ALBERT

Description: The Journal of Organic Chemistry DOI: 10.1021/jo5008948

Description: Abstract 3321 Background: Edoxaban is a direct inhibitor of factor Xa (FXa), and its efficacy as an oral anti-coagulant agent is less likely to be affected by food intake or drug-drug interaction. This profile of edoxaban suggests a good compliance in clinical use.However it is not clear whether genetic variations of FX influence the efficacy of edoxaban. Objectives: To investigate the possible inter-patient variability in the efficacy of edoxaban stemming from SNPs in the FX gene, we characterized the enzyme activity of FXa derived from wild type FX (FX-WT) and from FX with two known mutations, A152T (FX-A152T) and G192R (FX-G192R). The impact of FX mutations were also tested on the pharmacological activity of edoxaban. Methods: Among known FX SNPs in the NCBI dbSNP database, two non-synonymous SNPs are located inside mature FX, rs3211772 (allele frequency: 0.006) and rs3211783 (allele frequency: 0.022), corresponding to A152T and G192R. The former located inside the light chain and the latter located inside the activation peptide of FX. We selected these two SNPs and examined whether they might influence on the efficacy of edoxaban. We prepared recombinant FX proteins of FX-WT, FX-A152T and FX-G192R. We measured the enzyme activities of these FXa and the anti-FXa and anticoagulant effects of edoxaban on these FXa. Recombinant FX proteins were activated with Russell's viper venom factor × activator and FXa activity was measured using a chromogenic substrate S-2222. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured using FX-deficient plasma supplemented with recombinant FX proteins with a coagulometer CA-50. Results: Km values of FX-WT, FX-A152T and FX-G192R FXa were 0.55, 0.53 and 0.54 nM, respectively, Vmax of FX-WT, FX-A152T and FX-G192R FXa were 21.0, 21.8 and 21.4 mOD/min, respectively. PTs of plasma containing these mutations were 25.2 (FX-WT), 24.2 (FX-A152T) and 24.1 (FX-G192R) seconds. aPTTs of plasma containing the mutated FXs were 76.7 (FX-WT), 77.3 (FX-A152T) and 72.6 (FX-G192R) seconds. These data indicated that these mutations do not affect the basal FXa catalytic activity and coagulation activity. The Ki values of edoxaban for the mutated Fxas, the concentrations of edoxaban required to double the PT (PTCT2) and aPTT (aPTTCT2) in plasma containing the mutated FXs did not affected by two FX mutarions (Table 1). These data demonstrated that those mutations have no impact on the anticoagulant activity of edoxaban. Conclusions: Two FX mutations, A152T and G192R, do not affect the basal FXa catalytic activity and coagulation activity. Edoxaban acts equally on FX-WT, FX-A152T and FX-G192R. It is suggested that edoxaban has little inter-patient variability stemming from SNPs in FX gene. Disclosures: Noguchi: Daiichi Sankyo Co., Ltd.: Employment. Takahashi:Daiichi Sankyo Co., Ltd.: Employment. Ishihara: Daiichi Sankyo Co., Ltd.: Employment. Morishima:Daiichi Sankyo Co., Ltd.: Employment. Shibano:Daiichi Sankyo Co., Ltd.: Employment.

Description: BACKGROUND: The adenosine diphosphate (ADP) receptor P2Y12 plays a central role in platelet function, and this receptor is therapeutic target for thrombotic disorders. Previously, the P2Y12 gene was reported to have genetic polymorphisms, H1H2 haplotype (C139T, T744C, insertion 801A, and G52T) and C34T. The effect of these polymorphisms on the risk for arterial thrombosis, however, remains controversial. In this study, we screened the sequence variations in the P2Y12 gene, and performed case-control study to investigate the association between P2Y12 polymorphisms and coronary artery disease (CAD). METHODS AND RESULTS: Written informed consent was obtained from all study subjects who were Japanese males. Screening of polymorphisms in the P2Y12 gene comprising exon1, exon2, and intron1 was analyzed in 20 control subjects. As a result, new haplotypes were defined: haplotype A: 145C, 139C, 744T, and 1210T, haplotype B: 145T, 139T, 744C, insertion 801A, and 1210C. The G52T and C34T polymorphism were not linked with this haplotype. We next analyzed the relationship between these P2Y12 polymorphisms and susceptibility of CAD in a case-control study. One hundred and two patients with CAD were recruited at Keio University Hospital with a diagnosis of myocardial infarction or angina pectoris. CAD patients whose coronary lesions were confirmed by coronary angiography (identified stenosis ≥50%) were eligible for this study. To match CAD patients for sex and age at diagnosis of CAD, 257 healthy control subjects were recruited at their regular checkups. They had no clinical or laboratory evidence of past vascular disorders. Genotypes of the AB, C34T, and G52T polymorphisms were determined by single nucleotide primer extension-based method. We observed that the genotype frequencies of the AB (AA vs AB+BB), C34T (CC vs CT+TT), and G52T (GG vs GT+TT) did not differ between the CAD and control groups (p= 0.0635 for AB, p=0.2090 for C34T, and p=0.0816 for G52T). When study subjects were divided into the combination of two homozygous carriers (genotype combination), the AA and 34CC genotypes, and other genotypes (others), the frequency of genotype combination in CAD patients was significantly higher than that in controls (p=0.0058). Moreover, association of the genotype combination with the prevalence of CAD, adjusted for other risk factors (body mass index, smoking, hypertension, hyperlipidemia, and diabetes mellitus), was analyzed by a multiple logistic regression model, and adjusted odds ratio was 2.0 (95% CI, 1.1–3.5, p=0.0147) for the relation between CAD and the genotype combination. CONCLUSIONS: The combination of AA haplotype and 34CC genotype is highly associated with the risk of CAD. The present data point out the importance of genotypic combination, but not single specific polymorphism, to examine the association between CAD and P2Y12 polymorphisms.

