Moe Murata

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.

A novel prothrombin mutation in two families with prominent thrombophilia--the first cases of antithrombin resistance in a Caucasian population.

V. DJORDJEV IC ,* M. KOVAC,†‡ P . MIL J IC ,†§ M. MURATA, ¶ A. TAKAGI , ¶ I . PRUNER ,* D . FRANCUSK I , * T . KOJ IMA ¶ and D . RADOJKOVIC* *Institute of Molecular Genetics and Genetic Engineering, University of Belgrade; †Faculty of Medicine, University of Belgrade; ‡Hemostasis Department, Blood Transfusion Institute of Serbia; §Clinic of Hematology, University Clinical Center, Belgrade, Serbia; and ¶Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Japan

Molecular mechanisms of syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cells

Syndecan-4, a cell-surface heparan sulfate proteoglycan, can participate in inflammation and wound healing as a host defense molecule. Tumour necrosis factor (TNF)-α, one of the most potent proinflammatory cytokines, is known to upregulate syndecan-4 expression, but the precise mechanisms are unclear. To elucidate these mechanisms in detail, we examined syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cell. Of the two putative nuclear factor kappa-B (NF-κB) binding sites in the syndecan-4 gene (SDC4) promoter, deletion or mutation of one or both sites significantly diminished the effects of TNF-α. Electrophoretic mobility shift assays showed that p65 and c-Rel, but not p50, bound to these NF-κB binding sites, whereas pull-down assays showed binding of all three NF-κB components. Chromatin immunoprecipitation assays clearly showed that p65 and phosphorylated p65, but not p50 or c-Rel, bound to the SDC4 promoter. An NF-κB inhibitor, p65 knockdown and a transcriptional elongation inhibitor completely blocked the effect of TNF-α on SDC4 promoter activity and significantly, but not completely, blocked that on SDC4 mRNA expression. These data suggest that NF-κB p65 could be a key mediator of syndecan-4 upregulation by TNF-α through two binding sites in the SDC4 promoter, but other NF-κB-p65 independent pathways might also be involved through transcriptional elongation.

The first case of antithrombin-resistant prothrombin Belgrade mutation in Japanese.

Dear Editor, Antithrombin resistance (ATR) is a rare and novel hereditary risk factor of thrombophilia caused by prothrombin mutation. Prothrombin Yukuhashi mutation (c.1787G>T, p.Arg596Leu) found in Japanese thrombophilia pedigree resulted in mutant prothrombin with reduced clotting activity but resistant to inactivation by antithrombin [1–3]. Another variant, prothrombin Belgrade mutation (c.1787G>A, p.Arg596Gln) found in Serbia and India is also related to thrombophilia with the same mechanism [4, 5]. Here, we report the first Japanese family with ATR caused by prothrombin Belgrade mutation. The proband was a 22-year-old Japanese woman who had the first episode of DVT at the age of 15 years, followed by anticoagulant therapy with warfarin. Eight individuals of her maternal family members had one or more episodes of DVT (Fig. 1a). Interestingly, individuals in the third generation of this pedigree including this patient tended to have DVT in earlier age than those in the first and second generation. Protein S, protein C, and antithrombin deficiencies were not detected, and none of previous observations identified the causes of this hereditary thrombophilia. We conducted ATR kinetic assay [6] and F2 genome analysis and found that the relative residual thrombin activity after adding antithrombin of this patient was significantly higher than that of normally pooled plasma (Fig. 1b). These results indicated that thrombin formed from prothrombin of this patient was resistant to inactivation by antithrombin. Moreover, prothrombin Belgrade mutation was detected in this patient as well as in the other two family members (Fig. 1a, c). The mutation causes an Arg596-to-Gln replacement of prothrombin at one of antithrombin-binding sites of thrombin, resulting in impairment of thrombin inactivation by antithrombin [1, 3]. Her maternal aunt with this mutation has developed DVT at the age of 32, 40, and 47 years, although the mother of the patient had the mutation but no episodes of thromboembolism. The patient had DVT attack again even though she was warfarinized with PT-INR 2.5. We used heparin to treat DVT and warfarin to maintain PT-INR beyond 3.0. She had no bleeding episodes even if PT-INR elevated above 4. Neither pulmonary embolism by CT scanning nor pulmonary hypertension by cardiac catheterization was found; however, pulmonary perfusion defects were observed by ventilationperfusion lung scanning indicating small pulmonary embolism (Fig. 1d). ATR is a rare situation, and there are few data about optimal anticoagulant therapy. Only one report about effectiveness of anticoagulant therapy using high-dose LMWH for a pregnant woman with prothrombin Belgrade mutation was documented [7]. Diagnosis of unknown congenital risk factors of thromboembolism is challenging; however, ATR should be taken into consideration of a candidate for diagnosis. Whereas current conventional laboratory tests cannot detect ATR in the plasma, our novel laboratory test of ATR is useful even in warfarinized patients [6]. Further studies including more ATR patients * Mayuko Kishimoto mayutanne@med.nagoya-u.ac.jp

