Michio Sakai

Thrombosis from a Prothrombin Mutation Conveying Antithrombin Resistance

We identified a novel mechanism of hereditary thrombosis associated with antithrombin resistance, with a substitution of arginine for leucine at position 596 (p.Arg596Leu) in the gene encoding prothrombin (called prothrombin Yukuhashi). The mutant prothrombin had moderately lower activity than wild-type prothrombin in clotting assays, but the formation of thrombin-antithrombin complex was substantially impaired. A thrombin-generation assay revealed that the peak activity of the mutant prothrombin was fairly low, but its inactivation was extremely slow in reconstituted plasma. The Leu596 substitution caused a gain-of-function mutation in the prothrombin gene, resulting in resistance to antithrombin and susceptibility to thrombosis.

Decreased platelet aggregation of platelet concentrate during storage recovers in the body after transfusion.

BACKGROUND:  Previous reports have shown that storage decrease the ability of PLTs to aggregate in the form of PLT concentrate (PC). Nevertheless, there are few reports that have studied the PLT function in blood samples obtained from recipients after PLT transfusion. In this study, this issue was addressed by examining the ability of PLTs to aggregate after being transfused into the blood stream.

Genetic analysis of protein C deficiency in nineteen Japanese families : Five recurrent defects can explain half of the deficiencies

We have been studying the molecular basis of protein C deficiency. In this study, we determined the molecular defects of protein C deficiency in 19 Japanese families by using a strategy combining polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) analysis. We identified 10 missense mutations, 1 in-frame deletion, 1 frameshift deletion, 1 frameshift addition, and 1 splice site mutation, 5 of which were novel. From the results of genetic analysis of 67 Japanese families with protein C deficiency reported in this and previous studies, the recurrent defects including Phe139Val and Met365Ile substitutions and a Lys150 d letion, a G8857 deletion, and a splice site mutation of G3079A were only found in Japanese subjects and seemed to be a founder effect. In contrast, Arg169Trp, Arg286His, Val297Met, and Asp359Asn substitutions, all occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These 9 recurrent molecular defects were found in 43 families in total, accounting 64% of Japanese families with protein C deficiency. In particular, the recurrent defects of Phe139Val, Arg169Trp, Va1297Met, and Met36-4Ile substitutions and a G8857 deletion were found in 33 families in total, accounting for 49% of Japanese families with protein C deficiency. For the identification of the genetic defect in Japanese patients with protein C deficiency, screening of these recurrent defects by using restriction enzyme cleavage is a rational method.

The prevalence of neutralizing antibodies against adeno-associated virus capsids is reduced in young Japanese individuals.

Pre‐existing antibodies against adeno‐associated virus (AAV), caused by natural AAV infections, interfere with recombinant AAV vector‐mediated gene transfer. We studied the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 in healthy subjects (n = 85) and hemophilia patients (n = 59) in a Japanese population. For healthy subjects, the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 was 36.5%, 35.3%, 37.6%, 32.9%, and 36.5%, respectively, while that in hemophilia patients was 39.7%, 28.8%, 35.6%, 32.9%, and 27.4%, respectively. There was no difference in the prevalence of neutralizing antibody against each AAV serotype between the healthy subjects and the hemophilia patients. The prevalence of neutralizing antibodies against all AAV serotypes increased with age in both healthy subjects and hemophilia patients. High titers of neutralizing antibodies against AAV2 (≥1:224) and AAV8 (≥1:224) were more evident in older individuals (≥42 years old). Approximately 50% of all screened individuals were seronegative for neutralizing antibodies against each AAV tested, while approximately 25% of individuals were seropositive for each AAV serotype tested. The prevalence of seronegativity for all AAV serotypes was 67.0% (healthy subjects, 68.6%; hemophilia patients, 65.0%) and 18.6% (healthy subjects, 20.5%; hemophilia patients, 15.7%) in young (<42 years old) and older subjects (≥42 years old), respectively. The findings from this study suggested that young subjects are more likely to be eligible for gene therapy based on AAV vectors delivered via an intravascular route because of the low prevalence of antibodies to AAV capsids. J. Med. Virol. 86:1990–1997, 2014.

