Publications by authors named "Toshiro Shibano"

22 Publications

  • Page 1 of 1

Impact of nonsynonymous mutations of factor X on the functions of factor X and anticoagulant activity of edoxaban.

Blood Coagul Fibrinolysis 2015 Mar;26(2):117-22

aThe Keio-Daiichi Project on Genetics of Thrombosis, Keio University bBiological Research Laboratories cNew Drug Regulatory Affairs Department dResearch Oversight Function, R&D Division, Daiichi Sankyo Co., Ltd eLaboratory Medicine, Keio University School of Medicine, Tokyo, Japan.

Edoxaban is an oral direct factor Xa (FXa) inhibitor and its efficacy as an oral anticoagulant is less subject to drug-food and drug-drug interaction than existing vitamin K antagonists. Although this profile of edoxaban suggests it is well suited for clinical use, it is not clear whether genetic variations of factor X influence the activity of edoxaban. Our aim was to investigate a possible impact of single-nucleotide polymorphisms (SNPs) in the factor X gene on the functions of factor X and the activity of edoxaban. Two nonsynonymous SNPs within mature factor X, Ala152Thr and Gly192Arg, were selected as possible candidates that might affect the functions of FXa and the activity of edoxaban. We measured catalytic activities of wild type and mutant FXas in a chromogenic assay using S-2222 and coagulation times including prothrombin time (PT) and activated partial thrombin time (aPTT) of plasma-containing recombinant FXs in the presence and absence of edoxaban. Michaelis-Menten kinetic parameters of FXas, Km and Vmax values, PT and aPTT were not influenced by either mutation indicating these mutations do not affect the FXa catalytic and coagulation activities. The Ki values of edoxaban for the FXas and the concentrations of edoxaban required to double PT and aPTT were not different between wild type and mutated FXas indicating that both mutations have little impact on the activity of edoxaban. In conclusion, these data suggest that edoxaban has little interpatient variability stemming from SNPs in the factor X gene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MBC.0000000000000147DOI Listing
March 2015

Comparison of the effect of edoxaban, a direct factor Xa inhibitor, with a direct thrombin inhibitor, melagatran, and heparin on intracerebral hemorrhage induced by collagenase in rats.

Thromb Res 2014 Apr 6;133(4):622-8. Epub 2013 Aug 6.

R&D Planning Department, R&D Division, Daiichi Sankyo Co., Ltd., Japan.

Introduction: Intracerebral hemorrhage (ICH) is a major clinical concern with anticoagulation therapy. The effect of a new oral direct FXa inhibitor, edoxaban, was determined in a rat model of ICH and compared with a direct thrombin inhibitor, melagatran, and heparin.

Methods: To induce ICH, 0.1 U collagenase type VII was injected into the striatum of male Wistar rats under anesthesia with thiopental or halothane. Immediately after ICH induction, edoxaban, melagatran, or heparin were infused intravenously. Five hours after ICH induction, the brain was removed and ICH size was measured. To estimate the margin of safety, antithrombotic effects were evaluated in a rat venous thrombosis model.

Results: Edoxaban at 6mg/kg/h significantly increased ICH volume (1.8-fold) and prolonged prothrombin time (PT) 2.8-fold compared to the vehicle group. No deaths were observed with edoxaban. Melagatran at 1mg/kg/h increased ICH volume at 1mg/kg/h (2.8-fold) with 6.1-fold PT prolongation. At 3mg/kg/h, all rats died due to severe ICH (3.9-fold). Heparin at both 100 and 500U/kg/h significantly increased ICH. At 500U/kg/h, 5 out of 8 rats died. The doses required for 50% inhibition of thrombosis of edoxaban, melagatran, and heparin were 0.045mg/kg/h, 0.14mg/kg/h, and 55U/kg/h, respectively. The safety margins between antithrombotic and ICH exacerbation effects of these anticoagulants were 133, 7.1, and 1.8, respectively.

Conclusion: The safety margin of edoxaban was wider than that of melagatran or heparin. These results suggest that edoxaban may be preferable from the perspective of ICH exacerbation risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2013.07.015DOI Listing
April 2014

Comparison of antithrombotic and hemorrhagic effects of edoxaban, a novel factor Xa inhibitor, with unfractionated heparin, dalteparin, lepirudin and warfarin in rats.

