Factor VII deficiency due to compound heterozygosity for Leu-48Pro mutation and a novel Pro260Leu mutation.

We investigated the mechanisms responsible for factor VII (FVII) deficiency in a compound heterozygous Japanese patient with mutations both in the signal peptide and in the catalytic domain. FVII activity (FVII:C) and antigen (FVII:Ag) levels of the patient were 14.5% and 12.5% of those of the normal controls, respectively. In all, 2 heterozygous point mutations were identified in the patient: one was the mutation substituting Pro for Leu-48 in the prepeptide domain of FVII; the other one was a novel mutation substituting Leu for Pro260 in the catalytic domain. FVII activity and FVII:Ag levels in the condition medium that transiently coexpressed the 2 different FVII mutants in baby hamster kidney (BHK) cells were 4.81% and 5.18% of the wild-type FVII. Factor VII defect of the patient may be combined with both impairing endoplasmic reticulum (ER) targeting and altering FVII folding/biosynthesis, but cotransfection of 2 different FVII mutants may interfere with their expression in BHK cells.

Ex vivo coagulation test on tissue factor-expressing cells with a calibrated automated thrombogram.

A calibrated automated thrombogram is not affected by the turbidity of platelet and cell preparations because the measurement is based on fluorescence. To examine conditions that mimic the physiological state, we investigated thrombograms that show thrombin generation on tissue factor (TF)-bearing cells. An increase in the number of J82 cells did not affect the endogenous thrombin potential (ETP) of normal plasma, although the lag time (LT), the peak height, and the time to peak (ttPeak) did depend on cell concentration. When 5 parameters of coagulation factor-deficient plasmas were plotted on a radar graph, the thrombogram pattern of factor XI (FXI)-deficient plasma became slightly reduced. The thrombogram did not improve when washed normal platelets or washed normal platelets with adenosine diphosphate (ADP) were added. FVII-depleted plasma, FVIII-deficient plasma, and FIX-deficient plasma showed remarkably reduced peak heights, ttPeaks, and times to the end of thrombin generation (start tails). The thrombogram of FVII-depleted plasma was characterized by a remarkably prolonged LT, unlike the patterns of FVIII- or FIX-deficient plasma and FXI-depleted plasma. The ETP of FVIII- and FIX-deficient plasma, but not FVII-depleted plasma, improved significantly upon addition of washed normal platelets or washed normal platelets with ADP. The thrombograms of coagulation factor-deficient plasma containing TF-bearing cells differed from those for recombinant TF and phospholipid in the liquid phase. We suggest that thrombograms using TF-bearing cells can be a useful ex vivo test, because this experimental model may be analogous to most coagulation processes in vivo.

Japanese collaborative study to assess inter-laboratory variation in factor VII activity assays.

BACKGROUND: The clinical phenotype manifest by patients with factor VII (FVII) deficiency correlates poorly with that predicted by laboratory tests. Despite its importance, there are no data on the variability of inter-laboratory determinations of low to very low plasma FVII activity (FVII:C). METHODS: We distributed three FVII-deficient plasma samples, prepared by immunoaffinity chromatography, to 58 laboratories in Japan. All samples were assayed using standardized reference plasma as a calibrator. Recombinant thromboplastin was also supplied as a common reagent. RESULTS: In the case of sample A, which had a very low FVII:C, the use of standardized reference plasma and thromboplastin, lowered the variability of inter-laboratory measurements, when compared with the variability observed when samples were assayed using the respective laboratory's routine method. CONCLUSIONS: The data obtained indicated that results for samples with a very low FVII:C were greatly influenced by the number of plasma dilutions used in constructing a standard activity curve, and also by the type of calibrator and thromboplastin. Such variability was not seen for samples with moderate FVII:C. We conclude that it is necessary to develop a more sensitive and accurate FVII:C measurement system for the diagnosis and treatment of FVII deficiency.

Japanese collaborative study for fibrinogen assay: variability of the fibrinogen assay between different laboratories does not improve when a common calibrator is used.

Several national and local external quality assurance schemes have been developed to improve the plasma fibrinogen assay in Japan over the past 30 years. Now most commercial calibrant plasma may be calibrated against an International Standard preparation, in order to achieve agreement of results obtained by different laboratories. However, we have never achieved satisfactory results, according to an external quality control survey regarding the fibrinogen assay. Therefore, we distributed two kinds of fibrinogen standards to be used as common calibrators, along with three plasma samples, among 183 general laboratories in Japan. The results of this collaborative study showed that the assigned value for the commercially available calibrators remained problematic. Furthermore, it was concluded that the between-laboratory variability could not be improved beyond a certain degree of standardization, even if a common calibrator was used for the Clauss-derived assay carried out by an automatic coagulometer.

