A New Type of Ser Substitution for γ Arg-275 in Fibrinogen Kamogawa I Characterized by Impaired Fibrin Assembly

Abstract

SummaryA new type of substitution, Arg to Ser at γ275, has been found in a heterozygous dysfibrinogen derived from a 23-year-old woman with no major bleeding or thrombosis. By sequence analyses of the affected γ-chain and its gene, we found a single amino acid substitution of γ Arg-275 to Ser in an aberrant γ (274-302) residue peptide isolated from lysyl endopeptidase-digests of the patient’s fibrinogen. In agreement with this amino acid substitution, we identified a single nucleotide exchange of A for C at position 5728 in the γ-chain gene creating a codon (AGC) encoding Ser instead of the codon (CGC) encoding Arg at position γ 275. Like two other known types of mutants with a His or Cys substitution at this position, the functional abnormality was characterized by delayed fibrin polymerization, most likely due to impaired abutting of two D domains of adjacent fibrin monomers in the same strand of fibrin protofibrils. The structural derangement that affects the D:D association may not be so severe as compared with those of Cys and His mutants, possessing an additional disulfide-linked Cys molecule and an imidazole ring at the mutation site, respectively.

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of γ Ala327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism

“…[7][8][9][10] All samples were obtained with informed consent provided by the patient according to the Declaration of Helsinki, and the study was approved by the Bioethics Committee for Gene Analysis at Jichi Medical School. Clotting of patient-derived fibrinogen was compared with normal fibrinogen by the thrombin time method in the absence or presence of calcium.…”

“…The aggregation profiles of healthy subject-and patient-derived fibrin monomers were monitored at 350 nm according to the method of Gralnick as described previously. [7][8][9][10] To study effect of Tokyo V fibrin on normal fibrin polymerization, normal fibrin monomers and Tokyo V fibrin monomers at the same concentration were mixed at ratios of 9:1, 3:1, and 1:1, then processed for measuring fibrin monomer aggregation profiles as described earlier in this paragraph. For the control, normal fibrin monomers were mixed with buffer at ratios of 9:1, 3:1, and 1:1, then processed for measuring aggregation profiles of fibrin monomers.…”

“…8 Purified fibrinogen (1 mg/mL) was incubated with thrombin (1 U/mL) in the absence or presence of 2 mM calcium to investigate release of fibrinopeptides A and B and the cross-linking profiles of ␥ chains (␥-dimer formation) and ␣ chains (␣-polymer formation) as described previously. [7][8][9][10] To assess clottability of fibrinogen, purified fibrinogen (1 mg/mL) was incubated in Tris-buffered saline containing 1 U/mL thrombin in the presence of 1 U/mL factor XIII (FXIII) and 2 mM calcium. After incubation at 37°C for 60 minutes, fibrin clots were squeezed with bamboo sticks and the residual protein amount in supernatants was quantified by measuring absorbance at 280 nm.…”

“…10 PCR-amplified DNA fragments were isolated after agarose gel electrophoresis using a rapid gel extraction kit (Invitrogen Japan), and their nucleotide sequences were determined directly by the dideoxynucleotide termination reaction method using BigDye cycle sequencing kit and a DNA sequencer model ABI 310 (Applied Biosystems Japan, Tokyo, Japan) 10,11 or using Sequenase (US Biochemical, Cleveland, OH) and 35 S-dATP (Amersham Biosciences K. K., Tokyo, Japan). 7,9 Fibrin degradation by tPA-catalyzed plasmin digestion and tPA-mediated plasmin generation Purified fibrinogen (1 mg/mL) was incubated with tPA (1.25 U/mL), plasminogen (20 g/mL), FXIII (1 U/mL), and thrombin (1 U/mL) in the presence of 2 mM calcium. After incubation at 37°C, the reaction was terminated by incubating samples in buffer containing 2% SDS and 10 mM dithiothreitol (DTT) at 98°C for 2 minutes and samples were analyzed by SDS-PAGE under reducing conditions.…”

“…Based on these data, we performed peptide mapping of lysyl-endopeptidase digests of patient fibrinogen ␥ chain by high-performance liquid chromatography (HPLC) as described [7][8][9] and found a small aberrant peptide peak, suggesting that the abnormal ␥ chain polypeptide was a small part of the purified fibrinogen molecule. N-terminal amino acid sequencing 7-9 of the aberrant peptide found Phe (322), Glu (323), Gly (324), and Thr (327) in the peptide.…”