Osamu Takamiya

Resistance to activated protein C and Arg 506 Gln factor V mutation are uncommon in eastern Asian populations

We investigated the prevalence of the factor V (FV) Arg 506 Gln mutation in healthy subjects from three eastern Asian countries (Japan, n = 270; China, n = 113; and Korea, n = 93) and in 26 Japanese patients showing venous thromboembolic events. The patients were also examined for activated protein C (APC) resistance by using the Coatest APC resistance kit. The FV mutation was investigated by polymerase chain reaction and restricted enzyme digestion with MnlI RFLP assay of the FV gene. None of the patients showed APC resistance, while all subjects examined were homozygous for Arg at position 506 of the FV gene. Our results imply that FV mutation and APC resistance contribute little to venous thrombotic diseases in eastern Asia.

Electroimmunoassay of factor VII antigen

A clear precipitating arc of factor VII antigen appeared by immunoelectrophoresis using 125I-labelled anti-factor VII antibody incorporated in the Laurell plate to enhance the sensitivity. A plasma sample could be electrophoresed directly on 1% agarose containing 1% rabbit anti-human factor VII antiserum, 0.1% 125I-labelled anti-human factor VII specific IgG and 0.5% polyethyleneglycol 6000, and after washing, autoradiographed at -70 degrees C for 24 hr. A distinct rocket was seen from 45 ul standard reference plasma to 16-fold after auto-radiography. Of 4 patients with congenital factor VII deficiency, 3 patients had no detectable factor VII antigen, one patient had reduced factor VII antigen.

Studies on Vitamin K-Dependent Factor Deficiency during Early Childhood with Special Reference to Prothrombin Activity and Antigen Level

8 young infants aged 14 days to 5 months with vitamin K-dependent factor deficiency were studied with special reference to prothrombin activity and antigen level. Among them, 3 infants had congenital bile duct atresia and 5 were breast-fed babies with severe hemorrhagic tendency of unknown cause. In the patients with both congenital bile duct atresia and breast feeding the ratio of prothrombin activity to prothrombin antigen was lower than 0.1. Furthermore, the arc of prothrombin antigen in these patients demonstrated a faster anodal shift than did normal prothrombin antigen on crossed immunoelectrophoresis. This abnormal prothrombin antigen was not consumed after recalcification of patient plasma, and absorbed poorly on BaSO4. In addition, the abnormal prothrombin antigen disappeared from the patient plasma within a few days after parenteral administration of vitamin K. These results suggest that this abnormal prothrombin is PIVKA-II.

Combined factor VII and protein C deficiency found in a patient with peripheral pulmonary artery stenosis accompanied by progressive pulmonary hypertension and hemoptysis

A congenital deficiency of factor VII and protein C was found in a 21-year-old female suffering from recurrent and progressive attacks of dyspnea and hemoptysis over the last four years. She has been followed in our Department since the age of 17 under a diagnosis of peripheral pulmonary artery stenosis and pulmonary hypertension as confirmed by cardiac catheterization and angiography. Prolonged prothrombin time repeatedly examined during this time period prompted us to perform detailed coagulation studies. We found that factor VII and protein C were both half normal in activity as well as in antigen. Three other members of her immediate family were also found to be affected with this combined deficiency. Since the genes encoding factor VII and protein C are located in different chromosomes, the 13th and the second chromosomes, respectively, expression of the combined hereditary deficiency is a random and very rare association on the basis of frequencies of 1:50,000 for factor VII and 1:16,000 for protein C deficiencies.

Molecular mechanism of dysfunctional factor VII associated with the homozygous missense mutation 331Gly to Ser

We have identified a Japanese homozygous FVII deficiency associated with the mutation G331S (c184 [in chymotrypsin numbering]), and have determined the mechanisms responsible for the dysfunctional FVII variant by expressing the mutant recombinant FVII protein. In addition, the recombinant proteins FVIIG331D, G331W and G331F were expressed. The purified recombinant FVII proteins ran as a single chain form on SDS-PAGE having a molecular mass of approximately 50 Kda. The recombinant FVIIG331S expressed the level of the recombinant wild type FVII at 2.0%, and this mutant form was also similar to FVII in the patients plasma. However, the amidolytic activity of FVIIa using peptidyl substrate S-2288 differed little between the wild type and the four mutant FVII molecules. We suggest that the functional defect found in these mutants is not directly associated with peptidyl substrate recognition or catalysis. The Km values of FX and FIX for the mutant proteins were approximately 7.6- to 15-fold and 13- to 19-fold higher than those for the wild-type protein, respectively. Molecular modelling indicated that the side chain of the S331 mutant is oriented close to the side chain of D338 (c189) at the bottom of the specificity pocket of FVIIa. We show that the replacement of G331 with a serine likely results in a steric hindrance of macromolecular substrate binding, leading to a loss of FVIIa enzymatic activity.

Factor VII binding to tissue factor in plasma from warfarin-treated individuals

Using enzyme immunoassay, we measured the binding ability of artificial gamma-carboxyglutamic acid (Gla)-domainless-Factor VII (FVII) to tissue factor (TF) or of Factor VII in 44 patients stabilized by long term treatment with warfarin. Purified FVII digested with cathepsin G, that is Gla-domainless-FVII, showed a rapid loss in the binding ability of FVII to TF (FVII-TF binding). After adsorption with Al(OH)3 of plasma from 8 out of 44 warfarin-treated patients, no FVII clotting activity (FVII:c) was detected in the supernatant, whereas FVII antigen (FVII:ag) and FVII-TF binding remained 19.2% and 17%, respectively, as compared with those before adsorption. In the plasma from 44 warfarin-treated patients the FVII:c (mean +/- SD, 54.26 +/- 12.50%) was significantly lower than the FVII:ag (77.15 +/- 18.24%) (p < 0.001), although the FVII:c was significantly correlated with FVII:ag (r = 0.628). FVII-TF binding (68.27 +/- 21.16%) was significantly higher than FVII:c (p < 0.001), but not than FVII:ag (p > 0.05). The FVII-TF binding was significantly correlated with FVII:ag (r = 0.738), but somewhat poorly with FVII:c (r = 0.415). These results show that artificial Gla-domainless-FVII digested with cathepsin G loses both the clotting activity and the binding ability to TF. However, PIVKA-VII has little or no clotting activity but the binding ability to TF. This suggested that the low specific activity of FVII (FVII:c/FVII:ag) in the plasma of warfarin-treated patients would not entirely depend on the decreased FVII-TF binding.