Makoto Kaneko

Evaluation of safety of endoscopic biopsy without cessation of antithrombotic agents in Japan

BackgroundAlthough guidelines in Japan recommend the cessation of antithrombotic agents before endoscopic biopsy, the safety of biopsy without the cessation of these agents has not been evaluated to date in this country. Therefore, we aimed to assess the feasibility of biopsy without cessation of antithrombotic agents in Japan.MethodsThis was a prospective single-arm study from a single institution. From May 2010 to November 2011, 112 outpatients who were receiving antithrombotic agents because of their high-risk status for a thromboembolic event (after implantation of coronary stent, after valve replacement, or a previous history of thromboembolic event or heart failure due to atrial fibrillation) were enrolled. We evaluated the rate of severe bleeding complications within 2xa0weeks after endoscopy and the endoscopic bleeding time (EBT) after biopsy in patients who underwent biopsy for endoscopic findings requiring pathology assessment.ResultsAmong the 112 participants, 101 biopsies were performed for 48 and 12 outpatients who had had esophagogastroduodenoscopy and colonoscopy, respectively. All the biopsies provided enough specimens to evaluate pathologically. Hemostasis after biopsy was confirmed for all biopsies during endoscopic observation. No patients complained of any bleeding symptoms in the 2-week observation period after biopsy (0/101; 95% confidence interval [CI] 0–3.6%). Concerning the EBT (median 2.2xa0±xa01.8xa0min, range 0.5–9xa0min), there were no significant differences between patients receiving single antithrombotic agents and those receiving multiple agents (2.4xa0±xa01.4 vs. 2.1xa0±xa02.1xa0min), nor were there any significant differences between patients not receiving and receiving warfarin (2.3xa0±xa01.8 vs. 2.2xa0±xa01.8xa0min).ConclusionBiopsy without cessation of antithrombotic agents, as recommended in Western guidelines, can also be acceptable for Japanese people if performed carefully.

Hepatic stellate cell damage may lead to decreased plasma ADAMTS13 activity in rats.

ADAMTS13 is gaining attention, because its deficiency causes thrombotic thrombocytopenic purpura. Although its regulatory mechanism is not fully understood, we wondered if hepatic stellate cells (HSCs) play a role, because ADAMTS13 mRNA is exclusively expressed in the liver and primarily in HSCs. Plasma ADAMTS13 activity was markedly reduced in dimethylnitrosamine‐treated rats, where HSC apoptosis is an essential event, but not in carbon tetrachloride‐ or thioacetamide‐treated rats without HSC apoptosis. Furthermore, plasma ADAMTS13 activity was also reduced in 70% hepatectomized rats, where HSC loss occurs. These results suggest that HSC may be involved in the regulation of plasma ADAMTS13 activity.

Development of carboxymethyl cellulose nonwoven sheet as a novel hemostatic agent.

Carboxymethyl cellulose (CMC) is a plant-derived material that has high biocompatibility and water solubility. We developed a CMC nonwoven sheet as a hemostatic agent by carboxymethylating a continuous filament cellulose nonwoven sheet. The CMC nonwoven sheet was able to absorb water and dissolve in it. The rates of absorption and dissolution depended on the degree of carboxymethylation. After dissolving in blood, CMC accelerated clot development (possibly owing to the incorporation of CMC into fibrin fibers) and increased the viscosity of the blood, both of which would contribute to the improved blood clotting of an injured surface. In vivo experiments using a rat tail cutting method showed that a CMC nonwoven sheet shortened the bleeding time of the tail when applied to the cut surface. The hemostatic effect of the CMC nonwoven sheet was almost at the same level as a commercial hemostatic bandage. These results suggest that a CMC nonwoven sheet could be used as a novel sheet-type hemostatic agent.

Increased production of ADAMTS13 in hepatic stellate cells contributes to enhanced plasma ADAMTS13 activity in rat models of cholestasis and steatohepatitis

Although hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity. To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle alpha-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle alpha-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS13, was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle alpha-actin in the livers of rats fed a choline-deficient L-amino acid-defined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part, to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.

Lysophosphatidic acid protection against apoptosis in the human pre-B-cell line Nalm-6

Lysophosphatidic acid (LPA) promotes survival, growth, differentiation, and motility in a variety of cell types, and has been reported to act as a cell survival and growth factor in B lymphocytes. Autotaxin (ATX), through its lysophospholipase D activity, generates LPA from lysophosphatidylcholine (LPC). In this study, we investigated the effects of LPA and also the expression of ATX and LPA receptor, in the human pre‐B‐cell line Nalm‐6. It was found that LPA protects Nalm‐6 cells against both spontaneous and staurosporine‐induced apoptosis. Furthermore, ATX expression on the cell surface and ATX activity in the cell lysate were detected. No accumulation of LPA in the culture medium was, however, detected when the Nalm‐6 cells were cultured with LPC. The pre‐B cells were found to express the mRNA transcript for lipid phosphate phosphatase‐1 and LPA degradation was inhibited in the presence of the phosphatase inhibitor vanadate, it was surmised that LPA production in the culture medium may have been masked by LPA degradation by this ecto‐phosphatase. Abundant expression of LPA receptors, especially, LPA4, was detected by a real‐time polymerase chain reaction technique. Our results suggest an important and autocrine role of LPA in the survival of this well‐established model cell line, although the direct involvement of ATX in the production of LPA in these cells was not confirmed.

