Fibrinogens Kosai and Ogasa: Bβ15Gly→Cys (GGT→TGT) substitution associated with impairment of fibrinopeptide B release and lateral aggregation

Abstract

Summary. We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L À1, respectively) than when determined by the immunological method (2.87 and 2.72 g L À1 , respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bb15Gly!Cys (GGT!TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbo15Gly plus the existence of some additional disulfide-bonded forms.

Analysis of plasmin generation and clot lysis of plasma fibrinogen purified from a heterozygous dysfibrinogenemia, BβGly15Cys (Hamamatsu II)

“…The blood coagulation tests were performed on the propositus, and the immunologically determined fibrinogen concentration was measured by an automated latex photometric immunoassay [6]. Fibrinogen was purified from citrated plasma obtained from the propositus and from a normal control subject (NC) with informed consent.…”

“…Fibrinogen was purified from citrated plasma obtained from the propositus and from a normal control subject (NC) with informed consent. Purification and measurement of the fibrinogen concentration was performed as described [6]. The purity and characterization of the proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing (5% polyacrylamide gel) or reducing conditions (10% polyacrylamide gel) followed by immunoblot analysis and using with a rabbit antihuman fibrinogen antibody (Dako, Carpinteria, CA, USA) or a rabbit antihuman albumin antibody (Dako) with the reacting species being visualized with the aid of alkaline phosphatase-conjugated goat antirabbit IgG antibody (EY Laboratories Inc., San Mateo, CA USA).…”

“…The reactions were performed in a final volume of 100 l as described elsewhere [6]. Briefly, fibrinogen (90 l at 0.2 mg/ml) in N- [2-hydroxy-ethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) at pH 7.4 and 0.12M NaCl (referred to from now on as polymerization buffer) containing 1.0 mM CaCl 2 were mixed with human -thrombin (10 l at 0.5 unit/ml) (Enzyme Research Laboratories, South Bend, IN, USA).…”

“…In 2003, we reported two BGly15Cys variants, termed Kosai and Ogasa, which are characterized by the presence of albumin-binding variant fibrinogens and by impairments in both FPB release and lateral aggregation during fibrin polymerization [6]. To remove the complication of normal and/or normal-variant heterodimer molecules when assessing the function of plasma fibrinogen, we synthesized the recombinant variant fibrinogens B15Cys (B15C) and B15Ala and examined their functions in relation to the changes in their primary structure [7].…”

In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from γAsn319, Asp320 deletion dysfibrinogen, Otsu I

“…Samples for scanning electron microscopy (SEM) were prepared as described before [8]. Briefly, 10 µl of α-thrombin was added to 40 µl of fibrinogen solution from NC, Otsu and Otsu-1.34x and mixed by repeated pipetting.…”