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Hitoshi Ueda,1 Misato Toyoda,1 Morio Arai,2 Michio Sakai3 1Japan Medical Office, Takeda Pharmaceutical Company Limited, Tokyo, Japan; 2Takeda Development Center-Japan, Takeda Pharmaceutical Company Limited, Tokyo, Japan; 3Pediatrics Department, Munakata Suikokai General Hospital, Fukuoka, JapanCorrespondence: Hitoshi UedaHead of Rare Disease Medical Engagement, Japan Medical Office, Takeda Pharmaceutical Company Limited, 1-1, Nihonbashi-Honcho 2-chome, Chou-ku, Tokyo 103-8668, JapanTel +81 3-6837-6222Fax +81 3-6204-3801Email hitoshi.ueda@takeda.comIntroduction: The Baxject® II Hi-Flow device has previously been used to reconstitute the factor VIII products antihemophilic factor (recombinant) (ADVATE®) and rurioctocog alfa pegol (ADYNOVATE®).Methods: In this crossover study in healthy men, the convenience of an advanced device, Baxject III® with and without a nonslip sleeve, was compared with that of Baxject II Hi-Flow. The primary endpoint was the operational time for reconstitution; secondary endpoints included participants’ assessment of the usability of the devices for reconstitution and their preference for using each of the devices.Results: Twelve healthy adult men (mean ± standard deviation [SD] age: 36.7 ± 7.0 years) and 12 healthy elderly men (mean ± SD age: 70.3 ± 4.8 years) participated in the study. In the adult group, the mean operational time for reconstitution was shorter using Baxject III (mean ± SD: 19.7 ± 2.7 and 19.9 ± 5.2 seconds with and without a nonslip sleeve, respectively) than when using Baxject II Hi-Flow (49.6 ± 7.2 seconds, P < 0.0001 for both comparisons). Adult participants rated preference (P < 0.0001) and ease of reconstitution (P < 0.0001) as higher for Baxject III with a nonslip sleeve than for Baxject II Hi-Flow. Results were consistent regardless of age group or the use of the nonslip sleeve.Conclusion: Owing to the convenience of Baxject III, this device will improve the reconstitution process for patients with hemophilia treated with rurioctocog alfa pegol or antihemophilic factor (recombinant) at home.Keywords: coagulation factor VIII product, reconstitution device, crossover comparison study, hemophilia treatment, usability

Rurioctocog alfa (recombinant Factor VIII: AdvateⓇ) is available for the control of bleeding in patients with hemophilia A in Japan. To evaluate the immunogenicity, safety, and efficacy of prophylactic and on-demand use of rurioctocog alfa, postmarketing surveillance was conducted on 114 previously untreated Japanese patients aged 0–82 years with ≤ 3 exposure days under the conditions of routine clinical practice. A post-hoc comparison of mean annualized bleeding rates between patients in the regular prophylaxis group (7.4 bleeds/year) and in the on-demand treatment group (15.7 bleeds/year) using a negative binomial model found a statistically significant difference (P = 0.0164) in the subset of patients with severe hemophilia A. Favorable prophylactic and on-demand hemostatic efficacy (“excellent” or “good”) was shown in 71.4–88.5% across all treatment regimens. A total of 31 events of adverse drug reactions were reported. Of 114 patients, 21 (18.4%) developed de novo FVIII inhibitor; of these, 17 occurred within 50 exposures. One death was reported. A family history of positive inhibitors was significantly associated with inhibitor development (Fisher exact P value = 0.0004); no other risk factors were identified. Rurioctocog alfa was found to be well-tolerated and effective in previously untreated Japanese patients with hemophilia A in this postmarketing surveillance of routine clinical practice.

