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Aims: The efficacy of antiplatelet therapy may vary among different disease subtypes. Prasugrel is generally a more potent, consistent, and fast-acting platelet inhibitor than clopidogrel. This sub-analysis of the phase III comparison of PRAsugrel and clopidogrel in Japanese patients with ischemic STROke (PRASTRO-I) trial aimed to assess the differences in efficacy of these treatments for each stroke subtype. Methods: In the PRASTRO-I trial, a total of 3,753 patients with ischemic stroke were recruited from 224 centers throughout Japan and randomized (1:1) to prasugrel (3.75 mg/day) or clopidogrel (75 mg/day) for 96 weeks. For the sub-analysis, strokes were classified as large-artery atherosclerosis, small-artery occlusion (lacunar), stroke of other etiology, and stroke of undetermined etiology. The cumulative incidence of primary events (ischemic stroke, myocardial infarction, and death from other vascular cause) and hazard ratios (HRs) were calculated for each subgroup. Results: For patients with large-artery atherosclerosis, the primary event incidence was 3.8% in the prasugrel group and 4.8% in the clopidogrel group (HR 0.79; 95% confidence interval [CI] 0.45–1.41). For patients with small-artery occlusion, the incidence was 3.3% in the prasugrel group and 3.9% in the clopidogrel group (HR 0.82; 95% CI 0.45–1.50). For patients with stroke of undetermined etiology, the incidence was 4.6% in the prasugrel group and 3.0% in the clopidogrel group (HR 1.56; 95% CI 0.90–2.72). The incidence of bleeding was similar across subtypes. Conclusions: Although statistical significance was not reached, the efficacy of prasugrel was potentially different between stroke subtypes, warranting further studies.

CD5-positive diffuse large B-cell lymphoma (CD5+ DLBCL) is characterized by poor prognosis and a high frequency of central nervous system relapse after standard immunochemotherapy. We conducted a phase II study to investigate the efficacy and safety of dose-adjusted (DA)- EPOCH-R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) combined with high-dose methotrexate (HD-MTX) in newly diagnosed patients with CD5+ DLBCL. Previously untreated patients with stage II to IV CD5+ DLBCL according to the 2008 World Health Organization classification were eligible. Four cycles of DA-EPOCH-R followed by two cycles of HD-MTX and four additional cycles of DAEPOCH- R (DA-EPOCH-R/HD-MTX) were planned as the protocol treatment. The primary end point was 2-year progression-free survival (PFS). Between September 25, 2012, and November 11, 2015, we enrolled 47 evaluable patients. Forty-five (96%) patients completed the protocol treatment. There were no deviations or violations in the DA-EPOCH-R dose levels. The complete response rate was 91%, and the overall response rate was 94%. At a median follow up of 3.1 years (range, 2.0-4.9 years), the 2- year PFS was 79% [95% confidence interval (CI): 64-88]. The 2-year overall survival was 89% (95%CI: 76-95). Toxicity included grade 4 neutropenia in 46 (98%) patients, grade 4 thrombocytopenia 12 (26%) patients, and febrile neutropenia in 31 (66%) patients. No treatment-related death was noted during the study. DA-EPOCH-R/HD-MTX might be a first-line therapy option for stage II-IV CD5+ DLBCL and warrants further investigation. (Trial registered at: UMIN-CTR: UMIN000008507.)

Aim: Although high on-treatment platelet reactivity (HTPR) with dual antiplatelet therapy (DAPT) correlates with long-term adverse outcomes in patients undergoing percutaneous coronary intervention, the correlation in Japanese patients remains unclear. Therefore, we examined the relationship between platelet reactivity during DAPT with aspirin and clopidogrel and 1-year clinical outcomes following successful coronary stent implantation. Methods: A prospective, multicenter registry study (j-CHIPS) was conducted in patients undergoing coronary stenting and receiving aspirin and clopidogrel at 16 hospitals in Japan. A VerifyNow point-of-care assay was used to assess platelet reactivity, and a cutoff value to define HTPR was established. Results: Between February 2011 and May 2013, 1047 patients were prospectively enrolled, of which 854 patients with platelet function evaluation at 12–24 h after PCI were included in the final analysis. After 1 year of follow-up, the incidence of the primary endpoint (a composite of all-cause mortality, myocardial infarction, stent thrombosis, and ischemic stroke) was significantly higher in patients with HTPR than in those without (5.9% vs. 1.5%, p = 0.008), and HTPR showed a modest ability to discriminate between patients who did and did not experience major adverse cardiac and cerebrovascular events (area under the curve, 0.60; 95% confidence interval, 0.511–0.688, p = 0.039). HTPR status did not identify patients at risk for major or minor bleeding events. Conclusion: HTPR was significantly associated with adverse ischemic outcomes at 1 year after PCI in Japanese patients receiving maintenance DAPT, indicating its potential as a prognostic indicator of clinical outcomes in this high-risk patient population.

Introduction: The safety of prasugrel in elderly and/or low body weight Japanese patients with ischemic stroke who have a relatively high bleeding risk with antiplatelet therapy remains unknown. Objective: We aimed to investigate the safety and efficacy of long-term prasugrel monotherapy for stroke prevention compared with clopidogrel in elderly and/or low body weight Japanese patients with non-cardioembolic ischemic stroke. Methods: In this randomized, double-blind, comparative, phase III study, elderly (age ≥75 years) and/or low body weight (≤50 kg) Japanese patients with a previous history of non-cardioembolic ischemic stroke were assigned to a prasugrel 3.75 mg (PRA3.75) group, a prasugrel 2.5 mg (PRA2.5) group, or a clopidogrel 50 mg (CLO50) group and followed up for 48 weeks. The primary safety endpoint was the combined incidence of primary safety events, defined as life-threatening, major, and other clinically relevant bleeding. The efficacy endpoint was a composite of ischemic stroke, myocardial infarction, and death from other vascular causes. Results: A total of 654 patients (age 76.4 ± 7.3 years, body weight 55.6 ± 9.3 kg, women 43.9%) from 74 medical institutions within Japan were enrolled. The combined incidence (95% CI) of primary safety events was 4.2% (1.9–7.8%), 1.9% (0.5–4.7%), and 3.6% (1.6–6.9%) in the PRA3.75 group (n = 216), PRA2.5 group (n = 215), and CLO50 group (n = 223), respectively (hazard ratios [HR] PRA3.75/CLO50, 1.13 [0.44–2.93]; PRA2.5/CLO50, 0.51 [0.15–1.69]). The incidences of bleeding leading to treatment discontinuation (95% CI) were 2.3% (0.8–5.3%), 0.9% (0.1–3.3%), and 2.2% (0.7–5.2%) in the PRA3.75, PRA2.5, and CLO50 groups, respectively (HRs PRA3.75/CLO50, 1.01 [0.29–3.48]; PRA2.5/CLO50, 0.41 [0.08–2.12]). There was no significant difference in all bleeding events between groups. The incidence of ischemic stroke, myocardial infarction, and death from other vascular causes was lower, but not significantly so, in patients treated with prasugrel than in patients treated with clopidogrel: PRA3.75, 0.0% (0/216); PRA2.5, 3.3% (7/215); and CLO50, 3.6% (8/223; HRs PRA3.75/CLO50, 0.00 [0.00–0.00]; PRA2.5/CLO50, 0.90 [0.32–2.47]). Conclusions: Elderly and/or low body weight ­Japanese patients with previous non-cardioembolic ischemic stroke who received PRA3.75 showed similar results in terms of primary safety endpoint, and a numerically lower incidence of ischemic stroke, myocardial infarction, and death from other vascular causes, compared with those who received CLO50.