Thromb Res 2013 Aug 12;132(2):234-9. Epub 2013 Jun 12.

Biological Research Laboratories, R & D Division, Daiichi Sankyo Co., Ltd. Electronic address:

Background: Edoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants.

Objectives: To compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage.

Methods: Rats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail.

Results: In rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose-response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin.

Conclusions: These results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2013.05.020DOI Listing
August 2013

Impact of antithrombin deficiency on efficacy of edoxaban and antithrombin-dependent anticoagulants, fondaparinux, enoxaparin, and heparin.

Thromb Res 2013 Jun 11;131(6):540-6. Epub 2013 May 11.

Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.

Introduction: Oral factor Xa (FXa) inhibitors are a novel class of anticoagulants that, unlike heparins, are expected to demonstrate antithrombotic effects independent of plasma antithrombin (AT) concentrations. We utilized heterozygous AT-deficient (AT+/-) mice to determine the impact of AT deficiency on anticoagulant and antithrombotic effects of edoxaban, a direct FXa inhibitor, and compared with heparins (fondaparinux, enoxaparin, and unfractionated heparin [UHF]).

Materials And Methods: The effects of edoxaban and heparins on in vitro prothrombin time and activated partial thromboplastin time were measured in plasma obtained from wild type (AT+/+) and AT+/- male mice. To assess the antithrombotic effects of these anticoagulants in vivo, venous thrombosis was induced in the inferior vena cava by FeCl3 treatment. Potency ratios of antithrombotic effects in AT+/- compared with AT+/+ mice were analyzed by a parallel line assay.

Results: In vitro studies demonstrated that the clotting-time prolongation effects of edoxaban were not affected by heterozygous AT deficiency whereas those of AT-dependent anticoagulants were attenuated. In AT+/- mice, the antithrombotic effects of AT-dependent anticoagulants were less potent than those in AT+/+ mice. In contrast, edoxaban was equipotent in preventing thrombus formation in both wild-type and AT-deficient mice. The attenuated antithrombotic effects of fondaparinux, enoxaparin, and UFH in AT-deficient mice were restored by AT supplementation. Edoxaban exerts a comparable antithrombotic effect even in mice with low plasma AT antigen and activity to that in wild-type mice.

Conclusion: Edoxaban may potentially be prioritized over AT-dependent anticoagulants in patients with lower plasma AT concentration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2013.04.016DOI Listing
June 2013

The effects of warfarin and edoxaban, an oral direct factor Xa inhibitor, on gammacarboxylated (Gla-osteocalcin) and undercarboxylated osteocalcin (uc-osteocalcin) in rats.

Thromb Res 2013 Jan 19;131(1):59-63. Epub 2012 Sep 19.

Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.

Introduction: Osteocalcin plays a role in bone homeostasis. The vitamin K cycle is essential for the gamma-carboxylation of glutamic acid residues in osteocalcin. Some evidence suggests that long-term warfarin therapy, which inhibits the vitamin K cycle and prevents gamma-carboxylation, is associated with increased bone-fracture risk. The aim of this study was to determine the effects of warfarin and edoxaban, a direct factor Xa inhibitor, on the serum concentration of total, gamma-carboxylated (Gla-osteocalcin) and undercarboxylated osteocalcin (uc-osteocalcin) in rats.

Materials And Methods: Rats received orally administered warfarin or edoxaban, and 24h later serum and plasma were prepared. Osteocalcin level in serum was measured with ELISA. A Gla-osteocalcin was precipitated by the addition of hydroxyapatite, and the resulting supernatant was used for measuring uc-osteocalcin. Prothrombin time (PT) of plasma was also measured.

Results: Warfarin at 1mg/kg (a dose which prolonged PT 2.62-fold) markedly increased the serum level of uc-osteocalcin and slightly increased the total osteocalcin level compared with control in rats. Serum Gla-osteocalcin significantly decreased by warfarin. Edoxaban at 1mg/kg (an antithrombotic dose) and 54mg/kg (a dose which prolonged PT 2.25-fold) had no effects on total, uc-, and Gla-osteocalcin levels.