Severe factor VII deficiency with recurrent intracranial haemorrhages owing to double heterozygosity for a splice site mutation of an IVS4 and a novel nonsense mutation in exon 8 (Gln211-->Term).

Genetic analysis of a 10-month-old Japanese baby boy with recurrent intrathoracic bleeding, cerebral haemorrhages and gastrointestinal bleeding secondary to severe factor VII (FVII) deficiency revealed evidence of two distinct mutations of FVII: a splice site mutation of G-->A at nucleotide 6071 in the IVS4 splice site and a novel nonsense mutation (Gln211-->Term) in exon 8. His bleeding was difficult to control without prophylactic infusion of FVII. We detected a heterozygous splice site mutation of the IVS4 in his mother and a heterozygous nonsense mutation in exon 8 (Gln211-->Term) in his father. The parents' FVII levels are both 50% of normal controls. The FVII:C in plasma from the proband was < 1.5% of normal controls. FVII:antigen (Ag) was < 1% of normal controls, using a monoclonal antibody (mAb) hVII-B101/1 that specifically reacts with FVII epidermal growth factor 1 (EGF-1), and 5% of normal controls, using a rabbit polyclonal antibody against human FVII. After immunoadsorption with mAb hVII-B101/B1-Sepharose 4B, FVII levels of both the proband and his mother were 5% of normal controls; after immunoadsorption the FVII levels of normal subjects were < 1%. We hypothesize that secretion of a small amount of dysfunctional FVII lacking EGF-1 into the circulation accounts for this observation.

[A case positive for the inhibitor for coagulation factor V].

We report a 71-year-old man who exhibited hepatocellular carcinoma and the inhibitor for coagulation factor V (FV). The inhibitor was found when his coagulation screening tests revealed an abnormally prolonged prothrombin time (71.1 sec) and activated partial thromboplastin time (more than 120 sec) but normal values of fibrinogen (241 mg/dl), the thrombo test (84%) and hepaplastin test (71%). In addition, FV-coagulation activity of the patient's plasma showed less than 1% of the pooled normal plasma and inhibitory activity for FV of his plasma was 32 Bethesda units. This inhibitory activity was neutralized by the addition of anti-human immunoglobulin-gamma-chain serum. The patient was treated with a fibrin sealant including human thrombin when he underwent an partial hepatectomy (32 months before onset) and received 2 doses of thrombin orally (5 months and 2 weeks before onset) to stop bleeding from phlebeurysm. Several studies have reported that the inhibitor for FV was produced after treatment with bovine thrombin containing FV as a contaminant. These findings suggest that our patient may produce an immunoglobulin specific for FV after similar stimulation of human thrombin containing FV.

Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency.

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5' splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor-like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the triangle upEGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.

Coagulation factor VII Gln100 --> Arg. Amino acid substitution at the epidermal growth factor 2-protease domain interface results in severely reduced tissue factor binding and procoagulant function.

We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln100 --> Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X. We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant. In the sTF-FVIIa crystal structure the candidate residue Gln100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

Characterization of monoclonal anti-human factor VII antibody (B101/B1) that recognized three-dimensional structures near Arg at position 79 in the first EGF-like domain.

Murine monoclonal antibodies (designated hVII-B101/B1, hVII-DC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVII-DC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10(-10) M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced gamma-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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.

A simple method for detection of human factor IX inhibitor using ELISA.

We developed a simple ELISA for FIX inhibitors (FIXinh). The relationship between absorbance and various dilutions of plasma containing known FIXinh (28 Bethesda units/ml) was linear, ranging from 200 to 25,600-fold dilution on semi-log paper. Converted into the value of Bethesda Inhibitor Assay, ELISA can detect as low as 0.219 Bethesda units/ml. Absorbance for 30 normal individuals was 0.213 +/- 0.008, which corresponds to < 0.44 Bethesda units/ml. With ELISA, only the value for hemophilia B with FIXinh, which was revealed as positive by Bethesda Inhibitor Assay, was significantly higher than that of normal controls, hemophilia B without FIXinh, hemophilia A with FVIIIinh and systematic lupus erythematosus (SLE) with lupus anticoagulant (LA). The value in the patient with SLE, who had a high level of FVIIIinh (1000 Bethesda units/ml), was 3 Bethesda units/ml of FIXinh with Bethesda Inhibitor Assay, which was not significantly higher than that of normal subjects with ELISA. FIXinh in hemophilia B patients by ELISA for FIXinh was not affected by the presence of other anticoagulants.