Description: Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase, reportedly regulates the canonical Wnt and Hedgehog pathways. Evidence indicates that thrombopoietin, a major regulator of thrombopoiesis, induces several signaling pathways, such as MAPK, PI3K, and JAK/STAT. Here, we present data showing a novel role of GSK-3β in megakaryocyte (MK) and platelet production in an in vitro hematopoietic cell culture system. Primary human low-density bone marrow mononuclear cells (BMMNCs) were grown in serum-free media containing thrombopoietin and several inhibitors: PD98059, SB202190, LY294002 or TWS119 was used to inhibit MAPK, p38MAPK, PI3K, or GSK-3β, respectively. The MK or platelet cell count was examined by flow cytometry on day 12 using the relative value of CD41 (+)/propidium iodide (+) cells or platelet size CD41 (+) cells, respectively, versus 106 BMMNCs on day 0. When the cells were treated with PD98059, SB202190, or LY294002, the MKs and platelets decreased in number compared with the non-treated control cells. In contrast, MK and platelet production was increased by the presence of TWS119 in the cell culture [TWS119 (+)] as compared with its absence in the cell culture [TWS119 (−)]: MKs, 24965.3±5437.7 vs 5763.7±874.0, p=0.0038, platelets, 6645.8±792.0 vs 3276.0±298.1, p=0.0023. In addition, human CD34 (+) cells were obtained from BMMNCs and grown in the liquid culture system, and each small interference RNA (siRNA) for GSK-3β or negative control was transfected into the cells on day 3. The frequency of CD41 (+)/propidium iodide (+) cells in siRNA-GSK3β transfected-cells was 2-fold higher than that in siRNA-negative control transfected cells. The transcriptional profiling was then compared between BMMNCs in TWS119 (+/−) to study the mechanism underlying the effect of GSK-3β on MK and platelet production. This analysis using the Affymetrix U133 Plus 2.0 Gene Chip array, which covers approximately 50000 transcripts, demonstrated genes with significantly increased or decreased expression in TWS119 (+) relative to TWS119 (−) at day 8. Of those genes, some factors associated with cytoskeletal organization, such as Rho guanine nucleotide exchange factor and β tubulin cofactor A, had increased expression, while others, such as supervillin and α tubulin, had decreased expression. The BMMNC-derived MKs and platelets in TWS119 (+/−) were morphologically analyzed with electron microscopy. The size of MKs in TW119 (+) was larger in diameter than that of MKs in TWS119 (−). Also, there was abundant mature MKs in TWS119 (+) whereas most MKs in TWS119 (−) were immature at day 8. However, there was no difference in the ultrastructure of platelets obtained from the MKs in TWS119 (+) or TWS119 (−). The findings of the present study demonstrated that GSK-3β negatively regulates MK and platelet production. Alterations in the MK morphology and/or maturation time might contribute to this regulation.

Description: Recent studies of mice deficient in the thrombin receptor, protease-activated receptor 1 (PAR1), provided definitive evidence for the existence of a second thrombin receptor in mouse platelets. We recently identified a new thrombin receptor designated protease-activated receptor 3 (PAR3). The mRNA encoding a mouse homologue of PAR3 was highly expressed in mouse splenic megakaryocytes, making it a good candidate for the missing mouse platelet thrombin receptor. We now report that PAR3 protein is expressed on the surface of mouse platelets and that PAR3 antibodies partially inhibit activation of mouse platelets by thrombin but not U46619, a thromboxane receptor agonist. These observations suggest that PAR3 contributes to mouse platelet activation by thrombin.