Development of a new laboratory test to evaluate antithrombin resistance in plasma

INTRODUCTION We recently reported a variant prothrombin (p.Arg596Leu: prothrombin Yukuhashi) that confers antithrombin resistance to patients with hereditary thrombosis. To detect antithrombin resistance in plasma, we devised a laboratory test analyzing the kinetics of thrombin inactivation using antithrombin. MATERIALS AND METHODS After incubation with prothrombin activator components (phospholipids, CaCl2, and snake venom), samples were treated with excess antithrombin in the presence or absence of heparin for various time periods. Subsequently, H-D-Phe-Pip-Arg-p-nitoranilide was added and changes in absorbance/min (ΔA/min) were measured at 405 nm. RESULTS After 1 min inactivation using antithrombin and heparin, the relative residual thrombin activity of recombinant mutant prothrombin (34.3% ± 2.2%) was higher than that of the wild-type (6.3% ± 1.2 %). After 30 min without heparin, the relative residual thrombin activity of recombinant mutant prothrombin (95.8% ± 0.4%) was higher than that of the wild-type (10.1% ± 1.7%), indicating that this assay could detect antithrombin resistance of the variant 596Leu prothrombin. Moreover, warfarinized plasmas from 2 heterozygous patients with prothrombin Yukuhashi mutation clearly showed higher values of the relative residual thrombin activity than those from 5 thrombosis patients lacking the mutation in the presence or absence of heparin. CONCLUSIONS We have devised a laboratory test to detect antithrombin resistance in plasma by analyzing the kinetics of thrombin inactivation using antithrombin. This assay may be useful for detecting antithrombin resistance in plasma, even in warfarinized patients.

SVA retrotransposition in exon 6 of the coagulation factor IX gene causing severe hemophilia B

Abstract Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities of the coagulation factor IX gene (F9). Insertion mutations in F9 ranging from a few to more than 100 base pairs account for only a few percent of all hemophilia B cases. We investigated F9 to elucidate genetic abnormalities causing severe hemophilia B in a Japanese subject. We performed PCR-mediated analysis of F9 and identified a large insertion in exon 6. Next, we carried out direct sequencing of a PCR clone of the whole insert using nested deletion by exonuclease III and S1 nuclease. We identified an approximately 2.5-kb SINE-VNTR-Alu (SVA)-F element flanked by 15-bp duplications in the antisense orientation in exon 6. Additionally, we carried out exontrap analysis to assess the effect of this retrotransposition on mRNA splicing. We observed that regular splicing at exons 5 and 6 of F9 was disturbed by the SVA retrotransposition, suggesting that abnormal FIX mRNA may be reduced by nonsense-mediated mRNA decay. In conclusion, this is the first report of SVA retrotransposition causing severe hemophilia B; only five cases of LINE-1 or Alu retrotranspositions in F9 have been reported previously.