Conclusions: This study demonstrates that warfarin impaired the carboxylation of osteocalcin in rats. In contrast, edoxaban at or higher doses than needed for an antithrombotic effect sustained the circulating Gla-osteocalcin level. These findings suggest that edoxaban has no effects on the production of Gla-osteocalcin and thus, may have a lower risk of adverse effects on bone health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2012.08.304DOI Listing
January 2013

Comparison of antithrombotic and haemorrhagic effects of edoxaban, an oral direct factor Xa inhibitor, with warfarin and enoxaparin in rats.

Thromb Res 2012 Sep 29;130(3):514-9. Epub 2012 May 29.

Biological Research Laboratories, R & D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan.

Introduction: Factor Xa (FXa) is a key serine protease in the coagulation cascade and a promising target for a new antithrombotic agent. Edoxaban is an oral, selective and direct FXa inhibitor. The objective of this study was to compare the antithrombotic and haemorrhagic effects of edoxaban with clinically available anticoagulants, warfarin and enoxaparin, in rat models of thrombosis and haemorrhage.

Methods: Rats were treated with single oral administration of edoxaban, repeated oral dosing of warfarin for 4 days and single subcutaneous administration of enoxaparin before thrombosis or haemorrhage induction. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava for 60 min. Tail template bleeding time was measured after making an incision on the tail.

Results: Edoxaban at 0.3, 1 and 3mg/kg exerted dose-dependent and significant inhibition of venous thrombus formation. The 50% thrombus inhibition dose (ED(50)) was 1.9 mg/kg. At supra-therapeutic doses (10 and 20mg/kg), edoxaban significantly but moderately (less than 2-fold) prolonged bleeding time. Warfarin and enoxaparin also dose-dependently inhibited venous thrombosis and prolonged bleeding time. The ED(50) values of warfarin and enoxaparin were 0.12 mg/kg and 500 IU/kg, and the 2-fold bleeding time prolongation doses (BT2) were 0.16 mg/kg and 1700 IU/kg, respectively. The safety margin (ratio of BT2 to ED(50)) of edoxaban (>10.5) was greater than those of warfarin (1.3) and enoxaparin (3.4).

Conclusions: Edoxaban inhibited venous thrombosis comparably to warfarin and enoxaparin, and the attendant bleeding risk of edoxaban was lower than that of warfarin and enoxaparin in rats.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2012.05.008DOI Listing
September 2012

Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats.

Eur J Pharmacol 2012 Jul 21;686(1-3):74-80. Epub 2012 Apr 21.

Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo, Japan.

There are concerns that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. We have shown that low-dose administration of a direct thrombin inhibitor, melagatran, significantly worsens the coagulation status induced by tissue factor injection in rats. We compared the effect of inhibition of thrombin and factor Xa for their potential to aggravate tissue factor-induced coagulation in rats. Hypercoagulation was induced by the injection of 2.8 U/kg tissue factor after administration of melagatran, heparin and edoxaban in rats. Blood samples were collected 10min after tissue factor injection. Platelet numbers, thrombin-antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1mg/kg, i.v.) suppressed platelet consumption and thrombin-antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1mg/kg, i.v.) significantly enhanced platelet consumption and thrombin-antithrombin complex generation. In addition, although melagatran (3mg/kg, i.v.) improved coagulation status when tissue factor was given 5min after the drug administration, and 2, 4 and 8h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15-234ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190ng/ml or more) of the compound. In contrast, edoxaban and heparin did not show any exacerbation under these examination conditions. These results show that subtherapeutic concentrations of melagatran are associated with coagulation pathway activation, whereas factor Xa inhibition with edoxaban has a low risk of paradoxical hypercoagulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejphar.2012.04.031DOI Listing
July 2012

Reversal of anticoagulant effects of edoxaban, an oral, direct factor Xa inhibitor, with haemostatic agents.

Thromb Haemost 2012 Feb 21;107(2):253-9. Epub 2011 Dec 21.

Daiichi Sankyo Co., Ltd. – Biological Research Laboratories, Tokyo, Japan.

