Sumiyoshi Naito

Synthetic selective inhibitors of coagulation factor Xa strongly inhibit thrombin generation without affecting initial thrombin forming time necessary for platelet activation in hemostasis

Summary.  DX‐9065a and JTV‐803, synthetic selective inhibitors of activated factor X (FXa), have recently been demonstrated as strongly effective antithrombotic agents in animal thrombosis models, yet with a low risk of bleeding. The aim of the present study was to elucidate these characteristics. Using a chromogenic assay with purified coagulation factors, 73.9% of thrombin generation was suppressed by the addition of DX‐9065a (0.20 µm) and 75.7% by JTV‐803 (0.18 µm). Inhibition by argatroban (0.19 µm) was less (36.0%) and initial thrombin forming time (T50), the time required to generate 50% thrombin activity in vitro, which is considered important for platelet aggregation in hemostasis, was significantly prolonged by argatroban. In contrast, DX‐9065a and JTV‐803 had no apparent influence on T50, suggesting that initial thrombin was formed immediately, as in the control. We also investigated platelet aggregation in defibrinated plasma induced by tissue factor, to clarify whether initial thrombin contributes to hemostasis. Aggregation was not affected by the addition of either FXa inhibitor, whereas it was significantly reduced by argatroban. Our results suggest that initial thrombin, which is formed despite the presence of a FXa inhibitor, can activate platelets. We concluded that DX‐9065a and JTV‐803 are able to inhibit thrombin generation significantly without affecting the formation of initial thrombin for platelet activation, which may contribute to hemostasis through the preservation of normal bleeding time.

APTT reagent with ellagic acid as activator shows adequate lupus anticoagulant sensitivity in comparison to silica-based reagent

Summary.  Background:  Lupus anticoagulant (LA) is an antibody that interferes with phospholipid‐dependent coagulation reactions. Activated partial thromboplastin time (APTT) is widely used as a test for LA screening. APTT reagents are composed of activators, such as silica or ellagic acid, and phospholipids, and APTT reagents with silica are recommended for LA screening because of greater sensitivity. However, the effects of activators on LA activity have not been adequately investigated.

Factor Xa inhibitors: new anti-thrombotic agents and their characteristics.

Factor Xa (FXa) is a key enzyme that is positioned at the convergence of the intrinsic and extrinsic pathways in the blood coagulation cascade, and inactivation by a specific FXa inhibitor effectively prevents the generation of thrombin. Various types of low molecular weight (LMW) heparin, which function as semi-selective and indirect FXa inhibitors, are replacing unfractionated heparin (UFH) as agents for the prevention and treatment of venous thromboembolism (VTE), as well as in initial treatment for coronary events. Of those, heparinoid has been shown to be safer and more effective for the prevention of postoperative VTE than UFH, especially for treatment of heparin-induced thrombocytopenia (HIT). Further, synthetic pentasaccharide has been found to offer advantages over current thromboprophylactic regimens in a number of patients undergoing major orthopedic surgery. Other studies have shown that pentasaccharide is more effective for overall VTE in comparison with LMW heparin, though it was also associated with an increased rate of major bleeding. Synthetic, selective, and direct inhibitors to FXa, such as DX-9065a, are highly potent and orally bioavailable antithrombotic agents that have demonstrated an improved side effect profile, probably by allowing sufficient thrombin to remain for platelet activation and normal hemostasis, while preventing pathological thrombus formation. For thrombosis therapy, the most desirable type of antithrombotic agent is an orally active drug that has a broad range of effective doses and no hemorrhagic side effects. Presently, many types of direct inhibitors are in various stages of clinical trials and expected to provide significant benefits as compared to currently utilized therapy strategies.

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes.

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

Low levels of plasma protein S, protein C and coagulation factor XII during early pregnancy and adverse pregnancy outcome

It was the study objective to evaluate whether low levels of plasma protein S (PS) activity, free PS, protein C (PC) activity and coagulation factor XII (FXII) during early pregnancy are related to adverse pregnancy outcomes. Peripheral blood samples were obtained at 8-14 gestational weeks (GW) from a consecutive series of 1,220 women. The levels of plasma PS activity, free PS, PC activity, and FXII were measured. Cut-off values were defined as < 1st, < 5th, and < 10th percentiles of values obtained from 933 women whose pregnancies ended in normal deliveries without complications. PS activity of < 10th percentile yielded risks of pregnancy-induced hypertension (PIH) and severe PIH, while free PS level of < 5th percentile yielded a risk of pre-eclampsia. FXII level of < 1st percentile yielded a risk of premature delivery (PD) at < 34 GW. None was associated with PD at < 37 GW, fetal growth restriction or fetal loss. A multivariate analysis demonstrated that PS activity of < 10th percentile (odds ratio 5.9, 95 % confidence interval 1.7-18.1) and body mass index (BMI) ≥ 25 kg/m² (4.3, 1.1-13.3) were independent risk factors for severe PIH. Similarly, free PS level of < 5th percentile (4.4, 1.0-14.3) and BMI ≥ 25 kg/m² (4.0, 1.3-10.9) were independent risk factors for pre-eclampsia. In conclusion, women with low levels of plasma PS activity and free PS during early pregnancy might have increased risks of PIH, severe PIH or pre-eclampsia. Women with low FXII level might have an increased risk of PD at < 34 GW.

