A novel factor V compound heterozygous mutation associated with thrombosis (Y1961C; FV-Kanazawa, together with 1982_1983del).

BACKGROUND: ; Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV:C 6 IU/dL, FV:Ag 32 IU/dL), complicated with recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. AIM;: To clarify thrombotic mechanisms associated with this FV abnormality. METHODS AND RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using HEK293T cells, and analyses were targeted, therefore on the FV-Y1961C mutation. APTT-based clotting assays demonstrated that FV-Y1961C exhibited APCR, and that the reduced APC susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein (P)S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/PS-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor (TF)-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon). CONCLUSIONS: ; We identified a compound heterozygous. FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function, but impaired inhibition of TF-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.

A novel homozygous variant of the thrombomodulin gene causes a hereditary bleeding disorder.

We report a 19-year-old Vietnamese woman who experienced several life-threatening bleeding events, including ovarian hemorrhage. Blood analysis revealed a decreased fibrinogen level with markedly elevated fibrinogen/fibrin degradation products and D-dimer levels. Despite hemostatic surgery and administration of several medications, such as nafamostat mesylate, tranexamic acid, and unfractionated heparin, the coagulation abnormalities were not corrected, and the patient experienced repeated hemorrhagic events. We found that administration of recombinant human thrombomodulin (rhTM) remarkably improved the patient's pathophysiology. Screening and sequencing of the TM gene (THBD) revealed a previously unreported homozygous variation: c.793T>A (p.Cys265Ser). Notably, the Cys265 residue forms 1 of 3 disulfide bonds in the epidermal growth factor (EGF)-like domain 1 of TM. Transient expression experiments using COS-1 cells demonstrated markedly reduced expression of TM-Cys265Ser on the plasma membrane relative to wild-type TM. The TM-Cys265Ser mutant was intracellularly degraded, probably because of EGF-like domain 1 misfolding. The reduced expression of TM on the endothelial cell membrane may be responsible for the disseminated intravascular-coagulation-like symptoms observed in the patient. In summary, we identified a novel TM variant, c.793T>A (p.Cys265Ser). Patients homozygous for this variant may present with severe bleeding events; rhTM should be considered a possible treatment option for these patients.

Carrier frequencies of antithrombin, protein C, and protein S deficiency variants estimated using a public database and expression experiments.

BACKGROUND: Genetic deficiencies of antithrombin (AT), protein C (PC), and protein S (PS) are risk factors for venous thromboembolism. In the general population, the prevalence of heterozygous deficiency of AT, PC, and PS are reported as approximately 0.02%-0.2%, 0.2%-0.4%, and 0.03%-0.5%, respectively. The Exome Aggregation Consortium (ExAC) provides a public database containing reference data for over 60 000 exomes. OBJECTIVE: This study aimed to determine the frequency of AT, PC, and PS deficiencies using the ExAC database and transient expression experiments. METHODS: In total, 133, 157, and 221 variants of SERPIN1 (encoding AT), PROC (PC), and PROS1 (PS), respectively, were registered as missense and putative loss-of-function variants in the ExAC database. Variants with relatively high allele frequencies were selected and randomly sampled. Recombinant proteins were expressed in human embryo kidney 293 cells and their secretion and anticoagulant activities examined. RESULTS AND CONCLUSION: We assessed 9 AT, 4 PC, and 14 PS variants with relatively high allele frequencies and randomly sampled 12 AT, 15 PC, and 19 PS missense variants. All 21 AT variants showed normal or mildly reduced secretion, and 6 showed reduced total activity (specific activity × antigen level). Of the 19 PC variants, 11 showed impaired total activity. All 33 PS variants showed normal or mildly reduced secretion, and 4 showed reduced total activity. Based on allele frequencies in the ExAC database, we calculated the frequencies of AT, PC, and PS genetic deficiency as 0.36%, 0.63%, and 0.39%, respectively.

Real-world safety and efficacy data of ipilimumab in Japanese radically unresectable malignant melanoma patients: A postmarketing surveillance.