Edoxaban, an oral, direct factor Xa inhibitor, has a similar or low incidence of bleeding events compared with other anticoagulants in clinical trials. Therefore, agents to reverse the anticoagulant effects of edoxaban could be desirable in emergency situations. In this study, the reversal effects of haemostatic agents were determined on prothrombin time (PT) prolongation in vitro and bleeding time prolongation in vivo by edoxaban. PT using human plasma was measured in the presence of edoxaban at therapeutic and excess concentrations with the haemostatic agents, prothrombin complex concentrate (PPSB-HT), activated prothrombin complex concentrate (Feiba), and recombinant factor VIIa (rFVIIa). In rats, rFVIIa and Feiba was given during intensive anticoagulation with edoxaban. The haemostatic effect was evaluated in a model of planta template bleeding and a potential prothrombotic effect was evaluated in a venous thrombosis model. PPSB-HT, Feiba, and rFVIIa concentration-dependently shortened PT prolonged by edoxaban. Among these, rFVIIa and Feiba showed potent activities in reversing the PT prolongation by edoxaban. rFVIIa (1 and 3 mg/kg, i.v.) and Feiba (100 U/kg, i.v.) significantly reversed edoxaban (1 mg/kg/h)-induced prolongation of bleeding time in rats. In a rat venous thrombosis model, no potentiation of thrombus formation was observed when the highest dose (3 mg/kg) of rFVIIa was added to edoxaban (0.3 and 1 mg/kg/h) compared with the control. The present study indicated that rFVIIa, Feiba, and PPSB-HT have the potential to be reversal agents for edoxaban.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1160/TH11-09-0668DOI Listing
February 2012

Antithrombin-independent thrombin inhibitors, but not direct factor Xa inhibitors, enhance thrombin generation in plasma through inhibition of thrombin-thrombomodulin-protein C system.

Thromb Haemost 2011 Dec 20;106(6):1076-83. Epub 2011 Oct 20.

Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.

There is increasing concern that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. Previously, we demonstrated that at certain doses a direct thrombin inhibitor, melagatran, worsens the coagulation status induced by tissue factor (TF) injection in a rat model. We utilised an in vitro thrombin generation (TG) assay to determine if direct thrombin inhibitors could enhance TG in human plasma, and whether inhibition of the negative-feedback system [thrombin-thrombomodulin (TM)-protein C] contributed to the TG enhancement. TG in human plasma was assayed by means of the calibrated automated thrombography. In this assay, direct factor Xa (FXa) inhibitors such as edoxaban and antithrombin (AT)-dependent anticoagulants such as heparin did not increase, but simply suppressed TG. AT-independent thrombin inhibitors (melagatran, lepirudin, and active site blocked thrombin (IIai)) increased peak levels of TG (2.0, 1.6, and 2.2-fold, respectively) in the presence of 12 nM recombinant human soluble TM (rhsTM). Melagatran and lepirudin at higher concentrations began to suppress TG. In the absence of rhsTM, the enhancement of peak TG by melagatran decreased to 1.2-fold. Furthermore, in protein C-deficient plasma, AT-independent thrombin inhibitors failed to enhance TG. In addition, a human protein C neutralising antibody increased the peak height of TG in the presence of rhsTM. These results suggest that AT-independent thrombin inhibitors may activate thrombogenesis by suppression of the thrombin-induced negative-feedback system through inhibition of protein C activation. In contrast, direct FXa inhibitors are more useful than AT-independent thrombin inhibitors in terms of lower possibility of activation of the coagulation pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1160/TH11-06-0382DOI Listing
December 2011

Comparison of antithrombotic efficacy between edoxaban, a direct factor Xa inhibitor, and fondaparinux, an indirect factor Xa inhibitor under low and high shear rates.

Thromb Haemost 2011 Dec 22;106(6):1062-8. Epub 2011 Sep 22.

Biological Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.