Description: BACKGROUND: The adenosine diphosphate (ADP) receptor, P2Y12, plays a central role in platelet activation and its blocker clopidogrel reduces the incidence of cardiovascular events. Recently, the polymorphisms of the P2Y12 gene (H1 and H2 haplotype) was shown to be associated with increased ADP-induced platelet aggregation in healthy volunteers. In addition, the polymorphism (C34T) of the P2Y12 gene was reportedly associated with higher numbers of cerebrovascular events in patients with PAD receiving clopidgrel therapy. However, it is totally unknown if there is any association between polymorphisms combination and platelet reactivity. In this study, we aimed to investigate in detail about the association between ADP-induced platelet aggregation and the polymorphisms of P2Y12 gene, haplotype and C34T, in healthy volunteers. METHODS AND RESULTS: We determined P2Y12 genotype and platelet reactivity in 267 healty volunteers. Genotyping was performed using direct sequencing or a single nucleotide primer extension (SNuPE). In previous study, H1 and H2 haplotype were defined by 4 polymorphisms, C139T, T744C and insertion801A in intron1 and G52T in exon2, and there were in complete linkage disequilibrium. Result of genotyping of haplotype, however, G52T was found not to be in complete linkage disequilibrium. Allele frequencies of H1 and H2 were 0.83 and 0.17, respectively. For C34T, allele frequencies of 34C and 34T were 0.75 and 0.25, respectively. Platelet reactivity was evaluated by optical aggregometry in platelet-rich plasma. Aggregation was induced by 2 or 5 μmol/L ADP to evaluate maximal aggregation (%) and the area under the aggregate curve (AUC) for 6 min. Maximal aggregation induced by 2 μmol/L ADP was similar in homozygous carriers of H1 haplotype and carriers of H2 haplotype (p=0.262). AUC induced by 2 μmol/L ADP was lower in homozygous carriers of H1 haplotype than carriers of H2 haplotype (p=0.074). However, haplotype was not associated with platelet reactivity induced by 5 μmol/L ADP. For C34T, whereas, maximal aggregation and AUC induced by 2 or 5 μmol/L ADP was lower in homozygous carriers of 34C than carriers of 34T (p=0.009 and p=0.006 or p=0.002 and p=0.003, respectively). Furthermore, for the genotypic combination of haplotype and genotype (C34T), maximal aggregation and AUC induced by 2 or 5 μmol/L ADP was significantly lower in homozygous carriers of both H1 haplotype and 34C than others (p=0.003 and p

Description: [Background] α 2A 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 collagen/epinephrine (CEPI) cartridge in the platelet function analyzer (PFA)-100® system. [Methods and Results] Written informed consent was obtained from all study subjects who were genetically unrelated healthy Japanese males (n=255). Of the 255 subjects, an initial screening for the sequence variation(s) in the ADRA2A transcriptional region was performed on 44 subjects enrolled in 2003. Subsequently, relationship between ADRA2A polymorphisms and platelet function was investigated in 211 subjects enrolled between 2004 and 2005. In the screening, we observed 16 single nucleotide polymorphisms (SNPs) including 5 novel variations. We next examined the association between the ADRA2A polymorphisms and epinephrine-mediated platelet function using the PFA-100® system under high shear conditions (5000–6000/sec). According to the pilot study on the 16 SNPs, we focused on 1780A/G and 2732A/G (the first nucleotide of the open reading frame is nt#1, which corresponds to nt#31586326 of the GenBank NT_030059). We measured platelet function, as assessed by closure time (CT) using CEPI cartridge. A longer CEPI-CT indicates lower platelet function in the interaction with collagen and epinephrine. Because the manufacturer’s instructions for the PFA-100® report a mean CEPI-CT value of 132 s, study subjects were divided in two groups: a higher function group with a CEPI-CT 〈 132 s (n=90) and a lower function group with a CEPI-CT ≥ 132 s (n=121). The frequency of the 1780GG genotype in the lower function group was significantly higher than that in the higher function group (p=0.0478) whereas no association between the CEPI-CT and the 2372A/G polymorphism was observed (p=0.1164). We also observed the effect of the combination of 1780GG+2372AA genotypes for shorter CT (p=0.0319). A multiple logistic regression model to adjust the reported confounding factors (VWF: RCo, platelet count, and hematocrit) revealed an adjusted odds ratio of 2.10 (p=0.0274) for the genotypic combination or 1.82 (p=0.0757) for the 1780A/G alone. Plasma VWF:RCo level was also an independent predictor for CEPI-CT. These observations suggested the enhanced effect of the genotypic combination as compared with the 1780A/G alone. [Conclusion] Of the 16 sequence variations of the transcriptional region of the ADRA2A gene, the combination of the 1780A/G and 2372A/G polymorphism was associated with collagen/epinephrine-mediated platelet function by PFA-100® system.