Treatment with immune checkpoint inhibitors has improved prognosis among patients with cutaneous melanoma, but there are still unmet medical needs in Japan, especially for mucosal melanoma and acral lentiginous melanoma (ALM) subtypes. Ipilimumab, a fully human monoclonal antibody that specifically blocks cytotoxic T-lymphocyte-associated antigen 4 and potentiates antitumor T-cell response, was approved in Japan in 2015 for the treatment of radically unresectable malignant melanoma. This postmarketing surveillance (prospective, non-interventional, multicenter, observational study) evaluated the safety (occurrence of adverse drug reactions [ADR]) and efficacy (overall survival [OS]) of ipilimumab in a real-world setting in Japan. All patients with radically unresectable malignant melanoma undergoing treatment with ipilimumab in Japan during the registration period between August 2015 and February 2017 were enrolled. In total, 547 patients were analyzed; 67.5% were 60 years old or more, 85.7% had an Eastern Cooperative Oncology Group performance status of 0-1, 50.3% had melanoma of the skin (mainly of the ALM subtype) and 73.5% had negative BRAF mutation status. Most patients had experienced recurrence and received multiple treatments. The overall incidence of ADR and serious ADR was 69.5% and 40.8%, respectively. The most common ADR and serious ADR were liver disorder, colitis and diarrhea. The most common ADR of special interest were liver-related ADR (22.5%), skin-related ADR (22.1%), gastrointestinal-related ADR (20.3%) and endocrine system-related ADR (16.3%). Most of these events had recovered or were in remission by the last evaluation. The median OS was 7.52 months (95% confidence interval, 6.47-8.74). Median OS was 6.31 and 8.44 months in patients with mucosal melanoma and melanoma of the skin; 9.43 and 3.75 months in patients with and without ADR; and 10.32 and 6.11 months in patients with and without serious ADR, respectively. Ipilimumab was tolerable and showed efficacy in improving OS for these patients.

Protein S K196E mutation reduces its cofactor activity for APC but not for TFPI.

BACKGROUND: Protein S (PS) is an anticoagulant molecule that functions as a cofactor for activated protein C (APC) in the inactivation of activated coagulation factors Va (FVa) and VIIIa. It also serves as a cofactor for tissue factor pathway inhibitor (TFPI) in the efficient inhibition of factor Xa (FXa). The Lys196-to-Glu (K196E, Tokushima) mutation in the EGF-2 domain of PS is a genetic risk factor for venous thromboembolism (VTE) in the Japanese population. OBJECTIVES: To investigate the molecular basis of the thrombophilic phenotype of Japanese patients carrying the PS K196E mutation. METHODS: We expressed recombinant human PS wild-type (PS-K) and K196E-mutant (PS-E) in CHO cells, and purified them by Ni2+-affinity and anion exchange column chromatography. We investigated the anticoagulant functions of PS-K and PS-E by measuring APC cofactor activity, TFPI cofactor activity, affinity for the ß chain of complement component C4b-binding protein (C4BP), and cleavage by thrombin. RESULTS: PS-E had approximately 40% APC cofactor activity compared with PS-K in a clotting-based assay and a FVa inactivation assay. The TFPI cofactor activity of PS-E in the FXa inactivation assay was equivalent to that of PS-K in the absence and presence of coagulation factor V. The strengths of PS-E and PS-K binding to the ß chain of C4BP were comparable, and both were equally cleaved by thrombin. CONCLUSIONS: The PS K196E mutation increases the risk of VTE because of reduced APC cofactor activity but does not alter various other properties, including the TFPI cofactor activity.

Thrombophilia in East Asian countries: are there any genetic differences in these countries?

In recent years, genetic analyses of congenital deficiencies of three anticoagulant proteins, antithrombin, protein C (PC) and protein S (PS), in East Asian patients with venous thromboembolism (VTE) have greatly increased. The PS-K196E mutation is often identified in the Japanese population with an allelic frequency of 0.86 %, and a total of approximately 10,000 Japanese are estimated to be homozygotes. The heterozygotes show PS anticoagulant activities ranging from 40 to 110 %, and 16 % lower mean anticoagulant activity than that in wild-type individuals. Specific assay methods to identify carriers of this mutation have recently been developed. The mutation carriers are at risk of thrombosis during pregnancy but do not appear to be at risk for adverse pregnancy outcomes. To promote future research into this mutation and its relation to thrombosis, a thrombosis-prone mouse strain with the PS K196E mutation has been developed. We found the PS-K196E mutation and the heterozygous PS-deficiency in mice caused increased VTE, but did not cause aggravation of ischemic stroke, unlike factor V Leiden mutation. Importantly, the PS-K196E mutation is only identified in Japanese. This suggests that although East Asian populations including Japanese, Chinese, and Koreans are geographically and genetically close, the PS-K196E mutation seems to be Japanese-specific, suggesting that the mutation is a recent occurrence and fixed within the Japanese population. Some recurrent genetic mutations predisposing to VTE have been reported in Chinese and Korean populations. Although the genetic background for VTE is known to differ between populations with Caucasian descent and East Asian populations, some of the recurrent mutations differ even within the East Asian populations.

ELISA-Based Detection System for Protein S K196E Mutation, a Genetic Risk Factor for Venous Thromboembolism.