Rurioctocog alfa (recombinant factor VIII: Advate®) is available for the control of bleeding among patients with hemophilia A in Japan. To evaluate the perioperative safety and hemostatic efficacy of Advate®, a postmarketing surveillance was conducted in Japanese patients undergoing surgery in a real-world setting. A total of 74 surgical procedures performed in 58 subjects aged 0–75 years, including three females, were studied. A hemostatic efficacy rating of “excellent” or “good” was reported in 73/74 surgical procedures (98.6%). Perioperative bleeding was successfully controlled by Advate® in five subjects with positive FVIII inhibitors (2.4-9.1 BU/mL). Advate® was administered at higher initial bolus doses (114-385 IU/kg) and at higher rates by subsequent initial continuous infusion (8.3-15 IU/kg/hour) in the five subjects with inhibitor than in the subjects without inhibitor (n = 47; mean initial bolus dose: 53.4 IU/kg; subsequent mean initial continuous infusion: 3.8 IU/kg/h). Adverse drug reactions were reported in 7/74 (9.5%) procedures, two of which were the development of de novo FVIII inhibitors. Overall, the perioperative use of Advate® in a real-world setting was found to be safe and effective among Japanese patients with hemophilia A.

Rurioctocog alfa (recombinant factor VIII: Advate

Rurioctocog alfa (recombinant factor VIII: Advate

Rurioctocog alfa pegol (BAX 855) is a novel third-generation recombinant factor VIII whose active ingredient is chemically modified with polyethylene glycol. A global multicenter phase 2/3 study of the product in 137 patients (including 11 patients from Japan) with severe hemophilia A aged 12–65 years, reported an extended half-life and a good tolerability profile, as well as a significantly lower annualized bleeding rate in the prophylactic treatment arm than in the on-demand treatment arm. Using descriptive statistics, a post hoc analysis was performed to compare the pharmacokinetics, safety, and efficacy profiles of the product in the Japanese subpopulation and the overall population. Extended half-life was demonstrated in the Japanese subpopulation. The mean [standard deviation (SD)] annualized bleeding rates in the prophylactic treatment arm were 3.7 (4.7) for the overall population (n = 120) and 4.0 (3.4) for the Japanese subpopulation (n = 11). The proportion of bleeds reported as excellent or good was 94.9% (149/157) in the overall population, whereas that in the Japanese subpopulation was 92.3% (12/13). No FVIII inhibition or anaphylactic reaction was reported in the Japanese subpopulation. The post hoc comparisons demonstrated similar pharmacokinetic, safety, and efficacy profiles between the overall population and the Japanese subpopulation.

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

A defective platelet glycoprotein (GP) Ib/IX/V complex [von Willebrand factor (VWF) receptor] results in Bernard-Soulier syndrome (BSS), which is characterized by macrothrombocytopenia and impaired ristocetin- and thrombin-induced platelet aggregation. We found 2 independent BSS-variant families: Case I [compound heterozygous mutations, p.Glu331X and a frame shift by a deletion at c.1444delA of GPIbα (GP1BA) terminating at a premature stop codon (p.Thr452ProfsX58)], and case II [homozygous nonsense mutation at c.1723C > T, p.Gln545X]. Case I platelets expressed no GPIbα, resulting in absence of ristocetin-induced platelet aggregation (RIPA) and 50% reduction in thrombin-induced aggregation with no shape change. The mother's platelets had 50% the expression level of A-type GPIbα (4-repeated VNTR: variable number of tandem repeats, p.[Thr145Met; Ser399_Pro411[4]]); the father's platelets had the same expression level of C-type GPIbα (2-repeated VNTR, p.Ser399_Pro411dup) as the mother's platelets. The mother's RIPA was significantly higher than the father's. Thrombin-induced aggregation was normal in both parents. Case II platelets expressed a GPIbα with an abnormal cytoplasmic tail, p.Gln545X-truncated GPIbα, which complexed with GPIX and GPV on the cell surface; its expression level of the complex was normal. Case II platelets had reversible RIPA, with no ATP release, and weak thrombin-induced aggregation without shape change. These results suggest that a signaling process through the GPIbα cytoplasmic tail required for full platelet activation is defective in BSS variant case II and a length polymorphism of GPIbα is associated with a modified level of RIPA heterozygous BSS case I.