ObjectivesTo examine the incidence of stroke or systemic embolic events (SSEs) and bleeding events in untreated patients with non-valvular atrial fibrillation (NVAF) after widespread use of direct oral anticoagulant agents (DOACs).DesignMulticentre, non-interventional, observational, retrospective cohort study using real-world data in Japan (2016-2018).SettingThe Mie, Musashino University study of NVAF, which used the Mie-Life Innovation Promotion Center Database. This is a regional clinical database involving one university hospital and eight general hospitals in Mie Prefecture in Japan.ParticipantsJapanese patients with NVAF (n=7001).Primary and secondary outcomeThe incidence of SSEs and bleeding events.ResultsA total of 7001 patients with NAVF were registered, and 53.0% were treated with DOACs, 10.6% were treated with warfarin and 36.4% had no treatment. Additionally, 29.5% of patients with a CHADS2 (congestive heart failure, hypertension, age≥75 years, diabetes, previous stroke or transient ischemic attack) score of 3–6 were untreated. In the no treatment group, the SSE rates by the CHADS2 score (0, 1, 2 and 3–6) were 1.4%, 1.4%, 3.2% and 8.0%, respectively. The rates of bleeding events by the CHADS2 score (0, 1, 2 and 3–6) in the no treatment group were 0.7%, 1.0%, 1.2% and 2.9%, respectively. A multivariate analysis of SSEs in components of the CHADS2 showed that the adjusted HRs were 2.32 for heart failure, 1.66 for an age ≥75 years, 1.81 for diabetes mellitus and 5.84 for prior stroke or transient ischaemic attack.ConclusionsApproximately one-third of the patients do not receive any anticoagulation in the modern DOAC era in Japan. The SSE rate increases by the CHADS2 score. The SSE rate is low in patients with a CHADS2 score 1, the use of anticoagulant drug therapy is recommended because of a higher risk of stroke.

Summary Background The effect of prasugrel in terms of the prevention of recurrence of ischaemic stroke is unknown. We investigated the non-inferiority of prasugrel to clopidogrel for prevention of ischaemic stroke, myocardial infarction, and death from other vascular causes in Japanese patients with non-cardioembolic stroke. Methods In this phase 3 randomised, double-blind, non-inferiority trial, patients aged 20–74 years who had had a non-cardioembolic stroke in the previous 1–26 weeks were recruited from 224 hospitals in Japan. Eligible patients were randomly assigned (1:1) to receive prasugrel (3·75 mg/day) or clopidogrel (75 mg/day) orally for 96–104 weeks. Randomisation was stratified according to stroke subtype. The randomisation schedule was generated by an independent statistician who created a computer-generated random number sequence. Patients, investigators, and the funder were masked to treatment allocation. The primary endpoint was combined incidence of ischaemic stroke (fatal and non-fatal), myocardial infarction (fatal and non-fatal), and death from other vascular causes in the intention-to-treat population. The safety endpoint was incidence of bleeding events, comprising life-threatening bleeding, major bleeding, and clinically relevant bleeding. The safety analysis was done in the population excluding trial patients with serious Good Clinical Practice violations, and those who had not taken the trial drug. The predefined non-inferiority margin was an upper 95% CI limit for the risk ratio (RR) of 1·35. The trial was registered with the Japan Pharmaceutical Information Center (JapicCTI-111582). Findings Patients were recruited between Sept 1, 2011, and June 12, 2015. 3747 patients (797 [21%] women) were enrolled, with a mean age of 62·1 (SD 8·5) years. 3753 patients were randomly assigned to treatment and, of these patients, 1885 in the prasugrel group and 1862 in the clopidogrel group were confirmed to have taken the trial drug at least once, and six patients withdrew from the trial before administration of the trial drug. Thus, a total of 3747 patients were included in the full analysis set. 73 (4%) of 1885 patients in the prasugrel group and 69 (4%) of 1862 patients in the clopidogrel group reached the primary endpoint (RR 1·05, 95% CI 0·76–1·44). The incidence of bleeding events was not significantly different between treatment groups; life-threatening bleeding was observed in 18 (1%) patients in the prasugrel group and 23 (1%) patients in the clopidogrel group (RR 0·77, 0·41–1·42). Interpretation The non-inferiority of prasugrel to clopidogrel for the prevention of ischaemic stroke, myocardial infarction, and death from other vascular causes was not confirmed in Japanese patients with non-cardioembolic stroke. No safety concerns were identified. Funding Daiichi Sankyo.

Background and Purpose: High blood pressure increases bleeding risk during treatment with antithrombotic medication. The association between blood pressure levels and the risk of recurrent stroke during long-term secondary stroke prevention with thienopyridines (particularly prasugrel) has not been well studied. Methods: This was a post hoc analysis of the randomized, double-blind, multicenter PRASTRO-I trial (Comparison of Prasugrel and Clopidogrel in Japanese Patients With Ischemic Stroke-I). Patients with noncardioembolic stroke were randomly assigned (1:1) to receive prasugrel 3.75 mg/day or clopidogrel 75 mg/day for 96 to 104 weeks. Risks of any ischemic or hemorrhagic stroke, combined ischemic events, and combined bleeding events were determined based on the mean level and visit-to-visit variability, including successive variation, of systolic blood pressure (SBP) throughout the observational period. These risks were also compared between quartiles of mean SBP level and successive variation of SBP. Results: A total of 3747 patients (age 62.1±8.5 years, 797 women), with a median average SBP level during the observational period of 132.5 mm Hg, were studied. All the risks of any stroke (146 events; hazard ratio, 1.318 [95% CI, 1.094–1.583] per 10-mm Hg increase), ischemic stroke (133 events, 1.219 [1.010–1.466]), hemorrhagic stroke (13 events, 3.247 [1.660–6.296]), ischemic events (142 events, 1.219 [1.020–1.466]), and bleeding events (47 events, 1.629 [1.172–2.261]) correlated with increasing mean SBP overall. Similarly, an increased risk of these events correlated with increasing successive variation of SBP (hazard ratio, 3.078 [95% CI, 2.220–4.225] per 10-mm Hg increase; 3.051 [2.179–4.262]; 3.276 [1.172–9.092]; 2.865 [2.042–4.011]; 2.764 [1.524–5.016], respectively). Event rates did not differ between the clopidogrel and prasugrel groups within each quartile of SBP or successive variation of SBP. Conclusions: Both high mean SBP level and high visit-to-visit variability in SBP were significantly associated with the risk of recurrent stroke during long-term medication with either prasugrel or clopidogrel after stroke. Control of hypertension would be important regardless of the type of antiplatelet drugs. Registration: URL: https://www.clinicaltrials.jp ; Unique identifier: JapicCTI-111582.

This randomized double-blind crossover study aimed to investigate the influence of cytochrome P450 (CYP) 2C19 polymorphisms on the antiplatelet effects of prasugrel in patients with non-cardioembolic stroke treated with clopidogrel. Patients received clopidogrel 75 mg/day for > 4 weeks. Subsequently, patients received prasugrel 3.75 mg/day (group A; n = 64) or 2.5 mg/day (group B; n = 65) for 4 weeks followed by a 4 week switched-dose regimen. To assess the influence of CYP2C19 polymorphisms, patients were classified as extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs). The primary endpoint was P2Y12 reaction units (PRU) at the end of each 4 week treatment. A significant reduction in PRU was noted after treatment with prasugrel 3.75 mg/day compared with the pre-dose value (after treatment with clopidogrel) (p

BACKGROUND The aim of this study was to assess the echocardiographic characteristics of chronic hemodialysis (HD) patients with end-stage renal disease (ESRD) in a multicenter prospective cohort study.Methods and Results:Three hundred and fifteen patients with ESRD (67.9±10.6 years, 47.6% male) on chronic HD for ≥1 year were examined on transthoracic echocardiography, including Doppler-derived aortic valve area (AVA) measurement. Only 11.5% and 3.4% of all patients had normal left ventricular (LV) geometry and normal LV filling pattern, respectively. The majority of patients had aortic and mitral valvular calcification, and approximately 50% of all 315 patients had aortic valve narrowing with AVA

IntroductionThere is no proven therapy to reverse or ameliorate fetal growth restriction (FGR). Sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, has been reported to potentially play a therapeutic role in FGR, but this has not been established. Tadalafil is also a selective PDE5 inhibitor. We have demonstrated the efficacy of tadalafil against FGR along with short-term outcomes and the feasibility of tadalafil treatment. Based on the hypothesis that tadalafil will safely increase the likelihood of increased fetal growth in FGR, we designed this phase II study to prospectively evaluate the efficacy and safety of tadalafil against FGR.Methods and analysisThis study is a multicentre, randomised controlled phase II trial. A total of 140 fetuses with FGR will be enrolled from medical centres in Japan. Fetuses will be randomised to receive either the conventional management for FGR or a once-daily treatment with 20 mg of tadalafil along with the conventional management until delivery. The primary endpoint is fetal growth velocity from the first day of the protocol-defined treatment to birth (g/day). To minimise bias in terms of fetal baseline conditions and timing of delivery, a fetal indication for delivery was established in this study. The investigator will evaluate fetal baseline conditions at enrolment and will decide the timing of delivery based on this fetal indication. Infants will be followed up for development until 1.5 years of age.Ethics and disseminationThis study was approved by the Institutional Review Board of Mie University Hospital and each participating institution. Our findings will be widely disseminated through peer-reviewed publications.Trial registration numberUMIN000023778.