Protein S (PS) acts as a cofactor for activated protein C in the plasma anticoagulant system. PS Lys196-to-Glu (K196E) mutation is a genetic risk factor for venous thromboembolism in Japanese individuals. Because of the substantial overlap in PS anticoagulant activity between KK (wild-type) and KE (heterozygous) genotypes, it is difficult to identify PS K196E carriers by measuring PS activity. Here, we generated monoclonal antibodies specific to the PS K196E mutant and developed a simple and reliable method for the identification of PS K196E carriers. We immunized mice with a keyhole limpet hemocyanin-conjugated synthetic peptide with Glu196. The hybridoma cells were screened for the binding ability of the produced antibodies to recombinant mutant EGF-like domains of PS (Ile117-Glu283). We obtained three hybridoma cell lines producing PS K196E mutation-specific antibodies. We established a sandwich enzyme-linked immunosorbent assay (ELISA) system in which the PS K196E mutation-specific monoclonal antibody was used as a detection antibody. We measured human plasma samples by using this system and successfully discriminated 11 individuals with the KE genotype from 122 individuals with the KK genotype. The ELISA system using the PS K196E mutation-specific antibody is a useful tool for the rapid identification of PS K196E carriers, who are at a higher risk for venous thromboembolism.

Prevalence of dysmenorrhea and its correlating lifestyle factors in Japanese female junior high school students.

Dysmenorrhea is a common menstrual disorder experienced by adolescents, and its major symptoms, including pain, adversely affect daily life and school performance. However, little epidemiologic evidence on dysmenorrhea in Japanese adolescents exists. This cross-sectional study aimed to determine the prevalence of and identify factors associated with dysmenorrhea in Japanese female junior high school students. Among 1,167 girls aged between 12 and 15 years, 1,018 participants completed a questionnaire that solicited information on age at menarche, menstruation, and lifestyle, as well as demographic characteristics. Dysmenorrhea was defined based on menstrual pain using a Visual Analog Scale (VAS), with moderate or severe (moderate-severe) dysmenorrhea, which adversely affects daily life, defined as VAS ≥ 4, and severe dysmenorrhea defined as VAS ≥ 7. The prevalence of moderate-severe dysmenorrhea was 476/1,018 (46.8%), and that of severe dysmenorrhea was 180/1,018 (17.7%). Higher chronological and gynecological ages (years after menarche) were significantly associated with a higher prevalence of dysmenorrhea regardless of severity (P for trend < 0.001). In addition, short sleeping hours (< 6/day) were associated with moderate-severe dysmenorrhea (OR = 3.05, 95%CI: 1.06-8.77), and sports activity levels were associated with severe dysmenorrhea (P for trend = 0.045). Our findings suggest that dysmenorrhea that adversely affects daily activities is highly prevalent, and may be associated with certain lifestyle factors in junior high school students. Health education teachers should be made aware of these facts, and appropriately care for those suffering from dysmenorrhea symptoms, absentees, and those experiencing difficulties in school life due to dysmenorrhea symptoms.

Fluvastatin Upregulates the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells.

AIM: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are cholesterol-lowering drugs with a variety of pleiotropic effects including antithrombotic properties. Tissue factor pathway inhibitor (TFPI), which is produced predominantly in endothelial cells and platelets, inhibits the initiating phase of clot formation. We investigated the effect of fluvastatin on TFPI expression in cultured endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with fluvastatin (0-10µM). The expression of TFPI mRNA and antigen were detected by RT-PCR and western blotting, respectively. The effects of mevalonate intermediates, small GTP-binding inhibitors, and signal transduction inhibitors were also evaluated to identify which pathway was involved. A luciferase reporter assay was performed to evaluate the effect of fluvastatin on TFPI transcription. The stability of TFPI mRNA was estimated by quantitating its levels after actinomycin D treatment. RESULTS: Fluvastatin increased TFPI mRNA expression and antigen in HUVECs. Fluvastatin-induced TFPI expression was reversed by co-treatment with mevalonate or geranylgeranylpyrophosphate (GGPP). NSC23766 and Y-27632 had no effect on TFPI expression. SB203580, GF109203, and LY294002 reduced fluvastatin-induced TFPI upregulation. Moreover, fluvastatin did not significantly affect TFPI promoter activity. TFPI mRNA degradation in the presence of actinomycin D was delayed by fluvastatin treatment. CONCLUSIONS: Fluvastatin increases endothelial TFPI expression through inhibition of mevalonate-, GGPP-, and Cdc42-dependent signaling pathways, and activation of the p38 MAPK, PI3K, and PKC pathways. This study revealed unknown mechanisms of the anticoagulant effect of statins and gave a new insight to its therapeutic potential for the prevention of thrombotic diseases.