Intramural Hematoma of the Cecum as the Lead Point of Intussusception in an Elderly Patient with Hemophilia A: Report of a Case

Hemophilia A is a congenital bleeding disorder characterized by a deficiency of coagulation factor VIII. Intramural hematoma of the colon is a very rare complication of this disease. We report a case of intramural hematoma of the cecum serving as the lead point of intussusception in a 65-year-old man with hemophilia A. The patient presented with right-sided abdominal pain and bloody stool. Palpation of his abdomen revealed a fist-sized mass. Abdominal computed tomography (CT) showed a circular mass with concentric rings, consistent with an intussuscepted intestine. Because his activated partial thromboplastin time (APTT) was prolonged, we gave him a continuous infusion of factor VIII during and after surgery. Laparotomy revealed an irreducible colo-colic intussusception and we identified a cecal hematoma as the lead point. After an unsuccessful attempt at Hutchinsons maneuver, we performed right colectomy. We report this case to illustrate the necessity of monitoring APTT in patients with hemophilia A who undergo surgery.

Low Tissue Plasminogen Activator Relative to Plasminogen Activator Inhibitor-1 as a Marker of Cardiac Complication in Children With Kawasaki Disease

To determine whether the fibrinolytic system is related to the occurrence of cardiac complication in Kawasaki disease, we measured tissue plasminogen activator, plasminogen activator inhibitor-1, and related factors in the plasma of children with Kawasaki disease. Patients (mean age, 1.8 years) were classified into patients with cardiac complication (n = 9) and no complication (n = 14) groups echocardiographically. They underwent single, high-dose, intravenous γ-globulin infusion therapy. Blood was drawn just before and the day after the single high-dose intravenous γ-globulin infusion therapy (acute phase), and at early and late convalescent phases. Leukocytosis was normalized immediately after the single, high-dose, intravenous γ-globulin infusion therapy. C-reactive protein and fibrinogen were increased in the acute phase and normalized by convalescent phases. D-dimer fraction of fibrin degradation products changed in a similar manner. Tissue plasminogen activator and plasminogen activator inhibitor-1 were increased in acute phase. The tissue plasminogen activator/plasminogen activator inhibitor-1 ratio was lower in the complication group than in the no complication group throughout the observation period (0.095 versus 0.208 after single, high-dose, intravenous γ-globulin infusion therapy, p = 0.006; 0.094 versus 0.183 at late convalescent phase, p = 0.024). A low tissue plasminogen activator/plasminogen activator inhibitor-1 ratio can predict the propensity for cardiac complication, and can help the physician to decide whether additional therapies are necessary in acute phase Kawasaki disease.

Successful steroid pulse treatment in childhood acquired haemophilia with nephrotic syndrome

Summary.  We encountered a 2‐year‐old boy with acquired haemophilia, which rarely occurs in children, who was complicated with nephrotic syndrome. In mid‐August 2001, he was diagnosed to have nephrotic syndrome based on the presence of massive proteinuria and hypoalbuminaemia. Activated partial thromboplastin time (APTT) was normal at 42.4 s at that time. After starting prednisone administration of 2 mg kg−1 day−1, the proteinuria disappeared immediately. However, in early October the same year, subcutaneous ecchymosis and intramuscular bleeding occurred for no apparent reason, and from the examination results his APTT was 106.4 s, factor VIII (FVIII) activity was <1%, and the anti‐FVIII inhibitor titre was 6.9 BU ml−1. As a result, he was diagnosed to have acquired haemophilia. The anti‐nuclear antibody and anti‐phospholipid antibody were negative. With recombinant activated FVII, haemostasis was obtained, and after administering three courses of steroid pulse therapy (methyl prednisolone: 20 mg kg−1 day−1 × 3 days), the anti‐FVIII inhibitory activity disappeared. An analysis of the immunological and coagulation properties of his FVIII autoantibodies showed the anti‐FVIII inhibitory activity to be mediated by IgG1 antibody. In other words, his FVIII inhibitor was a Th1 dominant and it provided a good response to treatment. These findings correlate with those of previous reports. The patient thereafter frequently demonstrated a recurrence of nephrotic syndrome. As a result, he is presently being managed with cyclosporine. However, no recurrence of the anti‐FVIII titre has been observed.

