Yuki Takagi

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.

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.

Clinical and biochemical characterization of the prothrombin Belgrade mutation in a large Serbian pedigree: new insights into the antithrombin resistance mechanism

Essentials Prothrombin Belgrade mutation leads to antithrombin resistance. Clinical and biochemical phenotypes in a large family with this mutation were investigated. In carriers, we detected decreased factor II activity and increased endogenous thrombin potential. Prothrombin Belgrade mutation represents a strong prothrombotic risk factor.

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.

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.

In vitro exploration of latent prothrombin mutants conveying antithrombin resistance

INTRODUCTION Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). MATERIALS AND METHODS We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. RESULTS The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). CONCLUSIONS Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.

Progestin isoforms provide different levels of protein S expression in HepG2 cells

INTRODUCTION Use of combined oral contraceptives (COCs) results in acquired protein S (PS) deficiency, a well-established risk factor for venous thromboembolism (VTE). The risk of VTE due to COCs containing newer-generation progestins is double compared with COCs containing older-generation progestins, although there is little difference in estrogen contents between the generations. In contrast, progestin-only contraceptives do not confer an increased risk of VTE. In this study, we aimed to investigate how different isoforms of progestin in COCs affect the risk of VTE by measuring PS expression. MATERIALS AND METHODS The effect of progestin, levonorgestrel (LNG) or drospirenone (DRSP), on PS mRNA expression in HepG2 cells was measured using reverse transcription-quantitative PCR; PS level was determined using Western blot analysis. PROS1 promoter activity, PS mRNA stability, and de novo synthesis of PS mRNA were examined in HepG2 cells after treatment with progestin. RESULTS AND CONCLUSIONS In the presence of progestins, PS mRNA and protein expressions were significantly upregulated in HepG2 cells due to the augmentation of de novo PS mRNA expression modulated by RNA polymerase II (Pol II), thereby facilitating PS transcription elongation. Moreover, the transcription elongation inhibitor blocked progestin-mediated de novo PS mRNA expression. Conversely, progestin did not affect PROS1 promoter activity and PS mRNA stability. Pol II elongation efficiency in the newer-generation progestin (DRSP) treatment was not as strong compared with older-generation progestin (LNG), suggesting the difference in VTE risk between COC generations.

Distinct X chromosomal rearrangements in four haemophilia B patients with entire F9 deletion

Haemophilia B is an X‐linked bleeding disorder caused by a coagulation factor IX gene (F9) abnormality. Numerous F9 defects have been identified to date; however, only a few with an entire F9 deletion have been reported in detail.