Background: This comparison of PRAsugrel and clopidogrel in Japanese patients with ischemic STROke (PRASTRO)-I trial investigates the noninferiority of prasugrel to clopidogrel sulfate in the prevention of recurrence of primary events (ischemic stroke, myocardial infarction, and death from other vascular causes), and the long-term safety of prasugrel in Japanese patients with non-cardioembolic stroke. Research design and methods: This was an active-controlled, randomized, double-blind, double-dummy, parallel-group study conducted between July 2011 and March 2016 at multiple centers around Japan. Patients had to meet eligibility criteria before receiving 3.75 mg prasugrel or 75 mg clopidogrel orally once daily for a period of 96–104 weeks. Results: A total of 3747 patients were included in this trial; 1598 in the 3.75 mg prasugrel group and 1551 in the 75 mg clopidogrel group completed the study. During the study period, 287 (15.2%) patients in the prasugrel group and 311 (16.7%) in the clopidogrel group discontinued treatment. Baseline characteristics, safety, and efficacy results are forthcoming and will be published separately. Conclusions: This article presents the study design and rationale for a trial investigating the noninferiority of prasugrel to clopidogrel sulfate with regards to the inhibitory effect on primary events in patients with non-cardioembolic stroke.

IntroductionFew studies have examined the relationship between the pharmacodynamics of antiplatelet drugs and the risk of clinically significant bleeding following percutaneous coronary intervention (PCI) for treating acute coronary syndrome (ACS). We examined the associations between the pharmacodynamics of prasugrel and clopidogrel and the incidence of bleeding events in the acute and chronic phases after PCI.Materials and methodsWe performed a post hoc analysis of the PRASFIT-ACS (PRASugrel compared with clopidogrel For Japanese patIenTs with ACS undergoing PCI) study of patients in whom platelet reactivity was determined as P2Y12 reaction units (PRU; VerifyNow® P2Y12 assay) or vasodilator-stimulated phosphoprotein-phosphorylation reactivity index (VASP-PRI). Japanese patients were randomized to prasugrel (loading/maintenance dose: 20/3.75mg) or clopidogrel (300/75mg), both in combination with aspirin, for 24–48weeks. The bleeding outcome was a composite of major, minor, and clinically relevant bleeding.ResultsOverall, 66/685 (9.6%) and 65/678 (9.6%) of prasugrel- and clopidogrel-treated patients, respectively, experienced major, minor, or clinically relevant bleeding. PRU and VASP-PRI at 5–12h or in steady state conditions (at 4weeks) were not associated with the risk of bleeding in the acute (to day 3) or chronic (from day 4 to 14days after treatment discontinuation) phases of treatment, respectively. Less than 9% of patients with low on-treatment platelet reactivity (defined as PRU

Ezetimibe has been reported to provide significant incremental reduction in low-density-lipoprotein cholesterol (LDL-C) when added to a statin; however, its effect on coronary atherosclerosis has not yet been evaluated in detail. The aim of this study was to investigate the add-on effect of ezetimibe to a statin on coronary atherosclerosis evaluated by intravascular ultrasound (IVUS).In this prospective randomized open-label study, a total of 51 patients with stable coronary artery disease (CAD) requiring percutaneous coronary intervention (PCI) were enrolled, and assigned to a combination group (n = 26, rosuvastatin 5 mg/day + ezetimibe 10 mg/day) or a monotherapy group (n = 25, rosuvastatin 5 mg/day). Volumetric IVUS analyses were performed at baseline and 6 months after the treatment for a non-PCI site. LDL-C level was significantly reduced in the combination group (-55.8%) versus that in the monotherapy group (-36.8%; P = 0.004). The percent change in plaque volume (PV), the primary endpoint, appeared to decrease more effectively in the combination group compared with the monotherapy group (-13.2% versus -3.1%, respectively, P = 0.050). Moreover, there was a significant group × time interaction in the effects of the two treatments on PV (P = 0.021), indicating the regressive effect of the combination therapy on PV was greater than that of monotherapy for subtly different values of baseline PV in the two treatment groups. Moreover, percent change in PV showed positive correlations with percent change of LDL-C (r = 0.384, P = 0.015).Intensive lipid-lowering therapy with ezetimibe added to usual-dose statin may provide significant incremental reduction in coronary plaques compared with usual-dose statin monotherapy.

Background Few large-scale studies have examined the relationship between bleeding events not related to coronary artery bypass grafting (CABG), and the vascular access route used in acute coronary syndrome (ACS) or in elective treatment of coronary artery disease (CAD). Methods and results We compared the incidence of bleeding events occurring up to 3 days after percutaneous coronary intervention (PCI) or loading dose of prasugrel or clopidogrel in 2 studies of Japanese patients (PRASFIT-ACS, femoral and radial routes, n=683 and 531; PRASFIT-Elective, femoral and radial routes, n=135 and 508). Rates of periprocedural bleeding, bleeding not related to CABG, and puncture site bleeding were consistently lower in the radial access route group than in the femoral access route group in both studies. Risk factors for periprocedural bleeding included sex, body weight, age, and access route in PRASFIT-ACS (femoral access: hazard ratio [HR], 3.739; 95% confidence interval [CI]: 1.727-8.094; radial access: HR, 0.288; 95% CI: 0.128-0.65), and body weight, age, and access route in PRASFIT-Elective (femoral access: HR, 12.32; 95% CI 1.282->100; radial access: HR, 0.125; 95% CI: 0.013-1.205). Conclusions The incidence of periprocedural bleeding is lower with a radial access route than with a femoral access route for PCI in Japanese patients with ACS or those undergoing elective PCI for CAD.

This study investigated the effects and safety of eplerenone or thiazide diuretics in patients with hypertension and albuminuria (pretreatment urinary albumin/creatinine ratio ≥10 mg/gCr) treated with an angiotensin II receptor blocker. The primary end point was the mean percent change in the urinary albumin/creatinine ratio from baseline to 48 weeks. An efficacy analysis was performed in 195 patients (98 in the eplerenone group and 97 in the thiazide group). Systolic and diastolic blood pressures at 48 weeks were similar in the two groups. The mean percent change in the urinary albumin/creatinine ratio from baseline to 48 weeks was similar in the two groups (P=.804). In the safety analysis, the withdrawal rates for adverse events were similar in both groups. The antialbuminuric effects and safety of eplerenone therapy were similar to those of thiazide diuretics when combined with an angiotensin II receptor blocker in patients with hypertension and albuminuria.