Beta interferon therapy was given to seven chronic hepatitis C patients with haemophilia or other haemorrhagic disorders who had received clotting factor replacement therapy. Serum alanine aminotransferase (ALT) levels ranged from 82 to 275 UL(-1) and hepatitis C virus (HCV)-RNA ranged from 10(6) to 10(9) copies mL(-1) . HCV-genotypes were I+II in one patient, II in one, II+III in four and IV in one. Patients received 6 mega units (MU) daily of natural type beta interferon by intravenous infusion for 6 weeks. In three of seven patients, the protocol was modified to intermittent administration because neutrocytopenia (under 500 × 10(6) L(-1) ) developed in two patients and thrombocytopenia (under 50 × 10(9) L(-1) ) was observed in one during treatment. No modification was necessary with regard to daily and total dose. All patients received administration without any haemorrhagic complications. Six of seven patients showed improvement in serum ALT levels, and one of the patients showed normalization of ALT levels for 6 months after treatment. HCV-RNA disappeared in four patients by the end of treatment, although no one remained negative 6 months after treatment. The results of our study were similar to those reported in previous papers which described the use of alpha interferon in haemophiliacs. The reason none of the patients showed sustained loss of HCV-RNA after therapy might be associated with high HCV-RNA levels, characteristics of the HCV-genotype and prolonged duration of the disease.

Clotting factor concentrates prepared from human plasma are a potential route of parvovirus B19 (B19) infection in patients with coagulation disorders. However, it is not clear whether B19 transmits and persistently infects patients with haemophilia, especially those with HIV infection. We examined serological and virological markers of B19 in samples from 40 patients with haemophilia who had been receiving several brands of clotting factor concentrates. All of them were anti-B19 IgG seropositive and anti-B19 IgM seronegative. The levels of anti-B19 IgG were significantly higher in haemophiliacs than in healthy donors, whereas there was no difference between the level of anti-B19 IgG in haemophiliacs with HIV infection and those without HIV infection. Moreover, there was no difference between the level of anti-B19 IgG in haemophiliacs receiving recombinant factor VIII and that in those receiving plasma-derived clotting factors. Although by using polymerase chain reaction (PCR) B19 DNA was detected at very low levels (< 40 DNA copies mL(-1) , in 3 out of 40 haemophiliacs, persistent B19 infection was negligible.

We previously reported that platelet retention rates as measured with collagen-coated bead columns (the conventional column) reflect the processes of platelet adhesion and aggregation under low shear stress, and that this system could serve as an easy-to-use platelet aggregometry. With this column, platelet glycoprotein (GP) VI and GPIIb/IIIa, but not the GPIb-von Willebrand factor (VWF) interaction, play major roles in platelet activation. To develop a system that can better reflect the GPIb-VWF interaction under high shear stress, we designed a column containing small-sized beads (125-212 microm) coated with porcine collagen type I. As expected, the GPIb-VWF interaction played a crucial role in platelet retention rates at higher flow rates. Adenosine 5'-diphosphate, but not thromboxane A2, appears to support platelet activation in this system. The platelet retention rates among healthy individuals with the new columns are in the range wider than the conventional columns, and this diversity could be attributed to the broad range of the VWF antigen and/or its activity. It is suggested that this new column can serve as an easy-to-use method for evaluating the VWF antigen levels and its activity and for monitoring patients with thrombotic or bleeding disorders related to the VWF-GPIb interaction.

We describe a patient with haemophilia A and factor VIII inhibitor who underwent surgical excision of a large pseudotumour in the left femoral region. Haemostasis was successfully maintained with bolus doses of recombinant factor VIIa at 2-h intervals and anti-fibrinolytic therapy, and the pseudotumour was removed. Subsequently, the dose interval was gradually prolonged to 8 h for a total of 18 days. Although a spontaneous haemorrhage was observed on postoperative day 8, haemostasis was achieved by reducing the dosage interval. No adverse event occurred during the course of treatment.

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.

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.

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.