Effect of transfusion on the venous blood lactate level in very low-birthweight infants.

Background:  In recent years the blood lactate level can be easily and quickly measured with a small amount of blood, and the availability of an arterial blood lactate level has been reported as an indicator of oxygen deficit in adults. To determine whether venous blood lactate level can serve as such a marker for determining the indications for transfusion, blood lactate and hemoglobin level were monitored before and after transfusion.

A phase III clinical trial of a mixture agent of plasma‐derived factor VIIa and factor X (MC710) in haemophilia patients with inhibitors

MC710, a 1:10 protein weight ratio mixture of plasma‐derived activated factor VII (FVIIa) and factor X (FX), is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. We evaluated the haemostatic efficacy and safety of one to two administrations of MC710 in 21 joint, muscle, and subcutaneous bleeding episodes in 14 male patients, in a multi‐centre, open‐label, non‐randomized clinical trial.

Bernard-Soulier syndrome due to compound heterozygosity for a novel glycoprotein Ibβ mutation.

count remained at 50–100 × 10 9 /l. A detailed examination of thrombocytopenia was not performed due to difficulty in collecting a sufficient amount of blood for analysis. Thereafter, the patient did not experience spontaneous bleeding, even after she became active. At 3 years of age, she cut her finger on broken glass and the wound required 3 stitches; however, the bleeding from the wound stopped spontaneously. At 4 years of age, she was referred to our hospital by a family doctor due to thrombocytopenia, which was discovered by chance when she was examined after having caught a cold. The platelet count as determined by microscopic counting was as low as it had been just after her birth. Giant platelets were first pointed out by careful observation of peripheral blood smears (platelet diameter: 3.9 ± 1.1 μm, control subjects: 2.5 ± 0.3 μm; n = 31). No other hematological abnormalities were noted. Further examination of the macrothrombocytopenia was conducted. The platelet counts of her father and mother were 110–210 × 10 9 /l and 180–220 × 10 9 /l, respectively. Written informed consent was obtained from the parents, and the study was approved by the ethics committees of the University of Occupational and Environmental Health and Nagoya Medical Center. Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder characterized by thrombocytopenia, giant platelets, and absent ristocetin-induced platelet agglutination [1, 2] . The prevalence is estimated to be less than 1 in 1,000,000 [3] . Severe bleeding episodes in BSS patients are associated with trauma and surgical procedures; however, the severity and frequency of bleeding varies among individuals [4] . BSS is caused by defects in the platelet glycoprotein (GP) Ib/IX complex. More than 50 different mutations in the GPIbα, GPIbβ, and GPIX genes have been described so far [1, 2] . We herein report a BSS patient with a novel compound heterozygous mutation in the GPIbβ gene. The female patient was born at a gestational age of 35 weeks and 2 days, and her birth weight was 1,934 g, so she was admitted to the neonatal care unit of our hospital due to prematurity. She was the first child of nonconsanguineous parents. At the first blood examination, the platelet count was 41 × 10 9 /l and she was suspected to have neonatal alloimmune thrombocytopenia. She therefore received high-dose gamma globulin therapy, but her platelet count did not increase. During the hospital stay, she had no bleeding tendencies such as omphalorrhagia or bleeding from the site of venipuncture. Her platelet Received: December 18, 2012 Accepted after revision: March 19, 2013 Published online: September 19, 2013