Focal nerve enlargement is not the cause for increased distal motor latency in ALS: Sonographic evaluation.

OBJECTIVE: To elucidate the mechanism of focal conduction slowing in the median nerve in ALS. METHODS: The patients with ALS and CTS and normal control subjects were tested with sonography of the median and ulnar nerves. The cross-sectional areas (CSAs) and the wrist-forearm CSA ratios were compared with the parameters of nerve conduction study. RESULTS: The median motor distal latency was frequently prolonged in ALS and CTS. CSA and the wrist-forearm ratio of the median nerve were smaller in ALS than in CTS. The ulnar nerve sonography was similar in all the groups. CONCLUSIONS: Selective conduction slowing of the median nerve at the wrist in ALS is unlikely due to secondary compressive neuropathy, as seen in carpal tunnel syndrome. SIGNIFICANCE: Unique vulnerability of the median nerve in ALS may explain the selective conduction slowing.

Clinical and radiological findings of a cerebrotendinous xanthomatosis patient with a novel p.A335V mutation in the CYP27A1 gene.

We herein describe the case of a Japanese cerebrotendinous xanthomatosis (CTX) patient with a novel CYP27A1 gene mutation. The patient had been diagnosed with cataracts at 25 years of age and subsequently developed neurological symptoms in his forties, being referred to our hospital at 47 years of age. Upon admission, Achilles tendon xanthomas, cognitive impairment, dysphagia, dysarthria, dystonia, spasticity, muscle weakness and ataxia were observed. Brain MRI revealed abnormal signals in the dentate nuclei, periventricular white matter and pyramidal tract, and the serum cholestanol level was elevated. A CYP27A1 gene analysis identified compound heterozygosity for p.A335V, a novel mutation, and p.R405Q, a previously reported mutation. Making an early diagnosis of CTX is crucial, as the administration of chenodeoxycholic acid reverses metabolic derangement.

Antithrombin deficiency in three Japanese families: one novel and two reported point mutations in the antithrombin gene.

INTRODUCTION: Inherited antithrombin (AT) deficiency is associated with a predisposition to familial venous thromboembolic disease. We analyzed the AT gene in three unrelated patients with an AT deficiency who developed thrombosis. MATERIALS AND METHODS: We analyzed the SERPINC1 gene in three patients. Additionally, we expressed the three mutants in the COS-1 cells and compared their secretion rates and levels of AT activity with those of the wild-type (WT). RESULTS: We identified three distinct heterozygous mutations of c.2534C>T: p.56Arginine → Cysteine (R56C), c.13398C>A: p.459Alanine → Aspartic acid (A459D) and c.2703C>G: p.112 Proline → Arginine (P112R). In the in vitro expression experiments, the AT antigen levels in the conditioned media (CM) of the R56C mutant were nearly equal to those of WT. In contrast, the AT antigen levels in the CM of the A459D and P112R mutants were significantly decreased. The AT activity of R56C was decreased in association with a shorter incubation time in a FXa inhibition assay and a thrombin inhibition-based activity test. However, the AT activity of R56C was comparable to that of WT when the incubation time was increased. CONCLUSIONS: We concluded that the R56C mutant is responsible for type II HBS deficiency. We considered that the A459D and P112R mutants can be classified as belonging to the type I AT deficiency.

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells.

INTRODUCTION: Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects. MATERIALS AND METHODS: HUVECs were pretreated with 50 µM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 µM CORM-2 for 1 hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively. RESULTS: Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs. CONCLUSIONS: CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.

[Significant decrease in factor VII activity by tissue thromboplastin derived from rabbit brain in a patient with congenital factor VII deficiency (FVII Padua)].

Congenital factor VII (FVII) deficiency is a bleeding disorder that requires optimal hemostatic management for each case due to its wide variety of bleeding symptoms. We experienced a patient with inherited FVII deficiency who demonstrated different FVII activities depending on tissue thromboplastins used for assays. An 82-year-old woman without any episodes of abnormal bleeding was found to have different FVII activities of 1.4% and 32% when assayed using thromboplastins from rabbit brain and human placenta, respectively. DNA sequencing analysis revealed a homozygous missense mutation of G10828A (FVII Padua) that caused an amino acid substitution of Arg304 to Gln (R304Q). Carriers of 304Q alleles are usually clinically asymptomatic and do not require FVII replacement therapies even in cases of homozygotes. In case a prolonged prothrombin time or reduced FVII activity is detected, re-examination using thromboplastins of other sources can be helpful for preliminary diagnosis of R304Q, in order to prevent unnecessary FVII replacement therapies.