Edoxaban is an oral, direct factor Xa (FXa) inhibitor under late-phase clinical development. This study compared the antithrombotic efficacy of edoxaban with that of an indirect FXa inhibitor, fondaparinux, in in vivo venous and arterial thrombosis models and in ex vivo perfusion chamber thrombosis model under low and high shear rates in rats. Venous and arterial thrombi were induced by platinum wire insertion into the inferior vena cava and by application of FeCl₃ to the carotid artery, respectively. The perfusion chamber thrombus was formed by blood perfusion into a collagen-coated capillary at 150 s⁻¹ (low shear rate) and 1,600 s⁻¹ (high shear rate). Effective doses of edoxaban that reduced thrombus formation by 50% (ED₅₀) in venous and arterial thrombosis models were 0.076 and 0.093 mg/kg/h, respectively. In contrast, ED₅₀ of fondaparinux in the arterial thrombosis model (>10 mg/kg/h) was markedly higher compared to ED₅₀ in the venous thrombosis model (0.021 mg/kg/h). In the perfusion chamber thrombosis model, the ratio of ED₅₀ under high shear rate (1.13 mg/kg/h) to that under low shear rate (0.63 mg/kg/h) for edoxaban was 1.9, whereas that for fondaparinux was more than 66. While the efficacy of fondaparinux markedly decreased in arterial thrombosis and in a high-shear state, edoxaban exerted consistent antithrombotic effects regardless of flow conditions. These results suggest that shear rate is a key factor in different antithrombotic effects between edoxaban and fondaparinux.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1160/TH11-07-0451DOI Listing
December 2011

GSK-3β negatively regulates megakaryocyte differentiation and platelet production from primary human bone marrow cells in vitro.

Platelets 2011 13;22(3):196-203. Epub 2011 Jan 13.

The Keio-Daiichi Sankyo Project on Genetics of Thrombosis, Keio University School of Medicine, Tokyo, Japan.

Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the Wnt, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3β. GSK-3β is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for 12 days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/-) of TWS119, a GSK-3β inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (-). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3β on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (-) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/-) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (-). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/-), and platelet production was markedly decreased in TPO (-). This indicated that the GSK-3β inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3β inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (-) cells cultured in TWS119 (+/-) for 3 days. The results of the analysis indicated that GSK-3β inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the Wnt signaling and transforming growth factor-β signaling pathways. These observations suggest that GSK-3β inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/09537104.2010.541959DOI Listing
September 2011

Alpha 2A adrenergic receptor polymorphism is associated with plasma von Willebrand factor levels in a general population.

Blood Coagul Fibrinolysis 2008 Jul;19(5):395-9

The Keio-Daiichi Sankyo Project on Genetics of Thrombosis, Keio University, Tokyo, Japan.

High levels of plasma von Willebrand factor are a proposed risk factor for atherothrombotic disorders. Previously, ABO blood type and the von Willebrand factor -1793C/G polymorphism were shown to affect interindividual variations in plasma von Willebrand factor levels. We previously reported that polymorphisms of the alpha 2A adrenergic receptor, an epinephrine receptor, were associated with platelet function as assessed by platelet function analyzer-100. The measurement value of platelet function analyzer-100 has been reportedly associated with plasma von Willebrand factor levels. Also, it was demonstrated that epinephrine administration increases plasma von Willebrand factor levels in vivo. Thus, the present study investigated the association between plasma von Willebrand factor levels and genetic polymorphisms as follows: ABO blood type, von Willebrand factor -1051G/A (linked with -1793C/G), alpha 2A adrenergic receptor 1780A/G, and alpha 2A adrenergic receptor 2372A/G. Study subjects were genetically unrelated Japanese men (n = 277) recruited at their regular medical examinations. Genotyping of the polymorphisms was performed using the single nucleotide primer extension-based method. Plasma von Willebrand factor levels were measured as ristocetin-cofactor activities. The O blood type and alpha 2A adrenergic receptor 2372AA genotype were significantly associated with lower von Willebrand factor levels, though von Willebrand factor -1051G/A polymorphism did not affect them. In stratification analysis of the group according to blood type O and non-O, the significant association between the 2372AA genotype and lower plasma von Willebrand factor levels was observed in non-O subjects, but not O subjects. In conclusion, the alpha 2A adrenergic receptor 2372A/G polymorphism is associated with plasma von Willebrand factor levels in a general population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MBC.0b013e3283001d38DOI Listing
July 2008

Platelet responsiveness to in vitro aspirin is independent of COX-1 and COX-2 protein levels and polymorphisms.

