Purpose To investigate differences in plasma clot properties in patients with AF and CAD and compare the effect of warfarin and antiplatelets on clot structure in AF population. Methods We studied 270 patients and divided them into 3 groups: AF on warfarin (n=184), AF on antiplatelets (n=46) and CAD (n=40). Plasma samples were obtained from participants and centrifuged to prepare platelet poor plasma. Assays were performed in 96-well polystyrene microtiter plates. Reagents were diluted in standard buffer (10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid [HEPES], pH 7.4, 150 mM NaCl). Patient plasma samples (25 %) were incubated with tissue plasminogen activator (500 ng.mL-1) for 10 minutes at 37 Ê C before the addition of CaCl2 (7.5 mM).Either PPP reagent (2.5 %), aPPT reagent (2.5 %), or thrombin (0.5 U.mL-1) were then added to initiate coagulation. Polymerisation of fibrin in plasma was monitored (DOD340 nm) using a Synergy H1 hybrid multi-mode plate reader, readings were taken in 12 second intervals for up to 60 minutes. Results Comparisons between the 3 groups was performed using Kruskal-Wallis test, with Dunn's post-hoc analysis and Holm-Sidak adjustment. There were no significant differences in clot structure between 3 subgroups. The maximum rate of clot formation was significantly delayed in the warfarin subgroup with all reagents used (p<0.001) (table 1). Plasma clot susceptibility to fibrinolysis increased with warfarin compared to antiplatelets but was significant only with APPT and thrombin reagents (p<0.001 and 0.04 respectively). Conclusion Warfarin was effective in delaying clot formation compared to antiplatelets and also resulted in increased susceptibility of plasma clot to fibrinolysis. Conflict of interest None BS43ABSTRACT WITHDRAWN
nuclear fractionation and western blotting. Activation of the MRN DNA damage response pathway was confirmed by chIP qPCR and western blot analysis. Results Alizarin red, Alkaline phosphatase activity and RUNX2 and OCN qPCR confirmed development of calcification during H2O2 treatment, which was reduced with SIRT1 activation. Immunohistochemistry demonstrated enhanced DNA damage marker expression in diabetic vessels compared to non-diabetic control ITA tissue, while SMCs harvested from diabetic patients also showed a significant reduction in SIRT1 expression (p<0.01) and elevated p53 expression (p<0.01) compared to cells from non-diabetic patients. SIRT1 expression was reduced in cells following high glucose treatment, in conjunction with increased protein expression of the DNA damage marker, gH2A.X (p<0.05). The comet assay showed a significant increase in DNA damage in both osteogenic and high glucose conditions following H2O2 treatment, which also caused an increase in SIRT1 nuclear translocation. Activation of SIRT1 significantly reduced H2O2 induced DNA damage (p<0.01) and increased recovery after 3h (p<0.005). In addition, deacetylation of MRE11, RAD50 (p<0.01) and NBS1 (MRN) (p<0.005) correlated with SIRT1 activation, and an increase in their phosphorylation following H2O2 treatment. Conclusions This study demonstrates that SIRT1 protects against H2O2 induced DNA damage and subsequent calcification within a diabetic environment via activation of the MRN complex. The loss of SIRT1 within the calcified vessels of diabetic patients may contribute to a defective DNA repair mechanism, caused by the absence of SIRT1, resulting in a reduction of MRN deacetylation and thus activation of the DNA damage response, and could be an appropriate target for potential therapeutic intervention for vascular calcification.
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