Antithrombin-resistant prothrombin Yukuhashi mutation also causes thrombomodulin resistance in fibrinogen clotting but not in protein C activation

INTRODUCTION Prothrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)-protein C (PC) anticoagulant system have not been previously elucidated. MATERIALS AND METHODS We prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM. RESULT AND CONCLUSIONS rTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

High prophylactic LMWH dose successfully suppressed hemostatic activation in pregnant woman with a new prothrombin c.1787G > A mutation

Recently Miyawaki and coauthors identified a new prothrombin mutation, F2 c.1787G N T, in a Japanese family with juvenile thrombosis. The mutation led to impaired inhibition of mutant thrombin (p.Arg596Leu) by antithrombin, resulting in antithrombin resistance and an increased risk of thrombophilia [1]. The same mechanism was found in our patient characterized by a different change in thrombin (p.Arg596Gln) called the Belgrademutation [2]. Thiswas thefirst documented prothrombinmutation (F2 c.1787G N A) causing antithrombin resistance in a Caucasian. While the association of the prothrombin G20210A mutation with the risk of venous thrombosis and/or pregnancy complications is well documented [3–6], the role of the newly discovered F2 c.1787G N Amutation is still unexplored. Since there is no data about anticoagulant therapy during pregnancy in cases of F2 c.1787G N Amutation, we describe the course of pregnancy in a woman heterozygous for this mutation. Considering that thrombin generation (ETP) quantifies the amount of thrombin that can potentially be generated upon activation of the coagulation system, whereas D-dimer is a measure of ongoing thrombin generation [7] we used these tests, in order to clarify the effect of anticoagulant treatment during pregnancy in a heterozygous carrier of F2 c.1787G N Amutation. Our proband is the youngest member of the Serbian family, where the new prothrombin mutation was detected [2]. She developed her first deep venous thrombosis complicated with pulmonary embolism (PE) at the age of 31. She had been receiving hormonal therapy for 5 years for treatment of early breast cancer and the first thrombosis developed shortly after therapy ceased. Her two older sisters, who are also carriers of F2 c.1787G N A, already had recurrent thrombosis complicated with PE, while her mother died after the third delivery due to massive PE. Her oldest sister had three recurrent pregnancy losses; the last two upon anticoagulant therapy, while the third miscarriage was complicated with PE. We considered genetically related factor in our attempt to explain the background of severe thrombotic onsets at such a young age. Molecular testing showed the F2 c.1787G N A mutation, located in the last exon of the prothrombin gene resulting in the Arg596Gln replacement (prothrombin Belgrade) [2]. Plasma tests in accordance with previously published methods [8] revealed that she had antithrombin resistance diathesis (Supporting Information File). The proband was subjected to long-term anticoagulant therapy, originally heparin followed by warfarin that was used for about one year. In consultation with a hematologist, she was transferred to prophylactic LMWH before conception. LMWH dose was determined based on the body weight of 55 kg (nadroparin 5700 IU daily), adjusted related to anti-Xa results performedmonthly during the pregnancy. The

A complex genomic abnormality found in a patient with antithrombin deficiency and autoimmune disease-like symptoms

Hereditary antithrombin (AT) deficiency is an autosomal dominant thrombophilic disorder caused by SERPINC1 abnormality. In the present study, we analyzed SERPINC1 in a Japanese patient with AT deficiency and autoimmune disease-like symptoms. Direct sequencing and multiplex ligation-dependent probe amplification revealed that the patient was hemizygous for the entire SERPINC1 deletion. Single nucleotide polymorphism genotyping, gene dose measurement, and long-range polymerase chain reaction (PCR) followed by mapping PCR and direct sequencing of the long-range PCR products revealed that the patient had an approximately 111-kb gene deletion from exon 2 of ZBTB37 to intron 5 of RC3H1, including the entire SERPINC1 in chromosome 1. We also found a 7-bp insertion of an unknown origin in the breakpoint, which may be a combination of three parts with a few base-pair microhomologies, resulting from a replication-based process known as ‘fork stalling and template switching’. Because RC3H1, which encodes the protein roquin is involved in the repression of self-immune responses, the autoimmune disease-like symptoms of the patient may have resulted from this gene defect. In conclusion, we identified an entire SERPINC1 deletion together with a large deletion of RC3H1 in an AT-deficient patient with autoimmune disease-like symptoms.