A multicenter and open-labeled clinical trial of human recombinant factor VIIa (rFVIIa) was conducted in Japanese patients with severe hemophilia A or B with inhibitors. The trial consisted of 2 parts. In study 1, the pharmacokinetics, pharmacodynamics, and safety of a single dose of 120 microg/kg of rFVIIa were investigated in 8 patients. In the subsequent study 2, the hemostatic effect and safety of rFVIIa were evaluated during a 24-week period in 10 patients. In study 1, the mean maximum FVII-coagulant activity (FVII:C) was found to occur after 10 minutes; activity then decreased rapidly and returned to the baseline within 24 hours after a single intravenous infusion of rFVIIa. The mean half-life of FVII:C was 3.5 hours. The activated partial thromboplastin time and prothrombin time in the patients were immediately shortened but returned to the baseline within 24 hours after dosing. In study 2, 86 microg/kg to 120 microg/kg of rFVIIa (mean, 97 microg/kg) was administered 1 to 85 times to 10 patients. A total of 58.0% (91/157) of bleeding episodes were treated excellently or effectively, with 5 (3.2%) ineffective episodes. There was no apparent trend in the relationship of the hemostatic effect with bleeding sites, mean dose, or number of injections. The efficacy rate, however, was significantly higher (90.0%) in bleeding episodes treated within 3 hours than in those treated at longer intervals (31.0%). No treatment-related adverse events were observed, and there was no evidence of antibody formation to rFVIIa. In conclusion. rFVIIa is an effective and well-tolerated option for treatment of bleeding episodes in hemophilia patients with inhibitors.

術前検査にて先天性第XI因子欠損症と診断され,血漿交換により第XI因子補充を施行し周術期止血コントロールに成功した症例を経験した.症例は58歳男性,既往歴に止血異常はない.健診にて便潜血反応陽性を指摘され,精査にてS状結腸癌の診断を受け手術目的に入院した.術前凝固能検査にてAPTTの延長を認め,精査にて第XI因子活性が1.8%と低値を示し先天性第XI因子欠損症と診断された.術前血漿交換により第XI因子活性を35%にすることでS状結腸切除術の周術期に異常出血を回避できた.また本症例は術前禁食期間に直接ビリルビンの上昇を認め, Gilbert症候群を合併していた.先天性第XI因子欠損症は本邦では稀な疾患ではあるが,開腹手術に際しては術前血漿交換による第XI因子の補充が有用と考えられた.

血友病Aの補充療法に用いる第VIII因子製剤の投与量は, 製剤のパッケージに記された規格値から設定される. そこで, 製剤の実際の第VIII因子含有量と規格値の相違を検討した. 4社から提供された4種類の第VIII因子製剤, 各10ロットの第VIII因子活性を, 自動血液凝固測定装置で同時測定した. 各ロットの実測第VIII因子含有量は, 規格値の70%から196%と大きなばらつきを示した. 各製剤ブランドの10ロットごとの平均値は92%から129%, ロット間の変動係数は7.7%から23.5%と大きなメーカー間差が示された. これらの結果より, 適正な補充療法を行うためには, 第VIII因子活性測定法の標準化が必要であり, 第VIII因子製剤の実測力価が表示されることが望まれる.

Accidental exposure to blood-borne pathogens by contaminated needlestick puncture is a continuing problem for health care workers. We established an infection control center where infection control and follow-up programs are carried out for employees who suffer from accidental needlestick puncture. We analyzed 1062 needlestick accidents that occurred during an 8-year period from May 1990 to April 1998. Of these, 461 (43.4%) occurred in nurses and 392 (36.9%) in doctors; further, 668 (62.9%) occurred during normal needle use while 170 (16%) occurred during recapping. Needles contaminated with blood containing hepatitis C virus (HCV) accounted for 259 (24.4%) accidents. From these 259 incidents, two employees were infected with HCV and suffered acute hepatitis. Accidental exposure to contaminated blood was more likely between May and October and less likely between November and February.

第VIII因子 (FVIII) のトロンビンによる活性化過程におけるリン脂質の影響を検討した. フォスファチジルセリン (PS) とフォスファチジルコリン (PC) の混合リポソーム (PS/PC=20/80, モル比) は, 濃度依存性にトロンビンによるFVIII活性発現を最大12%にまで抑制したが, PCリポソームの影響はみられなかった. リポソームの存在下にFVIIIにトロンビンを添加し, 経時的にFVIII活性の変化を検討したところ, PS/PCリポソームの存在下では20分の経過中, 著明に活性化が抑制されたがPCリポソームでは対照と同様だった. トロンビン添加後の経時的なFVIII蛋白のフラグメントの変化をSDS-PAGEで解析した結果は, PCリポソームとPS/PCリポソーム存在下で差はみられず, 両者とも重鎖と軽鎖の経時的開裂が同様に示された. トロンビンによるFVIIIの最大活性化の直後にリポソームを添加し, その後の活性の変化をみると, PS/PCリポソームを添加したときに, PCリポソームに比較し, より急速な失活を認めた. 以上の結果より, FVIIIはPS/PCリポソームに結合した後では, トロンビンによる活性化FVIIIは生じるが, その安定性が低下し, 非酵素分解的な失活が促進されることが示された.