Thromb Res 2008 13;121(4):509-17. Epub 2007 Jul 13.

The Keio-Daiichi Project on Genetics of Thrombosis, Keio University, Tokyo 160-8582, Japan.

Aspirin's inhibitory effect on platelet function has been shown to be highly heterogeneous. However, due to the considerable individual variation in pharmacokinetics after aspirin intake, it has been difficult to investigate the mechanism of aspirin resistance empirically. Our objective was to examine whether platelet responsiveness to in vitro aspirin treatment could be affected by cyclooxygenase (COX)-1/2 protein levels in platelets or single-nucleotide polymorphisms (SNPs), which could possibly change specific activity of enzymes and/or aspirin susceptibility. Collagen/epinephrine closure time (CEPI-CT) of PFA-100 in blood from 178 healthy males was assessed with/without aspirin. Platelet COX-1 protein levels and the sequences of COX-1 gene exons were examined in three groups categorized by CEPI-CT: PR (Poor responders to aspirin), 10 people showing the shortest CEPI-CT under aspirin; GR-High or GR-Low (good responders to aspirin with high or low platelet basal reactivity), 10 people showing CEPI-CT over 300 s under aspirin and having the shortest or longest basal CEPI-CT, respectively. We analyzed the three groups, representing phenotypic extremes, aiming to increase statistical power to investigate the possible relevance of COXs to platelet response to aspirin. Western blot analysis revealed that COX-1 was abundantly expressed in platelets at comparable levels among the three groups, whereas COX-2 was undetectable. The frequencies of nonsynonymous COX-1/2 SNPs were unlikely to explain the difference in aspirin responsiveness considering the observed genotype frequencies and wide individual variation in platelet response. These results suggest that heterogeneity in platelet responsiveness to in vitro aspirin is independent of COX-1/2 protein levels and SNPs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2007.05.017DOI Listing
April 2008

Identification of ADRA2A polymorphisms related to shear-mediated platelet function.

Biochem Biophys Res Commun 2006 Sep 10;347(4):1001-5. Epub 2006 Jul 10.

The Keio-Daiichi Project on Genetics of Thrombosis, Keio University, Tokyo, Japan.

alpha2A adrenergic receptor (ADRA2A) on platelets interacts with epinephrine, which has a key role in regulating platelet functions. There is familial clustering of inter-individual variations in the epinephrine-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. In this study, we screened the sequence variations in the transcriptional region of ADRA2A gene and analyzed the relationship between the two common polymorphisms and platelet function using epinephrine/collagen cartridge in the platelet function analyzer-100 system, in a healthy Japanese male population (n=211). Among the identified 16 sequence variations including five novel variations, 1780GG genotype was associated with longer closure time which represents low platelet function under high shear-stress conditions (p=0.0478). We also observed enhanced effect of the combination of 1780GG and 2372AA genotypes on longer closure time (p=0.0319). These findings suggest that 1780A/G and 2372A/G polymorphisms are associated with platelet function in interactions with collagen/epinephrine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2006.06.180DOI Listing
September 2006

Increased basal platelet activity, plasma adiponectin levels, and diabetes mellitus are associated with poor platelet responsiveness to in vitro effect of aspirin.

Thromb Res 2007 21;119(4):517-24. Epub 2006 Jun 21.

The Keio Daiichi Project on Genetics of Thrombosis, Keio University, Tokyo, Japan.

Introduction: Aspirin is one of the most effective antiplatelet agents and is now commonly used to prevent vascular events. In some patients, however, recurrent vascular events have been demonstrated despite aspirin therapy. Our objective was to characterize individuals showing poor response to in vitro effect of aspirin, using PFA-100.

Methods: One hundred sixty-eight healthy male subjects were analyzed. We assessed platelet function tests, including PFA-100, whole blood aggregation, and optical platelet aggregation. Also measured were hemostatic and other parameters including von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), soluble vascular adhesion molecule-1 (sVCAM-1), high sensitive C-reactive protein (hs-CRP), and adiponectin. Poor responders were defined as having a collagen/epinephrine-induced closure time (CEPI-CT) under 250 s with PFA-100 when incubated with 10 microM aspirin, whereas good responders were defined as having a CEPI-CT of more than 250 s.