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.

Factor VIIShinjo: a dysfunctional factor VII variant homozygous for the substitution Gln for Arg at position 79.

We report a factor VII (FVII) variant, FVIIShinjo, characterized by normal FVII antigen levels and variable procoagulant activity using tissue thromboplastin from different sources. Normal FVII activity is obtained using human placenta thromboplastin but low activity using rabbit or bovine brain thromboplastin. Exons 2-8 and the intron-exon junctions of the FVII genes of the propositus were amplified by PCR from DNA extracted from peripheral white blood cells, and screened by single-strand conformational polymorphism (SSCP) analysis. DNA fragments showing aberrant mobility were cloned and sequenced. We detected a single-point mutation, a homozygous G to A transition at nucleotide position 6,055 in exon 4, which results in the substitution of Arg 79 by Gln in the first EGF-like domain. This mutation results in a loss of a site for the restriction endonuclease MspI. The Msp I digestion pattern of the PCR-amplified exon 3+4 fragments from each member of the family was determined. The Msp I haplotypes were consistent with this G to A transition being associated with reduced FVII activity as detected using thromboplastins from various species. We conclude that the Arg 79 to Gln substitution in the first EGF-like domain of FVII identified in the propositus is responsible for the inherited FVII abnormality in this Japanese family. We postulate that one of the sites of interaction between FVII and tissue thromboplastin includes Arg 79 in the first EGF-like domain of factor VII.

Genetic polymorphism (Arg353-->Gln) in coagulation factor VII gene and factor VII levels (coagulant activity, antigen and binding ability to tissue factor) in 101 healthy Japanese.

We studied the frequency of the Msp I genotype (Gln353) for factor VII in 101 young healthy Japanese individuals, and the levels of factor VII-related procoagulant activity (FVII:c), antigen (FVII:ag) and binding ability to tissue factor (FVII-TF binding). The frequency of the allele coding for the factor VII Gln353 was 0.05 among this group. Individuals with genotype M1M1 had an FVII:c of 103.52 +/- 17.85% (mean +/- SD), FVII:ag of 102.50 +/- 18.42% (mean +/- SD) and FVII-TF binding of 101.03 +/- 22.22% (mean +/- SD). Triglyceride levels of subjects with the M1M1 genotype were 84.34 +/- 44.97 mg dl-1 (mean +/- SD). FVII:c (50%), FVII:ag (45%) and FVII-TF binding (36%) from an individual with the M2M2 type was the lowest, with a triglyceride value of 39 mg dl-1. On the other hand, individuals with the genotype M1M2 had an FVII:c of 75.57 +/- 9.61% (mean +/- SD), FVII:ag of 72.43 +/- 8.54% (mean +/- SD) and FVII-TF binding of 68.14 +/- 14.91% (mean +/- SD). FVII:c, FVII:ag and FVII-TF binding in individuals with the M1M2 and M2M2 genotypes were significantly lower than those in individuals with the M1M1 genotype.

[Frequency of genetic polymorphism (Arg353-->Gln) and factor VII levels in Japanese healthy adults].

We studied factor VII levels and frequency for a G-to-A substitution resulting in the replacement of glutamine for arginine at codon 353 of factor VII by Msp I polymorphism in 101 Japanese healthy adults. An homozygote and seven heterozygotes for the factor VII Gln 353 allele was found in the samples. The frequency of Japanese was not different from those of populations of the United Kingdom, Europe and Afro-Caribbeans, but was different from that in Gurjarati Indians (0.25) previously reported by Humphries et al (Arch Pathol Lab Med 116:1322-1329, 1992). The homozygote had the lowest levels (factor VII activity; 50% and factor VII antigen; 45%) in all samples. The seven heterozygotes had levels of factor VII activity and factor VII antigen about 25% lower than those with only the arginine allele. There were no difference in the levels of factor VII between smoking and no smoking heterozygotes, and between those taking fatty and non-fatty diet.