Prevention of deep vein thrombosis (DVT) is important in patients undergoing major orthopedic surgery. Although the detection of an elevated D-dimer level is useful for predicting DVT, it is not efficacious in postoperative patients being treated with anti-Xa agents. The soluble platelet glycoprotein VI (sGPVI) level is a marker of activated platelets, but not bleeding. Therefore, sGPVI levels are usually examined as a predictor of DVT in such patients. In the present study, 83 orthopedic patients were treated with 30 mg of edoxaban for prophylaxis of DVT. Fourteen patients developed DVT and 17 patients discontinued the prophylaxis due to decreased hemoglobin levels. Plasma levels of sGPVI in the patients were significantly higher after surgery than before surgery. On day 1, the sGPVI levels increased, while the platelet counts decreased. There were no significant differences in D-dimer, soluble fibrin, or FDP levels in orthopedic patients with and without DVT before surgery and on days 1, 4, and 8. Plasma sGPVI levels were significantly higher in the patients with DVT than in those without DVT on days 1 and 4. Plasma levels of D-dimer were significantly higher in patients with withdrawal than in those without. However, there were no significant differences in sGPVI levels between those with and without withdrawal. As D-dimer levels are known to increase in patients with withdrawal, this parameter is not useful for evaluating the risk of DVT in these patients. In contrast, the sGPVI level is not increased in those with withdrawal and may therefore be useful for evaluating the risk of DVT in postoperative patients treated with an anticoagulant.

Background Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets. This study examined the soluble platelet glycoprotein VI (sGPVI) levels in patients with TMA to evaluate the activation of platelets in thrombotic states. Materials and Methods The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in patients with TMA. Results The plasma levels of sGPVI were significantly higher in postoperative patients, patients with TMA and those with disseminated intravascular coagulation (DIC) than in those without thrombosis. The plasma levels of sGPVI were the highest in patients with TMA without markedly reduced ADAMTS13 and those were significantly reduced after plasma exchange. Conclusion The measurement of sGPVI level is therefore considered to be important for the diagnosis and evaluation of TMA.

Quantitative analysis of itching in patients with itching dermatitis including atopic dermatitis (AD) is indispensable for the evaluation of disease activity and response to therapy. However, the objective evaluation system for itching is limited. We have developed a new objective and quantitative scratching behavior detection system using a wristwatch-type sound detector. The scratch sound detected on the wrist is recorded on a personal computer through a filtering, squaring and smoothing process by specific hardware. Subsequently, the data is automatically processed and judged for the scratching movement using specific software based on the periodicity and energy of the signal. Twenty-four measurements for healthy volunteers and those with AD by this system were evaluated by comparison with a simultaneously recorded video analysis system. The ratio of scratching time in sleeping time evaluated by these two systems was almost identical. The healthy subjects scratched their skin approximately 2 min during 6 h of sleeping time, while the mean scratching time of AD subjects was 24 min in their sleeping time. In contrast to the time-consuming video analysis system, this system takes only several minutes for evaluation of an overnight record. This scratch sound detection system is expected to serve as a new objective evaluation tool for itching dermatitis, namely, AD, and development of anti-itch therapies for dermatitis.

Background CYP2C19 loss-of-function genotype (*2 and/or *3 alleles) is related to low responsiveness to clopidogrel, which is a risk factor for ischemic cardiac events. The contribution of these genotypes to platelet reactivity in Japanese patients in a steady state receiving dual antiplatelet therapy after coronary stenting was evaluated. Methods and results A total of 155 Japanese patients were classified according to their CYP2C19 loss-of-function genotype. Platelet reactivity was assayed by plasma levels of soluble P-selectin and platelet-derived microparticles, light transmittance aggregometry induced by ADP (ADP-LTA), shear stress-induced platelet aggregometry, vasodilator-stimulated phosphoprotein phosphorylation (VASP) index and the VerifyNow-P2Y12 assay. Linear and logistic regression models were used to assess the associations between CYP2C19 loss-of-function genotype and high on-treatment platelet reactivity. In total, 62 patients (40.0%) were extensive metabolizers (EMs), 70 (45.2%) were intermediate metabolizers (IMs) and 23 (14.8%) were poor metabolizers (PMs). ADP-specific assays (ADP-LTA, the VASP index and VerifyNow-P2Y12) differed according to CYP2C19 genotype, with a significant gene-dose effect (PMs>IMs>EMs). CYP2C19 loss-of-function carrier status was associated with more frequent high platelet reactivity. CYP2C19 loss-of-function genotype alone could explain 12.2%, 14.3%, and 14.7% of the variability in the ADP-LTA, VASP and VerifyNow-P2Y12 assays, respectively. Conclusions CYP2C19 loss-of-function genotype is associated with more frequent high platelet reactivity, as assessed by ADP-specific platelet function tests, in Japanese patients.

Prasugrel is a third-generation thienopyridine that achieves potent platelet inhibition with less pharmacological variability than other thienopyridines. However, clinical experience suggests that prasugrel may be associated with a higher risk of de novo and recurrent bleeding events compared with clopidogrel in Japanese patients undergoing percutaneous coronary intervention (PCI). In this review, we evaluate the risk of bleeding in Japanese patients treated with prasugrel at the doses (loading/maintenance doses: 20/3.75 mg) adjusted for Japanese patients, evaluate the risk factors for bleeding in Japanese patients, and examine whether patients with a bleeding event are at increased risk of recurrent bleeding. This review covers published data and new analyses of the PRASFIT (PRASugrel compared with clopidogrel For Japanese patIenTs) trials of patients undergoing PCI for acute coronary syndrome or elective reasons. The bleeding risk with prasugrel was similar to that observed with the standard dose of clopidogrel (300/75 mg), including when bleeding events were re-classified using the Bleeding Academic Research Consortium criteria. The pharmacodynamics of prasugrel was not associated with the risk of bleeding events. The main risk factors for bleeding events were female sex, low body weight, advanced age, and presence of diabetes mellitus. Use of a radial puncture site was associated with a lower risk of bleeding during PCI than a femoral puncture site. Finally, the frequency and severity of recurrent bleeding events during continued treatment were similar between prasugrel and clopidogrel. In summary, this review provides important insights into the risk and types of bleeding events in prasugrel-treated patients. Trial registration numbers: JapicCTI-101339 and JapicCTI-111550.

Background/objectives Developmental changes of structures in neonatal and infant skin have not been well characterized. The purpose of this study was to clarify changes in skin structures during neonatal and infant growth in vivo. Methods Fifteen healthy, full-term neonates (seven girls, eight boys) were studied. The measurements were performed 4 to 7 days (neonate) and 1, 3, and 6 months after birth on the buttock, upper thigh, and ventral forearm skin using a confocal laser scanning microscope. Developmental changes in dermoepidermal junction structures, stratum corneum thickness, epidermal thickness, and microvascular development were investigated. Results A significant decrease in stratum corneum thickness was observed over the 3 months after birth. Dermal papillae were not observed in neonatal skin but were observed gradually over the next 3 months. Epidermal thickness, determined from the skin surface to the bottom of the epidermal layer, increased significantly from 4 to 7 days to 1 month of age, indicating the formation of dermal papillae and rete ridges. Complicated microvascular structures were observed in neonatal skin but disappeared gradually and were observed only at the dermal papillae at 3 months of age. Conclusions Our results reveal that infant skin is in a developmental stage structurally up to 3 months of age, paralleling skin functional and developmental maturation.

Immune thrombocytopenic purpura (ITP) is an acquired hemorrhage condition involving accelerated platelet consumption caused by antiplatelet autoantibodies. Although various therapeutic strategies are used to treat patients with ITP, the standard treatment method is steroid therapy. The most important problem with steroid administration may be a prolonged use tendency in many cases, because there are many refractory chronic patients. To elucidate the effects of glucocorticoid on bone mineral density (BMD) in patients with ITP, we retrospectively evaluated the relationship between BMD and the total dose of glucocorticoid or the mean daily dose given. We observed decreased BMD in 66.7% of the patients with ITP to whom glucocorticoid was given, although normal bone BMD was observed in 28.6% of patients with ITP treated without steroids. The mean level of BMD was markedly decreased in steroid-treated patients compared with nonsteroid-treated patients (P < .01). The relationship between BMD and the total dose of glucocorticoid (P = .023) or the mean daily dose revealed a negative correlation (P = .022). Administration of bisphosphonate revealed a significant increase in bone mass in patients at 6 and 12 months after the start of bisphosphonate treatment, despite the aggravation of thrombocytopenia. In conclusion, glucocorticoid-induced osteoporosis was observed in patients with ITP, similar to situation seen in patients with other diseases. Bisphosphonate may be an effective agent for the prevention and treatment of glucocorticoid-induced osteoporosis in patients with ITP scheduled to receive long-term steroid treatment.

The ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type I domain 13) related markers were measured in the plasma of healthy volunteers and thrombotic microangiopathy (TMA) patients including thrombotic thrombocytopenic purpura (TTP) to examine their efficacy in the diagnosis of TTP. The plasma levels of the ADAMTS13 antigen and ADAMTS13-factor XI complex were significantly lower in TMA patients with a significant decreased ADAMTS13 activity (and these patients were considered to have TTP) than in the healthy volunteers. The plasma levels of ADAMTS13 antigens closely correlated with those of ADAMTS13-factor XI complex. Autoantibody for ADAMTS 13 was also positive in almost all TTP patients. In addition, the ratio of von Willebrand factor (VWF)/ADAMTS13 activity was significantly high in TTP suggesting that this ratio might be more useful for the differential diagnosis of TTP than the ADAMTS13 assay alone. These findings suggest that ADAMTS13 related markers are useful for the diagnosis and analysis of TTP.

SummarySeveral studies have indicated that approximately 25% of patients treated with aspirin exhibit high on-treatment platelet reactivity (HTPR), which is potentially associated with cardiovascular events (CVEs). However, this association is still controversial, since the mechanisms by which HTPR contributes to CVEs remain unclear and a no standardised definition of HTPR has been established. To determine whether HTPR is associated with CVE recurrence and what type of assay would best predict CVE recurrence, we conducted a multicentre prospective cohort study of 592 stable cardiovascular outpatients treated with aspirin monotherapy for secondary prevention. Their HTPR was determined by arachidonic acid- or collagen-induced aggregation assays using two different agonist concentrations. Residual cyclooxygenase (COX)-1 activity was assessed by measuring serum thromboxane (TX)B2 or urinary 11-dehydro TXB2. Shear-induced platelet thrombus formation was also examined. We followed all patients for two years to evaluate how these seven indexes were related to the recurrence of CVEs (cerebral infarction, transient ischaemic attack, myocardial infarction, unstable angina, revascularisation, other arterial thrombosis, or cardiovascular death). Of 583 patients eligible for the analysis, CVEs occurred in 69 (11.8%). A Cox regression model identified several classical risk factors associated with CVEs. However, neither HTPR nor high residual COX-1 activity was significantly associated with CVEs, even by applying cut-off values suggested in previous reports or a receiver-operating characteristic analysis. In conclusion, recurrence of CVEs occurred independently of HTPR and residual COX-1 activity. Thus, our findings do not support the use of platelet or COX-1 functional testing for predicting clinical outcomes in stable cardiovascular patients.Supplementary Material to this article is available at www.thrombosis-online.com.

Primary immune thrombocytopenia (ITP) is an autoimmune disease mediated by the production of autoantibody against platelets. Rituximab, an anti-CD20 antibody, is reported to be useful for treatment of ITP. In Japan, however, robust evidence on this treatment has not been accumulated. Hence, we conducted this open-label phase III clinical trial to confirm the efficacy and safety of rituximab, administered at 375 mg/m² once per week at weekly intervals for 4 consecutive weeks in Japanese patients with chronic ITP, who had relapsed and were refractory to conventional therapy. The primary endpoint was defined as the percentage of patients with a platelet count above 50 × 10⁹/L at week 24 after the first dose of rituximab, which was 30.8% of 26 patients (95% confidence interval 14.3-51.8%). Although the lower confidence limit of primary endpoint failed to meet the pre-specified threshold of 20%, the clinical efficacy of rituximab is substantial in consideration of the 2% response rate in the placebo arm in other clinical studies in patients with chronic ITP. We conclude that rituximab is clinically useful and safe in the treatment of Japanese patients with chronic ITP, achieving the goal of maintaining platelet count and reducing risk of bleeding while minimizing treatment-related toxicity.

Background We examined the effects of cytochrome P450 2C19 (CYP2C19) polymorphisms on the efficacy and safety of prasugrel and clopidogrel in a post hoc analysis of the PRASugrel compared with clopidogrel For Japanese patIenTs with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) (PRASFIT-ACS) study. Methods Japanese ACS patients undergoing PCI were randomized (double-blind) to receive prasugrel (loading/maintenance dose: 20/3.75 mg) or clopidogrel (300/75 mg) plus aspirin for 24–48 weeks. Pharmacogenomic analyses were conducted in 773/1363 patients. P2Y12 reaction units (PRU) were determined using the VerifyNow® P2Y12 assay (Accumetrics, San Diego, CA, USA). CYP2C19 genotypes were classified as extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). Results Overall, 39.2% and 60.8% of patients in the prasugrel group and 35.2% and 64.8% of patients in the clopidogrel group were classified as EM and IM + PM, respectively. Among EM patients, PRU was significantly lower in the prasugrel group than in the clopidogrel group at 2–4 and 5–12 h after the loading dose, but was similar in both groups from week 4 onwards. Among IM + PM patients, PRU was significantly lower in the prasugrel group than in the clopidogrel group throughout the study. Among EM patients, the incidence of major adverse cardiovascular events (MACE) at 24 weeks was 11.8% in the prasugrel group and 11.9% in the clopidogrel group [hazard ratio (HR): 0.99, 95% confidence interval (CI): 0.50–1.96]. Among IM + PM patients, the incidence of MACE was 9.3% in the prasugrel group and 12.5% in the clopidogrel group (HR: 0.78, 95% CI: 0.45–1.35). The incidences of major, minor, and clinically relevant bleeding were similar between the two groups for each genotype. Conclusions Prasugrel showed more consistent antiplatelet effects than clopidogrel in Japanese ACS patients irrespective of the CYP2C19 phenotype.

BACKGROUND Anticoagulation is recommended as standard of care for venous thromboembolism (VTE) (pulmonary embolism [PE]/deep vein thrombosis [DVT]), for which unfractionated heparin (UFH) and warfarin are used in Japan. In the multi-regional AMPLIFY study, a fixed-dose regimen of apixaban alone was non-inferior to conventional therapy for treatment of PE/DVT and was associated with significantly fewer bleeding events. METHODS AND RESULTS Japan phase 3 study (AMPLIFY-J), randomized, active-controlled, open-label study in Japanese subjects with acute PE/DVT, was designed based on AMPLIFY. Key objectives were to investigate safety and efficacy of apixaban in symptomatic PE/DVT subjects during 24-week treatment. UFH/warfarin was used as control treatment. Apixaban was initiated at 10 mg twice daily for 7 days, followed by 5 mg twice daily for 23 weeks. All endpoints and imaging for thrombotic burden were assessed by an event adjudication committee. Eighty subjects were randomized, 33 subjects (41.3%) were aged

Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4+ T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers.Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 μg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4+ T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein.Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8+ and/or CD4+ T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8+ T-cell responses, two patients reacted to previously identified HER263-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells.Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8+ and/or CD4+ T-cell immune responses.

Plasma levels of granulocyte-derived elastase (GE-XDP), D-dimer, and soluble fibrin (SF) were examined in 177 patients with disseminated intravascular coagulation (DIC) of various etiologies. Plasma levels of GE-XDP and D-dimer, but not SF, were significantly high in patients with sepsis and solid cancer. The ratio of GE-XDP/D-dimer was significantly high in patients with trauma, burn, and sepsis, suggesting that fibrinolysis due to GE-XDP may be dominant in DIC. Plasma levels of GE-XDP and D-dimer, but not SF, were significantly high in patients with overt DIC and correlated with DIC score. Plasma levels of GE-XDP, but not SF, correlated significantly with D-dimer. Plasma levels of D-dimer, but not SF, correlated significantly with plasmin plasmin inhibitor complex (PPIC). Plasma levels of GEXDP and D-dimer, but not SF, were significantly high in nonsurvivors. Plasma levels of GE-XDP, but not SF, correlated significantly with sepsis-related organ failure assessment (SOFA) score. These results suggest that GE-XDP is a potentially useful marker for the diagnosis of overt-DIC and as a predictor of organ failure-related outcome.