Congenital factor XIII (FXIII) deficiency is a rare autosomal recessive bleeding disorder. To date 33 patients with FXIII deficiency have been reported in Japan. we examined the genetic defects in two unrelated Japanese families in which three patients had been diagnosed as congenital FXIII A subunit deficiency. Exons I-XV of the gene encoding the FXIII A subunit were individually amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced by the dideoxy termination method. The sequence analysis revealed a substitution from G to A at position 1468 in exon XI resulting in Gly461Arg in Family A, and a substitution from C to T at position 2068 in exon XIV resulting in Arg661stop in Family B. One patient of Family A was homozygote of the Gly461Arg mutation and his daughter and grandchild were heterozygote. Since this mutation did not generate or lose any restriction endonuclease recognition sites, we designed a mutagenic primer (F13E11Mut) that serves to generate a new EcoN restriction site in the amplified DNA from affected individuals, when 100 normal alleles were amplified by PCR using the F13E11Mut primer and digested by EcoN, the G to A mutation was not found in any of the normal alleles, and no other mutation was found in the remaining region of the gene encoding the FXIII A subunit. This suggests that Gly461Arg would cause a FXIII A subunit deficiency in Family A. Two patients in Family B were homozygote of the mutation Arg661stop and three other members of this family were heterozygote. This mutation has previously been reported by Mikkola et al. They observed this mutation in six of eight Finnish families and one Swedish family with congenital FXIII deficiency. Since the mutation is due to CpG dinucleotide change to TpG, a hot spot mutation site, the Arg661stop is considered to be a common cause of FXIII A subunit deficiency.

The association of factor VIII with von Willebrand factor (vWF) is important for protection of factor VIII against proteolytic degradation in plasma. Recently developed factor VIII preparations, namely monoclonal antibody-purified factor VIII (M-factor VIII) and recombinant factor VIII, contain free form of factor VIII that is expected to form complexes with endogenous vWF after venous injection in the patients with hemophilia A. To clarify the difference of free factor VIII and factor VIII-vWF complex in the commercial factor VIII concentrates, we developed a quantitative assay for free factor VIII in which the sample was added to the wells of vWF-coated microtiter plates. Bound factor VIII was detected by monoclonal antibody against factor VIII, followed by incubation with peroxidase conjugated anti-mouse IgG, then the substrate ABTS for measuring absorbance at 405nm. When the amount of free factor VIII antigen in one unit factor VIII activity of recombinant factor VIII was defined as one arbitrary unit, the ELISA detected free factor VIII as low as 0.016 unit/ml. Factor VIII was incubated with vWF or plasma from a patient with severe Hemophilia A for various concentrations, prior to the free factor VIII assay. At saturation, the stoichiometry was one factor VIII molecule per 50vWF monomers. Of the products of factor VIII-vWF complex concentrate, no free factor VIII was detected. While a M-factor VIII concentrate contained free factor VIII comprising 24% of total factor VIII activity. In a gel filtration experiment of M-factor VIII, 15% of the total factor VIII was eluted as free factor VIII and 77% of that was co-eluted with vWF in the void volume. The free factor VIII and the complex form of factor VIII were immunoisolated and were analyzed after SDS-PAGE by immunoblotting. The factor VIII molecular structure and the susceptibility of thrombin cleavage were indistinguishable between the two forms of factor VIII. These results suggested that free factor VIII present in the M-factor VIII concentrate forms complexes with vWF in hemophilic plasma and is involved in physiologic hemostasis.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Four patients with congenital factor XIII (FXIII) a subunit deficiency were treated with prophylatic FXIII substitution at a dose of 1, 000 or 1, 250 units every four weeks for 3 to 12 years to avoid the recurrence of severe bleeding, especially intracranial bleeding, which all had experienced. We evaluated the efficacy of this prophylactic therapy on the basis of both the clinical manifestation of hemostasis and of in vitro analysis of fibrin crosslinking in patient's plasma. Although the FXIII dosage was increased from 7, 837±4, 593units/yr to 12, 699±1, 336units/yr after the start of prophylaxis, the number of bleeding episodes were remarkably decreased from 4.15±1.54/yr to 0.23±0.19/yr with no life-threatening hemorrhage. Both γ-dimer and α-polymer formation was shown in fibrin obtained from patient's plasma taken 30 minutes after the administration of 1, 000units of FXIII, and γ-dimer crosslinking was still observed in fibrin from the plasma taken four weeks after the administration. These data suggest that intermittent prophylactic FXIII administration is valuable in the treatment of patients with congenital FXIII a subunit deficiency.

