Kagehiro Amano

Novel FV mutation (W1920R, FVNara) associated with serious deep vein thrombosis and more potent APC resistance relative to FVLeiden.

Factor V (FV) appears to be pivotal in both procoagulant and anticoagulant mechanisms. A novel homozygote (FVNara), a novel mechanism of thrombosis associated with Trp1920→Arg (W1920R), was found in a Japanese boy and was associated with serious deep vein thrombosis despite a low level of plasma FV activity (10 IU/dL). Activated partial thromboplastin time-based clotting assays and thrombin generation assays showed that FVNara was resistant to activated protein C (APC). Reduced susceptibility of FVaNara to APC-catalyzed inactivation and impaired APC cofactor activity of FVNara on APC-catalyzed FVIIIa inactivation contributed to the APC resistance (APCR). Mixtures of FV-deficient plasma and recombinant FV-W1920R confirmed that the mutation governed the APCR of FVNara. APC-catalyzed inactivation of FVa-W1920R was significantly weakened, by ~11- and ~4.5-fold, compared with that of FV-wild-type (WT) and FVLeiden (R506Q), respectively, through markedly delayed cleavage at Arg506 and little cleavage at Arg306, consistent with the significantly impaired APC-catalyzed inactivation. The rate of APC-catalyzed FVIIIa inactivation with FV-W1920R was similar to that without FV, suggesting a loss of APC cofactor activity. FV-W1920R bound to phospholipids, similar to FV-WT. In conclusion, relative to FVLeiden, the more potent APCR of FVNara resulted from significant loss of FVa susceptibility to APC and APC cofactor activity, mediated by possible failure of interaction with APC and/or protein S.

Identification and characterization of an adenine to guanine transition within intron 10 of the factor VIII gene as a causative mutation in a patient with mild haemophilia A

Haemophilia A is caused by various genetic mutations in the factor VIII gene (F8). However, after conventional analysis, no candidate mutation could be identified in the F8 of about 2% of haemophilia A patients. The F8 of a patient with mild congenital haemophilia A, in whom no candidate mutation was found in the exons or their flanking regions, was analysed in detail to identify the patients aetiological genetic abnormality. We also characterized anti‐FVIII antibody (inhibitor) development in this patient. Genomic DNA analysis revealed an adenine to guanine transition deep inside intron 10 (c.1478 + 325A>G) of F8 as a causative mutation. Analysis of the transcripts demonstrated that the majority of the patients transcript was abnormal, with 226 bp of the intronic sequence inserted between exon 10 and 11. However, the analysis also indicated the existence of a small amount of normal transcript. Semi‐quantification of ectopic F8 mRNA showed that about one‐tenth of the normal mRNA level was present in the patient. After the use of a recombinant FVIII concentrate, the presence of an inhibitor was confirmed. The inhibitor was characterized as oligoclonal immunoglobulin IgG4 directed against both the A2 domain and light chain of the FVIII molecule with type I reaction kinetics of inhibition of FVIII activity. When no mutations are found by conventional analysis, deep intronic nucleotide substitutions may be responsible for mild haemophilia. The inhibitor development mechanism of the patient producing some normal FVIII was thought to be of interest.

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

We identified 3 mutations in the factor V (FV) gene(F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.

The use of recombinant activated factor VII in patients with acquired haemophilia

Acquired haemophilia (AH) is a rare, often severe bleeding disorder characterised by autoantibodies to coagulation factor VIII (FVIII). Observational studies offer crucial insight into the disease and its treatment. Recombinant activated factor VII (rFVIIa, eptacog alfa activated) was available on an emergency and compassionate use basis from 1988 to 1999 at sites in Europe and North America. In 1996, rFVIIa was approved in Europe for the treatment of AH; it was licensed for this indication in the United States in 2006. Recombinant activated FVII is approved for first-line treatment of bleeding episodes and prevention of bleeding in surgical/invasive procedures in patients with AH. This review provides an up-to-date summary of the haemostatic efficacy of rFVIIa in patients with AH, from the first emergency and compassionate use programmes, to patient registries and a post-marketing surveillance study. In acute bleeding episodes, rFVIIa provided high and consistent rates of control, and available data showed that acute bleed control rates were higher for first-line rFVIIa versus salvage rFVIIa. In surgical procedures, rFVIIa also provided high rates of control. In patients with AH, rFVIIa has a high rate of haemostatic efficacy in acute and surgical bleeding episodes.