Thrombin induced a shape change of UT-7/TPO, a thrombopoietin-dependent human megakaryocytic cell line. Expression of myosin light chain (MLC) kinase was negligible in UT-7/TPO cells, while Rho-kinase and protein kinase C (PKC) were detected. Thrombin stimulated both monophosphorylation at Ser19 and diphosphorylation at Thr18 and Ser19 of 20 kDa MLC, as well as phosphorylation of myosin-binding subunit (MBS) and PKC-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI). The Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-(1-aminoethyl)-N-(4-phynidyl) cyclohexane-carboxamide dihydrochloride, monohydrade] strongly inhibited thrombin-induced shape change, MBS phosphorylation, and mono- and diphosphorylation of MLC. The PKC inhibitor GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) partially inhibited thrombin-induced shape change and MLC diphosphorylation even at the concentration that completely inhibited thrombin-induced CPI phosphorylation. In shape-changed UT-7/TPO cells induced by thrombin, phosphorylated MBS and CPI were colocalized with diphosphorylated MLC at pseudopods, whereas monophosphorylated MLC was mainly located in the cortical region. The accumulation of diphosphorylated MLC was blocked by preincubation with either Y-27632 or GF109203X. These results suggest that Rho-kinase is responsible for the induction of MLC phosphorylation in thrombin-induced shape change of UT-7/TPO cells and that myosin phosphatase inactivation through Rho-kinase-MBS and PKC-CPI pathways could be necessary for enhancement of MLC diphosphorylation which promote the pseudopod formation.

Vascular events caused by arteriosclerosis are the major cause of death in patients under hemodialysis (HD). Arteriosclerosis is associated with lipoprotein abnormalities such as increased serum levels of low-density lipoprotein (LDL), especially of modified LDL (M-LDL) and oxidized LDL (Ox-LDL). We examined the relationship between markers of arteriosclerosis, hemostasis, and lipid metabolism in patients with chronic renal failure, hyperlipidemia, and healthy volunteers. In patients under HD, the serum levels of total cholesterol, LDL, and triglyceride (TG) were decreased, but the serum levels of M-LDL were increased compared to HL and healthy volunteers. In patients with CRF, the serum levels of OxLDL in patients under HD were lower than in those under continuous ambulatory peritoneal dialysis or conservative therapy. The plasma levels of antithrombin and protein C were significantly lower and the plasma levels of thrombomodulin were significantly higher in patients under HD compared to those under conservative therapy. These data show that patients under HD were more in hypercoagulable state than those under conservative therapy. Among patients under HD, only the plasma levels of von Willebrand factor were significantly increased in patients with more than 30 U/L of Ox-LDL compared to those with less than 30 U/L of Ox-LDL. There was no significant difference in the tests of arteriosclerosis among M-LDL values and OxLDL values. These findings suggest that abnormalities of lipid are not the main risk factor for arteriosclerosis disease in patients under HD.

We measured the plasma level of fibrinogen in 560 patients with disseminated intravascular coagulation (DIC) and evaluated its relationship with outcome and with other hemostatic markers. Forty-seven percent of patients had >200 mg/dL of plasma fibrinogen and 24% had

Antiplatelet therapy is widely used with proven benefit for the prevention of further ischemic complications in patients with coronary heart disease and stroke. Treatment guidelines for acute coronary syndrome and percutaneous coronary intervention now recommend the use of oral antiplatelet agents including clopidogrel in combination with aspirin (dual antiplatelet therapy: DAPT) for the prevention of recurrent ischemic events. The limitations of DAPT with clopidogrel include the potential for low response to clopidogrel identified through platelet reactivity or genetic testing, or slower return to normal platelet activity in patients who received clopidogrel prior to emergent or planned surgical procedures. This review will discuss the pharmacologic properties and clinical studies about new antiplatelet drugs including prasugrel, ticagrelor and PAR-1 inhibitors.

Protein kinase C (PKC)-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1delta catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A(2) analog STA(2) resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca(++) ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2-loaded platelets with no change in cytosolic Ca(++). The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC(20)) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC(20) with similar IC(50) values. These findings suggest that the activation of PKC by PMA induces MLC(20) phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA(2)-induced MLC(20) phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA(2)-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA(2)-induced MLC(20) phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA(2)-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC(20) phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase-mediated regulation of myosin phosphatase, during agonist-induced platelet secretion. (Blood. 2001;97:3798-3805)

Before hemodialysis (HD), plasma levels of tissue factor (TF), free-TF pathway inhibitor (TFPI) and thrombo modulin (TM) were significantly higher in patients with HD than in healthy volunteers. Plasma levels of (T-F) TFPI and plasmin plasmin inhibitor complex (PPIC) were significantly higher in patients with HD than in healthy volunteers. During HD, plasma levels of TF and (T-F) TFPI were not significantly increased, but plasma levels of total TFPI and free TFPI at 1 hour after and at the end of HD were significantly increased, compared with levels before start of HD. Plasma level of PPIC 1 hour after start of HD was significantly higher than before start of HD, and plasma levels of thrombin antithrombin com plex (TAT). PPIC. D-dimer. TM, and protein C (PC) at the end of HD were significantly higher than before start of HD. In patients with thrombosis complications, plasma TF levels were significantly higher than in patients without thrombotic com plications during HD. Plasma levels of PC were significantly lower in patients with thrombotic complications than in patients without thrombotic complications. There was no significant difference between both groups during HD in hemostatic pa rameters, with the exception of TF and PC. Hemostatic abnormalities existed in patients with HD; espe cially, increased TF and decreased PC might cause thrombotic complications.

This study evaluated hemostatic data in 28 patients with newly diagnosed acute promyelocytic leukemia (APL) and 15 patients with relapsed APL. Activated partial thromboplastin time and prothrombin time were prolonged at initial onset of APL. Plasma level of fibrinogen was significantly decreased in patients with initial disease of APL, but it was not decreased significantly during the relapse of APL. Plasma fibrin and fibrinogen degradation products levels were significantly increased and platelet counts significantly decreased in both groups. Plasma levels of antiplasmin significantly decreased at initial onset but not during relapse. Plasma levels of antithrombin were within normal range in patients with initial disease but significantly decreased in those with relapse. Plasma levels of D-dimer, soluble fibrin monomer (sFM), plasmin-plasmin inhibitor complex (PPIC), and thrombin antithrombin complex (TAT) levels were significantly high in both groups. Plasma levels of PPIC, sFM, and D-dimer were significantly higher at initial onset of APL than during relapse. However, there was no significant difference in DIC score between patients with initial onset and those with relapse; plasma levels of tissue factor (TF) significantly increased in both groups, but they were significantly higher at initial onset of APL than during relapse. TF and tissue type plasminogen activator (t-PA) antigen levels in leukemic cell lysate were significantly increased in both groups, and they were significantly lower during relapse than at initial onset. Hemostatic abnormalities occurring in patients with relapsed APL might be the result of the decrease of TF and t-PA in leukemic cells. These findings suggest that DIC in APL patients with relapse might not be caused only by TF and t-PA and thus should be treated with different therapy from patients with initial onset of APL.

Fas, a member of the tumor necrosis receptor superfamily, is 36 kD surface protein containing a single transmembrane region and induces apoptosis by Fas-Fas ligand binding. Soluble Fas (sFas) is produced as the form lacking 21 amino acid residues containing the transmembrane domain by alternative splicing. We found that the plasma sFas levels of 33 patients with thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), 19 patients with disseminated intravascular coagulation (DIC), and 10 non-DIC patients with multiple organ failure (MOF) were significantly higher than those of 21 non-DIC patients without organ failure and those of 25 healthy volunteers. The plasma sFas ligand levels of the TTP/HUS patients, the DIC patients, and the non-DIC patients with MOF were significantly higher than those of the non-DIC patients without organ failure and those of the healthy volunteers. The plasma sFas levels were significantly correlated with the plasma sFas ligand levels in all subjects. The plasma thrombomodulin (TM) levels were increased significantly in the TTP/HUS patients, the DIC patients, and the non-DIC patients with MOF compared with the levels of the non-DIC patients without organ failure and the healthy volunteers. The plasma sFas antigen levels were correlated significantly with the plasma TM levels in all subjects. These findings suggest that the increases of sFas and sFas ligand that cause apoptosis might be related to the vascular endothelial cell injuries in TTP and DIC with organ failure.