Verification of the guidelines for lupus anticoagulant detection: usefulness of index for circulating anticoagulant in APTT mixing test.

INTRODUCTION Lupus anticoagulant (LA) is an antibody that interferes with one or more in vitro coagulation reactions, which are dependent on interactions with protein-phospholipid complexes. For LA diagnosis, a mixing test is considered useful for differentiating the inhibitor from a factor deficiency. However, the usefulness and the index of circulating anticoagulant (ICA) in a mixing test with activated partial thromboplastin time (APTT) has not been adequately investigated, and there is scant information regarding the effects of warfarin, heparin, and hemophilia plasma on ICA. We evaluated the usefulness of ICA by investigating the correlation of that index with international normalized ratio (INR), heparin concentration, and factor VIII activity in hemophilia patients. MATERIALS AND METHODS We examined samples from 28 patients positive for LA, 23 receiving warfarin, 19 receiving unfractionated heparin, and 29 with hemophilia A, as well as 61 normal samples. APTT-SLA, Actin FSL, APTT-SP, and PTT-LA were used as reagents in this study. RESULTS The correlation coefficient values between ICA and INR, heparin concentration, and factor VIII activity ranged from 0.031-0.342, 0.764-0.843, and 0.564-0.754, respectively, with the 4 reagents. The ICA values for the LA-positive samples were significantly higher than for the normal, warfarin, heparin, and hemophilia samples with all APTT reagents. Samples with a high heparin concentration above approximately 0.5U/ml showed ICA values greater than 15. CONCLUSION ICA was able to distinguish LA-positive samples from the normal, warfarin, and hemophilia samples, but not heparin samples. ICA calculated from APTT clotting time is useful for LA diagnosis.

Increase in plasma thrombin-activatable fibrinolysis inhibitor may not contribute to thrombotic tendency in antiphospholipid syndrome because of inhibitory potential of antiphospholipid antibodies toward TAFI activation

The causes of thrombosis in antiphospholipid syndrome (APS) remain unknown, though several hypotheses in regard to hypofibrinolysis have been proposed. To clarify the mechanism, we measured plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) in APS patients. Both the TAFI antigen (TAFI:Ag) level measured with an ELISA, and thrombin-thrombomodulin-dependent TAFI activity (TAFI:Ac) were elevated in 68 APS patients as compared with those in 66 healthy controls, though they were lower than those in 46 patients with autoimmune diseases. As for the influence of antiphospholipid antibodies (aPL) on TAFI levels, the mean TAFI:Ac level in 39 SLE patients positive for APS was significantly lower than that in 27 SLE patients without APS, whereas there was no difference in TAFI:Ag between those groups. Furthermore, purified IgG from patients positive for aPL, and monoclonal aPL (EY2C9 and 23-1D) inhibited the activation of TAFI in a concentration dependent manner. These results suggest that aPL inhibits TAFI activation by affecting the function of thrombomodulin-thrombin complex through phospholipids. Although TAFI in plasma is elevated in autoimmune diseases including APS, we concluded that an elevated level is not likely a risk factor for thrombosis in APS patients, because of the inhibition of TAFI activation by aPL.

Index of circulating anticoagulant cut-off value establishment in activated partial thromboplastin time mixing test for lupus anticoagulant diagnosis

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The production of coagulation factor VII by adipocytes is enhanced by tumor necrosis factor-α or isoproterenol.

Background:A relationship has been reported between blood concentrations of coagulation factor VII (FVII) and obesity. In addition to its role in coagulation, FVII has been shown to inhibit insulin signals in adipocytes. However, the production of FVII by adipocytes remains unclear.Objective:We herein investigated the production and secretion of FVII by adipocytes, especially in relation to obesity-related conditions including adipose inflammation and sympathetic nerve activation.Methods:C57Bl/6J mice were fed a low- or high-fat diet and the expression of FVII messenger RNA (mRNA) was then examined in adipose tissue. 3T3-L1 cells were used as an adipocyte model for in vitro experiments in which these cells were treated with tumor necrosis factor-α (TNF-α) or isoproterenol. The expression and secretion of FVII were assessed by quantitative real-time PCR, Western blotting and enzyme-linked immunosorbent assays.Results:The expression of FVII mRNA in the adipose tissue of mice fed with high-fat diet was significantly higher than that in mice fed with low-fat diet. Expression of the FVII gene and protein was induced during adipogenesis and maintained in mature adipocytes. The expression and secretion of FVII mRNA were increased in the culture medium of 3T3-L1 adipocytes treated with TNF-α, and these effects were blocked when these cells were exposed to inhibitors of mitogen-activated kinases or NF-κB activation. The β-adrenoceptor agonist isoproterenol stimulated the secretion of FVII from mature adipocytes via the cyclic AMP/protein kinase A pathway. Blockade of secreted FVII with the anti-FVII antibody did not affect the phosphorylation of Akt in the isoproterenol-stimulated adipocytes.Conclusion:Obese adipose tissue produced FVII. The production and secretion of FVII by adipocytes was enhanced by TNF-α or isoproterenol via different mechanisms. These results indicate that FVII is an adipokine that plays an important role in the pathogenesis of obesity.

Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes.

Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.