospray ionization mass spectrometry (ESI-MS) analysis of isolated Aa and Bb chains. However ESI-MS revealed a mass of 48 345 D for the isolated g chains, 31 D less than the measured mass of control chains (48 376 D). Since normal and abnormal g chains were not resolved, this implies a 60-62 D mass decrease in 50% of the molecules. A 60 D decrease was confirmed when DNA sequencing indicated heterozygosity for a mutation of Tyr→Cys at codon 280 of the g chain gene.Fibrin monomer polymerization revealed a delayed lag phase and reduced final turbidity and although factor XIIIa crosslinking of fibrinogen was normal, it is likely that this delay is due to impaired D:D self association. Recent crystallographic studies show residues g280 and g275 make contact across the D:D interface, suggesting a similar mechanism for the polymerization defects in fibrinogens Banks Peninsula and Tokyo II (g275Arg→Cys).

Structure and function of human fibrinogen inferred from dysfibrinogens

“…Interestingly, each vArg-275 residue in the DD fragment has a different set of contacts, i.e., with vTyr-280 in one direction and with vSer-300 in the other. To support this finding, a V Tyr-280 to Cys substitution has been reported in association with defective fibrin polymerization in fibrinogen Banks Peninsula [52]. Impaired D:D association may also take place in variant fibrinogens with a mutation located close to these residues, including vGly-268 to Glu (Kurashiki I) [53], vAsn-308 to Lys (Kyoto land others) [54] or to lie (Baltimore III) [55], and vMet-310 to Thr (Asahi) [56].…”

Determination of the Prevalence of Fibrinogen Banks Peninsula Mutation (γ280Tyr→Cys) by PCR

“…We present a novel PCR/restriction digestion assay for the dection of the new Á280Tyr→Cys mutation of fibrinogen recently described by Fellowes et al [3], which is associated with mild bleeding [5], although it does not appear to cause severe disease. Unfortunately, no samples from patients with this defect were available.…”

“…During the final stage of the coagulation cascade fibrinogen is converted to an active form by thrombin-mediated cleavage of small peptides. A large number of familial dysfibrinogenemias have been described so far [1,2], and Fellowes et al [3] recently found a new mutation of Tyr→ Cys at position 280 of the Á chain of fibrinogen to cause fibrinogen Banks peninsula. Electrospray ionization mass spectrometry was used to identify this mutation and the genetic basis was verified by polymerase chain reaction (PCR), amplifying and sequencing exon 8 of the Á chain.…”

Epigenetic, polymorphic and mutational (Αα167Arg→Lys) contribution to a functionally abnormal fibrinogen

“…The mature 340 kDa protein is a dimer of three polypeptide chains (Aa Bb c) 2 arranged in a linear D-E-D structure. The central E domain, formed from the N-termini of all six chains, is connected to the globular D domains by a coiled coil of all three chains.…”

“…It can arise from either gene mutation [2] or post-translational events [3]. The congenital condition can be precipitated by polymorphisms or personal mutations, and acquired changes can be caused by liver disease [4] or drug-induced epigenetic events [5].…”