Human antithrombin III (AT-III) is the major inhibitor of blood coagulation and hereditary deficiency of this glycoprotein is a well known risk factor for familial venous thromboembolic disease. In order to identify the genetic defect in Japanese patient with type Ia AT-III deficiency, we have analyzed seven exons of the AT-III gene using polymerase chain reaction (PCR) and direct sequencing techniques. Comparing the nucleotide sequence found in the patient with the nucleotide sequence of the AT-III gene which was reported by Olds et al (Biochemistry 1993), three differences in the sequence, at position 67 (5′ untranslated region), 2745 (exon2), and 7626 (exon4), were detected. The substitution from G to A was observed at position 7626 in exon 4. This nucleotide substitution is silent and has been previously reported as a polymorphism. The frequency of this polymorphism in the analysis of 100 alleles from 50 Japanese normal individuals was 0.46(G)/0.54(A). Neither of the substitutions, G to A at position 67 in the 5′ untranslated region or C to T at position 2745 in exon 2, was found in the 100 normal alleles. Although the C to T substitution at position 2745 was detected in all 5 patients who were diagnosed as AT-III deficiency by the coagulation test, the G to A substitution at position 67 was detected in only 3 out of 5 patients. These findings suggest that the C to T transition, substituting alanine 94 for valine, may cause AT-III deficiency in this family. Furthermore, the G to A transition at position 67 is probably a low frequency polymorphism.

Continuous intravenous infusion of factor VIII (FVIII) concentrate has been reported to be effective and safe to achieve a stable plasma level of FVIII activity as well as reduction of FVIII consumption. We have used continuous infusion of a monoclonal-antibody purified FVIII concentrate (M-FVIII) for the treatment of 6 patients with hemophilia A, 5 of whom underwent major surgery and 1 with subcortical cerebral hemorrhage. The age of the patients were ranged from 16 to 68. Duration of continuous infusion was 3 to 27 days. In vitro study of the FVIII activity in the M-FVIII showed that it retained over 80% of initial FVIII activity for the period of 24 hours at room temperature in a plastic syringe. The patients required bolus shot of 63.4±29.4U/kg followed by 4.25±1.49U/kg/hr continuous infusion to keep the initial plasma factor VIII level of 100% that result in 102.3±19.0% of actual activity in the plasma of over 2 hours after starting the infusion. Subsequently, dose of continuous infusion was reduced stepwise so that the achieved plasma level of FVIII gradually decreased to 60% in 7 to 10 days after the operation, followed by bolus infusion. All the patients showed excellent hemostasis with no remarkable adverse reactions during and after the operation. 28% of the total dose of M-FVIII was reduced as compared with theoretical dose of bolus infusion of twice a day for the first 3 days. We conclude that the use of continuous infusion of M-FVIII is practical and effective replacement therapy especially for hospitalized patients with hemophilia A for elective surgery.