Activity and expression of four major protein serine/threonine (Ser/Thr) phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), protein phosphatase type 2B (PP2B) and protein phosphatase type 2C (PP2C) were evaluated in normal peripheral leukocytes, and in various leukemic cells from patients with acute myelogenous leukemia (AML), common acute lymphocytic leukemia (cALL), or chronic lymphocytic leukemia (CLL). PP1 was the most abundant phosphatase in blood cells, and relative abundance of each phosphatase was: PP1PP2APP2B approximately = PP2C. PP1 activity and its expressions were higher in blasts of AML-M4 and -M5 than in cells of AML-M1, cALL and CLL. PP2A activity and its expression were higher in blasts of AML-M3, -M4 and -M5 than in cells of AML-M1, cALL and CLL. Activity and expression of both PP1 and PP2A in normal monocytes were highest, and PP2A activity in normal neutrophils was lowest among normal leukocytes. PP2B activity and its expression were higher in blasts of AML-M2, -M3 and normal lymphocytes. PP2C activity and its expression were relatively constant in various leukemic cell types. Activities of PP1 and PP2A of AML blasts correlated positively with the expression of CD11b, whereas activities of PP1 and PP2B correlated negatively with the expression of CD7. Thus, each phosphatase was ubiquitously but differently expressed in various leukemic cell types and in normal leukocytes. These data also suggest that expressions of PP1, PP2A and PP2B are relatively low in leukemic blasts arresting at the stage of early pluripotent stem cells, and are differently modulated during the course of myelomonocytic commitment and maturation.

In de novo t(8;21) AML which shows terminal neutrophilic differentiation, the BCL-2 expression was found to be significantly lower than that in types of other AML regardless of the phenotypic differentiation status. An inverse correlation between BCL-2 expression and the S/G2/M population cells was observed in AML. The S/G2/M population in t(8;21)AML was larger than in the other types of AML. In t(8;21)AML, spontaneous apoptosis after a 12-h liquid culture was prominent, and the autonomous DNA synthesis after a 72-h liquid culture was low. G-CSF and IL-5 promoted the colony formation of t(8;21)AML cells. The data suggest that, in vivo, the low BCL-2 in t(8;21)AML induced entry of cells from the G0/G1 phase to S phase, but the cells easily die by apoptosis, in vitro. The low BCL-2 expression and the supportive effects of G-CSF and IL-5 in t(8;21)AML is thought to be a key phenomenon which might be related to the formation of the in vivo blood picture, such as prominent neutrophilic differentiation and eosinophilia. Cellular extracts from t(8;21)AML cell line Kasumi-1 bound to both the AML1 and CRE binding sites in the bcl-2 promoter, but none of the cellular extracts from de novo t(8;21)AML bound to either of these sites. The DNA binding activity of transactivators in de novo t(8;21)AML is different from that in Kasumi-1 cells probably due to the phosphorylation status.

Circulating platelets are susceptible to various stimuli in vessels, and a certain portion of platelets may thereby form native microaggregates (NMAs), which can be visualized by a bother scanning electron microscopy (SEM). We developed an easy method to evaluate the existence and extent of NMAs and the subsequent spontaneous platelet aggregation (SPA) induced by not any agonist but stirring force with a novel platelet aggregometer (PA-200; Kowa, Tsukuba, Japan) employing a particle-counting method based on laser light scattering. In this system, platelet-rich plasma obtained by a delicate centrifugation was infused in a cuvette to replace the content and to avoid stirring shear. An insertion attachment was designed in a coaxial shape to maintain a constant sample volume. Our data indicated that NMAs, tentatively defined as a “small” division by their light scattering (25–400 of the intensity threshold), could be detected by not only a complicated SEM but also by our simple system with the PA-200 in a few minutes. No correlation was found in the comparison of the existence of NMAs and the SPA generation, in aliquot platelets, respectively. These results suggested that NMAs and SPA might be mutually different cluster and have meanings as representations of platelet status.

Various hemostatic and vascular endothelial cell markers were measured in patients with disseminated intravascular coagulation (DIC), non-DIC, or thrombotic thrombocytopenic purpura (TTP) and in healthy volunteers to examine the relationships between the hemostatic abnormalities or vascular endothelial cell injuries and the patients' outcomes. Although the plasma levels of soluble fibrin monomer, thrombin-antithrombin complex, plasmin-plasmin inhibitor complex, and D-dimer were significantly increased in the DIC patients, there were no significant differences in these markers between the DIC patients who survived and those who died, suggesting that these markers might not be directly related to the patient outcome. The plasma thrombomodulin (TM) levels in the DIC and TTP patients were significantly higher than those in the healthy volunteers, and the plasma TM levels in the patients who died were significantly higher than those in the patients who survived. These findings showed that the TM level reflected the outcome, and that the outcome of the diseases underlying DIC and TTP might depend on vascular endothelial cell injuries. The plasma protein C and antithrombin activities were markedly reduced in the DIC, non-DIC, and TTP patients who died compared to those who survived. These findings suggest that reduced plasma antithrombin and protein C activities are useful markers of systemic vascular endothelial injuries. Although the plasma tissue factor (TF) levels were significantly increased in the DIC patients, there was no significant difference in the plasma TF levels between the DIC patients who died and those who survived. In conclusion, we found that the outcome of the diseases underlying DIC and TTP is related to vascular endothelial cells, and that plasma TM, antithrombin, and protein C are useful markers for systemic vascular endothelial cell injury.

SummaryAlterations in platelet aggregability may play a role in the pathogenesis of qualitative platelet defects associated with cardiopulmonary bypass (CPB). We circulated fresh heparinized whole blood through tubing sets coated with heparin (C group, n = 10) and through non-coated sets (N group, n = 10) as a simulated CPB circuit. Shear stress (108 dyne/cm2)-induced platelet aggregation (hSIPA), plasma von Willebrand factor (vWF) activity and platelet glycoprotein (GP) Ib expression were measured, before, during, and after this in vitro set up of circulation. In the two groups, the extent of hSIPA significantly decreased during circulation and was partially restored after circulation. Decreases in the extent of hSIPA were significantly less with use of heparin-coated circuits. There was an equivalent reduction in plasma vWF activity, in the two groups. Expression of platelet surface GP Ib decreased significantly during circulation and recovered after circulation. Reduction of surface GP Ib expression during circulation was significantly less in the C group than that in the N group. Decrease in surface GP Ib expression correlated (r = 0.88 in either group) with the magnitude of hSIPA, in the two groups. The progressive removal of surface GP Ib was mainly attributed to redistribution of GP Ib from the membrane skeleton into the cytoskeleton. Our observations suggest that use of heparin-coated circuits partly blocks the reduction of hSIPA, as a result of a lesser degree of redistribution of GP Ib.

In patients with coronary artery disease (CAD), there is an increasing therapeutic need among interventional cardiologists to conduct dual antiplatelet therapy (DAPT) whose duration is shorter than current guideline-recommended 6-12 months after the implantation of drug-eluting stents. However, no clinical grounds sufficient to rationalize the need are available.To define the optimal duration of DAPT and to examine the safety and efficacy of the Endeavor zotarolimus-eluting stent (E-ZES) in real-world Japanese patients with CAD.The present prospective, nonrandomized, multicenter, controlled study is uniquely designed to examine the analysis set to be formulated after integrating two different databases consisting of the following two study arms: the 3-month DAPT arm, in which 1,210 patients were consecutively enrolled at 106 medical institutions; and the 12-month DAPT arm, in which 1,210 patients will be consecutively extracted from the Endeavor Japan post-marketing surveillance at 60 medical institutions. The primary endpoint is "net adverse cardiac and cerebrovascular events-death, myocardial infarction, cerebrovascular accident, and major bleeding)" at 12 months after implantation. The secondary endpoints are as follows: major adverse cardiac events at 1, 3, 6, 9, and 12 months after implantation; target vessel revascularization and target lesion revascularization at 9 and 12 months after implantation; and stent thrombosis, DAPT compliance, and bleeding events at 12 months after implantation. Noninferiority in the E-ZES's profiles between the study arms will be investigated.The present study will provide insight into the optimal duration of DAPT after the E-ZES implantation in individual, real-world patients with CAD.