Prognostic Factors in Elderly Patients with Acute Myelogenous Leukemia: A Single Center Study in Japan

We retrospectively analyzed data of 47 patients aged 60 years or older, hospitalized in our institution with the diagnosis of acute myelogenous leukemia (AML), and searched for prognostic factors. Induction with anthracyclines significantly correlated with better complete remission (CR) rate (P =0.0016) and overall survival (OS) (P <0.001). Another factor significantly affecting CR rate was higher age (>70 years) (P =0.042). Therapy-non-related factors predictive for shorter OS in univariate analyses were age older than 70 years (P =0.003), percentage of blasts in bone marrow more than 80% (P =0.048), serum lactate dehydrogenase level higher than 250 u2009 U u2009 l m 1 (P =0.032). In stepwise cox proportional hazard regression model, all the four factors predictive for poor OS remained to be independently and significantly prognostic for shorter OS. Only two patients receiving anthracyclines died within 30 days and the frequency was not different from that in patients not receiving anthracyclines. The use of anthracyclines as induction therapy is recommended even in the elderly patients.

Causes of Thrombocytopenia in Chronic Hepatitis C Viral Infection

We retrospectively studied 89 patients with chronic hepatitis C virus (HCV) infection, including 50 chronic hepatitis (CH) cases, 18 liver cirrhosis (LC) cases, and 21 LC with hepatocellular carcinoma (LC + HCC) cases, with regard to various factors related with thrombocytopenia. The platelet count decreased with the stage advancement of liver diseases. Multiple regression analysis revealed that splenomegaly and von Willebrand factor (vWF) were explanatory variables that correlated with thrombocytopenia. Splenomegaly appears to be the most responsible factor, although there are a considerable number of thrombocytopenic cases without splenomegaly, suggesting other factors may also be responsible. The vWF level is inversely correlated with the platelet count. Soluble thrombomodulin, a marker of endothelial dysfunction, increases with the advancement of liver fibrosis. It is positively correlated with vWF and inversely with the platelet count. Our present results imply that vascular endothelial dysfunction is also involved in thrombocytopenia during chronic HCV infection.

Bevacizumab and Aflibercept Activate Platelets via FcγRIIa.

PURPOSEnTo confirm the formation of a drug-growth factor complex and investigate the effects of three VEGF inhibitors in the activation of platelets.nnnMETHODSnGrowth factors and individual drugs were mixed and incubated. Scattered light intensity was measured by dynamic light scattering (DLS) to monitor the formation of drug-growth factor complex. Blood samples were obtained from 16 subjects (5 AMD patients and 11 healthy volunteers). Platelets obtained from the platelet-rich fraction by centrifugation were washed and resuspended in HEPES/Tyrode buffer. Platelet aggregability was assessed using a light transmission aggregometer in the presence of VEGF inhibitors and growth factors.nnnRESULTSnIn DLS study, a mixture of bevacizumab and VEGF-A showed one peak with a relatively gentle slope, indicating a heterogeneous mixture of multimeric bevacizumab-VEGF-A complexes. In aggregation study, no detectable aggregation was observed in the presence of ranibizumab, while significant aggregation was observed in the presence of VEGF-A and bevacizumab in five cases (one AMD patient and four healthy volunteers), VEGF-B and aflibercept in two cases (two volunteers), and placental growth factor (PlGF) and aflibercept in one case (one volunteer). No aggregation was observed when FcγRIIa antibody was added beforehand.nnnCONCLUSIONSnA complex composed of bevacizumab or aflibercept, but not ranibizumab, and growth factors activates platelets via FcγRIIa.

Mechanisms of platelet retention in the collagen-coated-bead column.

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.

Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells

BackgroundThe migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P.MethodsS1P was measured using two-step lipid extraction followed by high-performance liquid chromatography (HPLC) separation while other phospholipids were determined by an enzymatic assay. The expression of S1P and lysophosphatidic acid receptors on Jurkat T cells was examined by RT-PCR and flow cytometry. Jurkat cell migration by S1P and the supernatant of activated platelets (SAP) was evaluated by a modified Boyden’s chamber assay.ResultsS1P1 receptor was confirmed to be expressed on Jurkat T cell by RT-PCR and flow cytometry. S1P at 10-100xa0nM induced strong Jurkat cell migration, which was inhibited by the S1P1 (and S1P3) antagonist VPC23019 and the Gi inactivator pertussis toxin (PTX). We found that the supernatant (releasate) of human platelets activated by collagen stimulation, which contains S1P abundantly, induced Jurkat cell migration and that the migration was inhibited by VPC23019 and PTX. In addition, human serum, into which platelet contents (including S1P) are fully released, induced the Jurkat cell migration, which was also inhibited by VPC23019.ConclusionsOur findings suggest that platelet-derived S1P induces Jurkat T cell migration possibly via S1P1. S1P may be a key molecule involved in the responses triggered by platelet-T cell interactions, including atherosclerosis.