To determine the difference in reactivity of factor VIII (FVIII) inhibitor to FVIII/von Willebrand Factor (vWF) complex and FVIII free of vWF, an autoantibody to FVIII light chain was tested. A patient (1-3) suffered from autoimmune hemolytic anemia with autoantibody to FVIII. Epitope specificity of the patient's IgG (I-3 IgG) was shown to be the C2 domain of FVIII light chain (2170-2332) by Western blotting using recombinant FVIII deletions expressed in Escherichia coli. The inhibitory effect on FVIII procoagulant activity (VIII:C) of I-3 IgG was tested against a conventional FVIII concentrate; Haemate P, a monoclonal antibody-purified FVIII concentrate; Hemofil M, and a recombinant FVIII (rFVIII); Kogenate. I-3 IgG showed only 1.3 BU/mgIgG for Haemate P, in contrast to 20 BU/mgIgG for both Hemofil M and Kogenate. The ratio of VIII:C/vWF:Ag in Haemate P and Hemofil M was 1/3.43 and 1/0.01, respectively, while Kogenate did not contain vWF. The inhibitory effect of the I-3 IgG was then compared with Kogenate and its complex with vWF. The inhibitory effect was decreased against the rFVIII by forming a complex with vWF from 22 BU/mgIgG to 0.5 BU/mgIgG. Fab from the I-3 IgG had the same effect. In addition, vWF showed a protective effect on FVIII inactivation by the I-3 IgG in a dose dependent manner. Fifty-nine percent of residual VIII:C was retained in the presence of 8 U/ml of vWF after 1 hr incubation with I-3 IgG. These results suggested that vWF could compete with the I-3 IgG for binding to FVIII.

Factor XI deficiency is a rare hereditary coagulation disorder and mostly its bleeding manifestation is excessive bleeding after surgery and trauma. Hemostasis of a 69-year-old Japanese woman with factor XI deficiency in total hip joint replacement was successfully achieved by plasma replacement therapy. Plasma exchange was took place preoperatively with 2800ml of fresh frozen plasma. Factor XI level of the patient plasma showed 65% of normal immediately after the plasma exchange and 57% at the operation. Subsequently, fresh frozen plasmas were transfused to maintain factor XI level at 40% for 24 hours after surgery then 30% for the next 3 days. Factor XI level and APTT were monitored and no excessive bleeding was observed during and after the operation.

This paper evaluates the effect of iodinated X-ray contrast media on plasma clotting time, plasma thrombin generation and thrombin activity. Investigated materials consisted of diatrizoate, iothalamate and ioxaglate as ionic contrast media (ICM), and iopamidol, iohexol, ioversol and iotrolan as nonionic contrast media (NICM). Both the prothrombin time and the activated partial thromboplastin time of normal pooled plasma were markedly prolonged in the presence of contrast media in a dose-dependent manner. All the contrast media also inhibited thrombin generation in plasma, as assessed in the def ibrinated plasma with a chromogenic substrate, S-2238. Although these contrast media did not affect the thrombin amidolytic activity, they interfered with thrombin-clotting of fibrinogen by affecting either thrombin-fibrinogen interaction or fibrin polymerization, or both. The anticoagulant activities of the contrast media were more evident in 3 ICM than in 4 NICM used in this study. These results suggested that NICM could not sufficiently suppress the thrombin generation in the catheter-syringe system because of this lesser degree of anticoagulant activity than that of ICM. In a separate set of experiment, heparin cofactor activity of antithrombin III was not impaired in the presence of these contrast media, suggesting that heparin would be an effective agent to prevent the thromboembolic complications during angiography using NICM.

SummaryBernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder, which is caused by deficiency or decrease of the platelet GPIb/IX/V complex. Analysis of two patients with BSS by How cytometry of the blood revealed different expression patterns of the components of the GPIb/IX/V complex. In case 1, GPIX was completely absent but residual amounts of GPIbα and GPV were detectable; in case 2, GPIbα was completely absent. We amplified the coding regions of GPIbα, GPIbß, GPV, and GPIX from the patients’ genomic DNA with the polymerase chain reaction (PCR) and sequenced the PCR products. In case 1, we identified a point mutation in the GPIX coding region that changes the codon for tryptophan-126 (TGG) to a nonsense codon (TGA). In case 2, we found a deletion of nucleotide within seven adenine repeats at the position of 1932 to 1938 in the coding region of GPIbα, which causes a frame shift that results in 58 altered amino acids and a premature stop codon. These genetic changes alter the transmembrane domain of GPIX or GPIbα and, therefore, would prevent proper insertion of the proteins in the plasma membrane. Thus, abnormality of a single component protein (GPIX or GPIbα) alters the assembly of the GPIb/IX/V complex and causes heterogenous surface expression of GPIbα, GPV and GPIX.

Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patient's platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patient's platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patient's platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.