In extracts of human platelets, three isoenzymes of cyclic nucleotide phosphodiesterase (PDE), namely, PDE2, PDE3, and PDE5, were identified; activities of PDE1 and PDE4 were not detected. In human platelets, the cGMP-hydrolytic activity was about six times higher than the cAMP-hydrolytic activity, and PDE5 and PDE3 are the major phosphodiesterase isoenzymes that hydrolyze cGMP and cAMP, respectively. PDE5 exhibited organ-specific expression in humans, and platelets were among the tissues richest in PDE5. A novel inhibitor of PDE5, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl ] piperidine-4-carboxylate sesquihydrate (E4021), was a potent and highly selective inhibitor of human platelet PDE5. However, E4021 (up to 10 microM) did not inhibit 9,11-epithio-11,12-methano-thromboxane A2-induced platelet aggregation, in vitro. E4021 plus SIN-1 (3-morpholino-sydnonimine), at concentrations that had little effect individually, inhibited aggregation. These results suggest the unique distribution of phosphodiesterase isoenzymes in human platelets and the PDE5 inhibitors might be useful as a new class of antiplatelet drugs.

Cyclosporin A and FK506, at concentrations that inhibited phosphatase activity of calcineurin in HL-60 cellular lysates, augmented the proliferation of leukemic HL-60 cells. These immunosuppressants did not affect 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced monocytic differentiation of HL-60 cells, but did abrogate the 1,25(OH)2D3- induced inhibition of HL-60 cell growth. Treatment with 20 nmol/L 1,25(OH)2D3 led to a progressive increase in calcineurin phosphatase activity in subcellular fractions from HL-60 cell extracts, the increase in this activity appeared to parallel the phenotypic and functional changes of HL-60 cells during monocytic differentiation induced by 1,25(OH)2D3. Immunoblot analysis indicated that increase in calcineurin activity was concordant with the increased expressions of calcineurin catalytic subunit isozymes, calcineurin A alpha (CNA alpha), and calcineurin A beta(CNA beta), and a regulatory calcineurin B subunit (CNB) proteins, which were preceded by a coordinate increase in the levels of CNA alpha, CNA beta and CNB mRNAs. The expression of calmodulin remained unaltered throughout 1,25(OH)2D3-induced monocytic differentiation. These results suggest that calcineurin activation has a net negative effect on HL-60 cell proliferation, and that the increased expression of calcineurin may be involved in 1,25(OH)2D3- induced inhibition of HL-60 cell proliferation.

SummaryThe association of protein Ser/Thr phosphatase type 1(PP1) and type 2A (PP2A) with the cytoskeleton (Triton X-100 insoluble residue) during human platelet activation was investigated. In unstimulated platelets, 40% of total PPl-like activity was present in the Triton-insoluble cytoskeleton, while only 10% of the total PP2A-like activity was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fold increase in PPl-like activity and a 7-fold increase in PP2A-like activity, respectively, in the cytoskeletal fraction, under aggregating conditions. Immunoblot analysis revealed that thrombin treatment increased association of PP1 catalytic subunit isozymes (PPlα, PPlβ, PP1γ) and PP2A catalytic subunit with the cytoskeleton, with concomitant decrease of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated PP1 and PP2A depended on the dose of thrombin which could activate platelets. Agonist-induced redistribution of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a prostaglandin I2 analog). Interaction of PP2A with cytoskeletal proteins strongly correlates with aggregation, whereas the association of PP1 with cytoskeleton can be detected upon platelet activation, even in the absence of aggregation. Co-extraction of protein kinase C and myosin light chain kinase with the cytoskeleton eventually translocated to the cytoskeleton, but only during aggregation. These results suggest that differential translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation, and their association with cytoskeletal proteins may regulate phosphorylation levels together with protein kinases in platelets.

Two potent inhibitors of protein phosphatase type 1 (PP1) and type 2A (PP2A), calyculin A (CAL-A) and okadaic acid (OKA), inhibited human platelet aggregation induced by thrombin, collagen and 9,11-epithio-11,12-methano-thromboxane A 2 (STA 2 ). IC 50 values of CAL-A and OKA for STA 2 -induced aggregation were 53 nM and 3.5 μM, respectively. These drugs also inhibited thrombin-induced [ 14 C]serotonin secretion of platelets. CAL-A and OKA elicited phosphorylation of certain proteins with an apparent M r (× 10 −3 ) of 200, 60, 50 and 20 light chain of myosin (MLC). Agonist-induced 47,000 M r protein phosphorylation was strongly inhibited by these compounds, whereas phosphorylation of 20,000 M r MLC was enhanced. The increase in 50,000 M r protein phosphorylation by CAL-A and OKA was observed in the presence of agonists, and the 50,000 M r phosphorylation may be involved in the inhibition of platelet activation by these compoundes. Subcellular analysis of the phosphatase activity in human platelets showed that MLC phosphatase activity was present mainly (approx. 78%) in the cytosolic fraction. Chromatography of human platelet extract on heparin-Sepharose resolved two peaks of MLC phosphatase activity: PP2A in 0.1 M NaCl eluate and PP1 in 0.5 M NaCl eluate. PP2A and PP1 isozymes (PP1α PP1α and PP1δ) have also been identified in human platelets, by cross-reactivity with polyclonal antibodies against (PP2A and PP1 isozymes, respectively. These results suggest that PP1 and/or PP2A may play an important role in the process of platelet activation by regulating levels of phosphorylation of certain proteins.

Activity of protein phosphatase measured in the absence of divalent cations was decreased by 50% during all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation into the granulocytic phenotype. Treatment of HL-60 cells with ATRA led to a dramatic decrease in the amount of protein phosphatase type 2A (PP2A) protein, whereas that of protein phosphatase type 1 (PP1) protein was relatively constant, as detected by immunoblotting with antibodies specific to PP1 and PP2A. The decreased phosphatase activity may be mainly due to a decrease in the expression of the PP2A protein. The mRNA level of PP2A beta was markedly decreased within 5 h after addition of ATRA, but there was only a slight increase in the mRNA level of PP2A alpha. Selective down-regulation of PP2A beta mRNA clearly preceded the cell differentiation induced by ATRA treatment. Thus, PP2A is down-regulated during ATRA-induced differentiation of HL-60 cells into granulocytes.

We searched for a possible role for protein kinase C in the growth of human erythroid progenitor cells, using pharmacologic approaches. Two protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine, dose-dependently inhibited the growth of immature erythroid progenitor cells (BFU-E) induced by interleukin 3 (IL-3) plus erythropoietin (Ep) or granulocyte macrophage colony-stimulating factor (GM-CSF) plus Ep whereas a weaker analog of H-7, N-(2-guanidinoethyl)-5-isoquinoline sulfonamide (HA-1004), had no effect on the number of BFU-E. These three compounds had no effect on the growth of mature erythroid progenitor cells (CFU-E) stimulated by Ep. The culture of accessory cell-depleted bone marrow demonstrated that the effects of these compounds on colony formation do not appear to be mediated by accessory cells. The potential of these compounds to inhibit the GM-CSF-dependent growth of KG-1 cells correlated well with the extent of their inhibitor of protein kinase C activities from KG-1 cells. Thus, the protein kinase C system is apparently involved in the growth of BFU-E, supported by IL-3 or GM-CSF. The growth signal for CFU-E transduced by Ep may be achieved through other systems.

A 19-year-old man with common acute lymphoblastic leukemia (ALL) developed necrotizing leukoencephalopathy (NL) after extensive chemotherapy and irradiation. Clinicians should exercise care in the treatment of leukemia, since chemotherapy has shown a trend of becoming more intensive, and the survival time of patients with leukemia is being extended.