Results And Conclusions: PFA-100 tests revealed that 40 subjects (24%) were poor responders (PR) and 128 (76%) were good responders (GR). Poor responsiveness was significantly associated with (1) higher basal platelet activities in PFA-100, as well as in whole blood aggregation and aggregometer;(2) increased level of adiponectin (8.8+/-4.1 micro g/mL [PR] vs 7.3+/-2.9 micro g/mL [GR], p=0.010);and (3) the presence of diabetes mellitus (17.5% [PR] vs 4.7% [GR], p=0.009). Importantly, whereas 24% of the subjects showed insufficient inhibition in PFA-100 when incubated with 10 microM aspirin, almost all subjects showed maximum inhibition with 30 microM aspirin. These observations suggest that higher doses of aspirin might overcome aspirin resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2006.04.004DOI Listing
April 2007

Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions.

Br J Haematol 2005 Feb;128(4):533-9

Department of Medicine, Keio University, Tokyo 160-8582, Japan.

Interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case-control studies indicated that two GPIb alpha polymorphisms, (145)Thr/Met and/or variable number (1-4) tandem repeats of 13 amino-acid sequences, are associated with arterial thrombosis. The (145)Met-allele and the 3R- or 4R-allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIb alpha fragments and tested them in vitro. The dissociation constants of ristocetin-induced (125)I-labelled VWF binding to two forms of soluble recombinant GPIb alpha [(1)His-(302)Ala, either (145)Thr (145T) or (145)Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full-length GPIb alpha beta/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized-VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2141.2004.05349.xDOI Listing
February 2005

Identification of novel mutations in ADAMTS13 in an adult patient with congenital thrombotic thrombocytopenic purpura.

Blood 2004 Oct 4;104(7):2081-3. Epub 2004 May 4.

New Product Laboratories II, Daiichi Pharmaceutical, Tokyo, Japan.

Congenital thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is associated with an inherited von Willebrand factor-cleaving protease (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type I domains 13]) deficiency. In this study, we identified novel mutations in the ADAMTS13 gene in a patient with TTP. The patient was a 51-year-old Japanese male who exhibited TTP symptoms at frequent intervals. The ADAMTS13 activity during acute episodes was less than 3% that of normal. The enzyme activities of the patient's father and mother were both 46%, and both parents were asymptomatic. Genetic analysis revealed that the patient was a compound heterozygote for 2 mutations. One mutation was a missense mutation in the metalloprotease domain (A250V, exon 7), and the other was a guanine to adenine substitution at the 5' end of intron 3 (intron 3 G-->A). In vitro expression studies revealed that the A250V mutation markedly reduced ADAMTS13 activity and the intron 3 G-->A mutation caused abnormal mRNA synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2004-02-0715DOI Listing
October 2004

Detection of von Willebrand factor-cleaving protease (ADAMTS-13) in human platelets.

Biochem Biophys Res Commun 2004 Jan;313(1):212-6

Department of Internal Medicine, Keio University, Tokyo 160-8582, Japan.

The hemostatic activity of von Willebrand factor (vWF) is strongly dependent on its multimeric structure, with the highest activity in 'unusually large' multimers secreted from endothelial cells. The multimeric structure is regulated by a plasma protease, vWF-cleaving protease (vWF-CP, or ADAMTS-13). ADAMTS-13 mRNA is variably expressed in liver and other tissues. Because 15-25% of circulating vWF is stored in platelets, the presence and function of ADAMTS-13 in platelets are important issues. Here we report ADAMTS-13 expression in human platelets. Western blot analysis and flow cytometric analysis on permeabilized platelets revealed the presence of ADAMTS-13 protein. Real-time PCR demonstrated that ADAMTS-13 mRNA is present in platelets of six healthy volunteers, with little quantitative difference. The presence of ADAMTS-13 in platelets may imply the functional role of this enzyme in the local regulation of platelet function at the site of vascular injury or thrombus formation, and provide a useful tool for the analysis of structure and function of this enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2003.11.111DOI Listing
January 2004

Low molecular weight G-proteins of rho-family mediate relaxations to bradykinin in porcine coronary arteries.