A Phase II clinical trial of a mixture of plasma-derived factor VIIa and factor X (MC710) in haemophilia patients with inhibitors: haemostatic efficacy, safety and pharmacokinetics/pharmacodynamics.

MC710, a mixture of plasma‐derived activated factor VII and factor X at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In a Phase II trial, we evaluated the haemostatic efficacy and safety of single doses of MC710, and investigated pharmacokinetic and pharmacodynamic parameters in nine joint bleeding episodes in six male haemophilia patients with inhibitors. This trial was a multi‐centre, open‐label, non‐randomized study of two doses (60 and 120 μg kg−1 as FVIIa dose), allowing the re‐administration of different MC710 dosages to the same subjects. Haemostatic efficacy was assessed by evaluating reduction in pain and swelling, as well as increase in range of motion in a bleeding joint. The results of the study showed that in nine bleeding episodes, seven treatments were rated as ‘excellent’ or ‘effective’ according to investigators rating system of efficacy at 8 h after administration. No serious or severe adverse events were observed after administration; furthermore, measurement of several diagnostic markers revealed no signs or symptoms of disseminated intravascular coagulation (DIC). The haemostatic potential of MC710 was confirmed at doses of 60 and 120 μg kg−1 in this trial. MC710 is thus expected to be a safe and efficacious novel bypassing agent for controlling bleeding in haemophilia patients with inhibitors.

Two double heterozygous mutations in the F7 gene show different manifestations

Summary. We sequenced the factor VII gene (F7) in two unrelated Japanese patients with factor VII (FVII) deficiency. In the first (an asymptomatic 46‐year‐old man with FVII activity and antigen levels of 1·2% and 21% of normal respectively), novel E25K and H348Q mutations were identified in the doubly heterozygous state. In transiently transfected HEK293 cells, the level of FVII‐E25K mutant activity in the culture media was significantly lower than that of FVII wild type, whereas the antigen levels of both proteins were similar. This suggests that the E25K mutation is associated with a dysfunctional FVII molecule. In the second patient (a 47‐year‐old woman with FVII activity and antigen levels of less than 1% and 6% respectively), an IVS4+1 mutation and a novel −96C to T transition were detected in the double heterozygous state. In electrophoretic mobility shift assays, the −96T mutation was shown to disrupt binding of Sp1.

Five novel and four recurrent point mutations in the antithrombin gene causing venous thrombosis.

We analyzed the antithrombin (AT) gene in 9 unrelated Japanese patients with thrombotic disease. All 7 exons, the splice junctions, and the 5′-flanking region of the AT gene were amplified by polymerase chain reaction and sequenced directly. Nine different point mutations, all in the heterozygous state, were identified. Five novel (M-32T, M89K, L146H, Q159X, and L409P) and 2 previously reported (R132X and R359X) point mutations were identified in patients with type 1 deficiency.Two different missense mutations, R393C and R393H, located in the protease reactive site were detected in patients with type 2 deficiency. No other sequence abnormalities in the AT gene were detected by direct sequencing. None of the mutations was present in 100 alleles from 50 unrelated Japanese control subjects. Although type 1 deficiency was diagnosed in patient 7 on the basis of approximately 50% AT antigen and activity levels, the data indicated that the novel L409P mutation is a type 2 pleiotropic effects (PE) deficiency because its location in the C-terminal portion of the reactive site is similar to the locations of reported PE type mutations, and it is highly conserved among other serpins.

Treatment of acute bleeding in acquired haemophilia A with recombinant activated factor VII: analysis of 10-year Japanese postmarketing surveillance data.

Patients with acquired haemophilia A (AHA) have autoantibodies against factor VIII (FVIII), and may develop spontaneous bleeding that requires treatment with FVIII inhibitor bypassing agents such as recombinant activated FVII (rFVIIa, NovoSeven®). However, data regarding the use of rFVIIa are limited.

