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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.

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.

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.

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.

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.

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.

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.

Summary. Flow cytometric studies demonstrated that the platelets from a patient with Bernard-Soulier syndrome expressed substantial amounts of both GPIX and GPV, although in reduced amounts.

Introduction: Factor (F) VIII genotype has been shown to be a risk factor for inhibitor development, where different mutation types are associated with different degrees of risk (Oldenburg J, et al.Haemophilia 2006;12 Suppl 6:15–22). However, it is not clear why some patients with a specific mutation develop inhibitors while others with the same mutation do not, including those with high-risk defects such as null mutations. Patients with severe hemophilia A (HA) with low-risk mutations are thought to produce some functional or non-functional FVIII that is sufficient to induce immune tolerance (Oldenburg J, et al.Haemophilia 2002;8 Suppl 2:23–29). In the present study, the distribution of F8 gene mutations in 81 patients fully exposed to FVIII with severe HA and no inhibitors is compared to data on mutation distribution among severe HA patients with/without inhibitors. Materials & Methods: Turoctocog alfa (NovoEight®) is a new recombinant FVIII product with a truncated B-domain. Previously treated patients (PTPs) with severe HA participated in the pivotal trials guardian™1 (n=150; 12–65 yrs) or guardian™3 (n=63; Results: Eighty-one patients enrolled in the guardian™ trials had F8 gene mutation analyzed. For 52 patients Null mutations (inversions, nonsense mutations, and large deletions) were present in 66% and 75% of patients with severe HA and severe HA with inhibitors, respectively (table 1). In guardian™, 57% (46/81) of patients had null mutations despite never having developed an inhibitor. The difference in distribution of null mutations in the 3 populations is mainly accounted for by large deletions and nonsense mutations, while distribution of inversions was similar. It should be noted that in guardian™, 10/11 patients with missense mutations had these located outside the C1/C2 domain, which reportedly has a lower frequency of inhibitors compared with missense mutations at the C1/C2 domain (Oldenburg J, et al. 2006). Conclusions: Eighty-one patients in the guardian™ trial with severe hemophilia A had their genotype analyzed and all of them were inhibitor-free over their lifetime. Interestingly, the prevalence of high-risk mutation types does not seem to differ markedly from previously published data in patients with inhibitors, probably indicating the relevance of other factors in inhibitor development. Table 1. Distribution of F8 gene mutation types guardian™ Additional studies guardian™ patients (N) Distribution (%) in guardian™ patients (severe HA without inhibitors) Distribution (%) in patients with severe HA and inhibitors (Salviato R, et al. 2007)* Distribution (%) in patients with severe HA with/without inhibitors (Oldenburg J, et al. 2006) Total, N 81 81 76 753 Intron 22 inversion 33 40.7 44.7 45.0 Intron 1 inversion 3 3.7 1.3 2.5 Nonsense 8 9.9 18.4 13.5 Large deletion 2 2.5 10.5 5.0 Missense 11 13.6 5.2 14.5 Small del/ins/dupl 16 19.8 10.5 16.0 Splice site 4 4.9 5.2 3.5 Unidentified/ not enough information 4 4.9 3.9 0.0 Total 81 100.0 100.0 100.0 *Only patients with severe HA included. Disclosures Matytsina: Novo Nordisk A/S: Employment. Arai:Novo Nordisk Pharma, Ltd: Employment. Oldenburg:Baxter, Bayer, Biogen Idec, Biotest, CSL-Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background. Decompensated hepatitis C virus (HCV)-related cirrhosis is the main indication for liver transplantation. We report the first successful living-related liver transplantation in a 49-year-old hemophilia A patient with end-stage HCV-related cirrhosis using a graft obtained from his 20-year-old daughter, an obligate carrier. Methods. The donor’s autologous fresh-frozen plasma rich in factor VIII (FVIII) by treatment with 1-deamino-8-D-arginine vasopressin was prepared before the operation. At induction, 1-deamino-8-D-arginine vasopressin was given to the donor to increase plasma FVIII level. In addition, autologous fresh-frozen plasma containing high FVIII concentrate was infused intraoperatively. The right lobe was harvested from the donor and transplanted orthotopically. The recipient was treated postoperatively with recombinant FVIII and immunosuppressive agents. Results. The donor did not receive recombinant FVIII or allogenic blood during perioperative periods. No bleeding was encountered in the donor perioperatively. The recipient showed a steady increase in FVIII activity postoperatively and was discharged 40 days after transplantation. Ribavirin and interferon-α were provided for 3 months postoperatively to prevent potential recurrence of HCV infection. Serum HCV-RNA by RT-PCR became negative after such treatment. Conclusions. End-stage liver disease in patients with hemophilia A can be an indication for living-related liver transplantation. Furthermore, a graft from a living-related donor with hemophilia A carrier seems to be suitable provided such individuals receive adequate support for coagulopathies.

Background — We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Methods and Results — Whole blood obtained from 2 patients with GP VI–deficient platelets and the effects of the Fab of anti–GP VI antibody (Fab/anti–GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor γ-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s −1 ) or high (1500 s −1 ) wall shear rate, either in the case of blood containing GP VI–deficient platelets or in the presence of Fab/anti–GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI–associated Fc receptor γ-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI–deficient platelets. Conclusions — GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.

We described a 10-year-old girl with SLE complicated with anticardiolipin antibody, who showed bleeding tendency since infant. Her platelet was 6 x 10(3)/microliter. The APTT was remarkably prolonged. The platelet count was normalized after steroid treatment. A false positive Venereal Disease Research Laboratory (VDRL) test, lupus anticoagulants and anticardiolipin antibodies (IgG and IgM) were positive. Her symptoms and the laboratory data fulfilled the criteria of antiphospholipid syndrome. Renal biopsy was performed because of proteinuria, hematuria and the positive of anti-nuclear antibody. The pathology of kidney showed that of lupus nephritis (diffuse membranous glomerulonephritis), and she was diagnosed SLE. She showed no laboratory findings of hyperfibrinolysis and no symptoms of thrombosis under the steroid treatment for 3 years.

Most factor VIII inhibitors are developed at an early age and in patients with severe type of hemophilia A. We report a case of newly developed factor VIII inhibitor in a 60-year-old patient with mild hemophilia A who had been treated with several kinds of factor VIII concentrates. The patient was treated with a total of 103,580 units of recombinant factor VIII concentrate by continuous and bolus infusions for the open surgery of sigmoid colon cancer. On the 95th postoperative day, the patient had right low limb muscle bleeding and was infused with 1,000 units of recombinant factor VIII concentrate for three days. Subsequently, the level of factor VIII inhibitor in the patient's plasma was 2 Bethesda units (BU)/ml. Since then numerous subcutaneous hemorrhages developed, but an adequate hemostatic effect was not obtained even with the administration of a high dose of recombinant factor VIII concentrate. The patient was switched to bypass therapy using human plasma-derived factor VIIa concentrate, which showed a favorable hemostatic effect.