Acta Pharmacol Sin 2003 Nov;24(11):1070-6

Center for Experimental Therapeutics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

Aim: To determine whether or not low molecular G-proteins are involved in the endothelium-dependent relaxations to bradykinin.

Methods: The effects of botulinum ADP-ribosyltranferase C3 were studied in porcine coronary arteries and endothelial cells.

Results: Incubation of membrane fractions isolated from endothelial cells with the enzyme and 32P-NAD resulted in the ribosylation of the proteins with molecular weight of 24-25 kDa. Radio labelling of these proteins was suppressed in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS), a hydrolysis-resistant analog of GTP. In the isolated arteries, ADP-ribosyltransferase C3 attenuated the relaxations to bradykinin during contractions with prostaglandin F2alpha in the presence of tween 80 (non ionic detergent), but not in the absence of tween 80.

Conclusion: Low molecular weight G-proteins of the Rho family contribute to the mechanism of relaxation induced by bradykinin.
View Article and Find Full Text PDF

Download full-text PDF

Source
November 2003

Combined sodium and calcium channel blockade in prevention of lethal arrhythmias.

J Cardiovasc Pharmacol 2003 May;41(5):665-70

Department of Physiology, Cardiovascular Diseases Section, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

Anti-arrhythmic compounds with multiple actions reduce arrhythmic death risk in post-myocardial infarction (MI) patients. Sudden death prevention, however, may rely more on implantable defibrillators than anti-arrhythmic drugs due to ineffective pharmacologic intervention. Widespread use of implantable defibrillators should not obscure the need for development of new anti-arrhythmic drugs. This study tested the hypothesis that combined blockade of I(Na) and I(Ca(L)) prevents ischemia-dependent ventricular fibrillation (VF) in conscious dogs after MI. I(Na) and I(Ca(L)) blockade was accomplished with levosemotiadil in 11 dogs known to be at high risk for VF during 2 min of coronary occlusion during submaximal treadmill exercise 30 days after MI. Negative chronotropic effect of levosemotiadil was examined using the heart rate response to isoproterenol and comparing it with response to propranolol. Levosemotiadil prevented VF in 64% (7 of 11) of the high-risk animals. Heart rate responses to myocardial ischemia and to graded doses of isoproterenol were blunted by the high dose of levosemotiadil. Propranolol prevented VF in 73% (8 of 11) of the dogs. Levosemotiadil had approximately one half the beta-blocking activity of propranolol. The combination of I(Na) and I(Ca(L)) channel blockade coupled with partial beta-adrenergic blockade was equally effective in preventing VF as propranolol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/00005344-200305000-00001DOI Listing
May 2003

Rational design and evaluation of new lead compound structures for selective betaARK1 inhibitors.

J Med Chem 2002 May;45(11):2150-9

Discovery Research Laboratories and New Product Research Laboratories II, Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd., 16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan.

Beta-adrenergic receptor kinase 1 (betaARK1) and cyclic adenosine 5'-monophosphate-dependent protein kinase A (PKA) have structurally similar adenine-binding pockets but have different physiologic functions. To obtain specific betaARK1 inhibitors, a two step rational drug design process was used. First, a search was conducted on three-dimensional models of commercially available compounds to find compounds that fit the adenine-binding pocket of betaARK1. Second, a comparative docking study that focused on the differences between the adenine-binding pockets of the two enzymes was used to evaluate the binding specificity of each compound that inhibited betaARK1 activity. The results of these analyses yielded three betaARK1-selective inhibitor leads from 11 candidates, a hit rate for selectivity of 27%. Although the IC50 values of these compounds for betaARK1 ranged from only 1.3 x 10(-4) M to 5.6 x 10(-4) M, the compounds did not inhibit PKA at concentrations up to 1.0 x 10(-3) M. Thus, the present study shows the usefulness of a rational drug design strategy in finding specific kinase inhibitors for proteins with similar drug target binding sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm010093aDOI Listing
May 2002