Emicizumab-mediated haemostatic function in patients with haemophilia A is down-regulated by activated protein C through inactivation of activated factor V

Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti‐FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once‐weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab‐mediated haemostasis remain to be explored. We investigated the role of APC‐mediated reactions in these circumstances. APC dose‐dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII‐deficient plasmas, and in normal plasmas preincubated with an anti‐FVIII antibody (FVIII‐depleted). FVIIIa‐independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC‐induced down‐regulation of emicizumab‐dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII‐depleted FV‐deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab‐related TG in FVIII‐depleted FVLeiden plasma was decreased by APC more than that observed with native FVLeiden plasma. The findings indicated that emicizumab‐driven haemostasis was down regulated by APC‐mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.

Factor VIII haplotypes of Japanese population show similarity to those of Caucasian populations

Haemophilia A is the most common inherited bleeding disorder which is X-linked recessive. This disease is caused by a quantitative or qualitative abnormality of plasma factor VIII (FVIII), which is affected by a genetic mutation located in the coagulation FVIII gene. Diagnosis and replacement therapy strategies in haemophilia A patients are well established; however, some issues remain to be addressed. The most important issue concerning replacement therapy in haemophilia A patients is the development of inhibitors (alloantibody) against FVIII. This development leads to marked attenuation in the effectiveness of replacement therapy, leading to a substantial deterioration in the quality of life of patients. In general, the incidence of inhibitor development in treatment-naive patients with haemophilia A is estimated to be 20–30% [1]. However, it has been shown that its incidence in the Black population is markedly higher, about twice as high as in other racial groups [2]. The question of why this inhibitor develops with such high incidence in the Black population remains undetermined. Recently, Viel et al. reported that six wild-type FVIII proteins, the H1–H6 haplotypes, had different prevalence rates among racial groups [3]. They speculated that a mismatch of the FVIII haplotype between the FVIII concentrate and its recipients, particularly in immunodominant epitopes, caused an increase in the frequency of inhibitor development. In particular, they focused on four different amino acid polymorphisms (R484H, R776G, D1241E and M2238V), based on a non-synonymous single nucleotide polymorphism. By using a combination of those amino acids, they classified the FVIII protein into six haplotypes, namely, H1: RRDM, H2: RREM, H3: RREV, H4: HREM, H5: RRDV and H6: RGEM. It is presumed that amino acid positions 484 and 2238, located in the A2 and C2 domains, respectively, are components of the immunodominant epitope of FVIII. In Caucasian participants, positions 484 and 2238 have been observed as R and M , respectively, and have only shown the haplotypes of H1 and H2. However, Viel et al. confirmed the presence of H in place of R at position 484, or V in place of M at position 2238, in approximately 25% of Black participants. Thus, the haplotype frequencies in Black participants (H1: 0.354; H2: 0.374; H3: 0.222; H4: 0.040 and H5: 0.010) are different from those in Caucasian participants. Furthermore, they also analysed haplotypes in ethnic Chinese participants, and observed the H6 haplotype, but not the H3, H4 or H5 haplotype. We set out to analyse the haplotypes of FVIII proteins among 106 unrelated Japanese subjects at our institution (63 with haemophilia A and 43 with haemophilia B as a control group), as shown in Table 1. The study was approved by the Ethics Committee of Tokyo Medical University and written informed consent was obtained from each patient. The studies were carried out in accordance with the principles of the Declaration of Helsinki. Among the Japanese participants, only the H1 and H2 haplotypes (H1: 0.896 and H2: 0.104) were found at frequencies not significantly different from the Caucasian frequencies (H1: 0.926 and H2: 0.074). Despite the Chinese being geographical neighbours of the Japanese, the H6 haplotype which was observed in approximately 8% of Chinese was not detected in any Japanese. Currently in Japan, approximately 80% of haemophilia A patients receive replacement therapy using the recombinant FVIII concentrates Kogenate (Bayer) or Advate (Baxter). Data regarding the incidence of inhibitor development in Japanese patients with severe haemophilia A were previously presented as poster presentations on post-authorisation safety studies for Kogenate or Advate at the 22nd Congress of the International Society on Thrombosis and Hemostasis (ISTH) (2009) and the Hemophilia World Congress (World Federation of Hemophilia) (2010) by Fukutake et al., who reported that the incidence was similar to that in Caucasians. Although the role of the haplotypes of FVIII proteins as a risk factor for inhibitor development is not yet determined, the similarities of haplotype incidence may partially explain the similarities in inhibitor incidence between Japanese patients and Caucasian patients. Further studies are needed to clarify the role and characteristics of FVIII haplotypes in Japanese population.