, 2005; Eichinger, 2008; Ofosu, 2006). The increased production and action of thrombin may even be stronger in persons with deep vein thrombosis and/or pulmonary embolism, acute coronary syndrome, myocardial infarction (Smid et al., 2011) or ischemic stroke (Faber et al., 2003). In view of the important
role ascribed to thrombin in the establishment and progression of both venous and arterial thrombosis, thrombin selleck compound inhibition is the key for novel, successful antithrombotic pharmacotherapy (Bijak and Bobrowski, 2010). Researches carried out in the last years provide evidence that polyphenol compounds are able to inhibit the activity of many enzymes including serine proteases (Cuccioloni et al., 2009a). In our in vitro previous studies, we have shown that polyphenol-rich extracts from black chokeberry and grape seeds have anticoagulant (Bijak et al., 2011) and antithrombin (Bijak et al., 2013b) properties. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, the most important serine protease in plasma hemostasis, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Materials and methods Reagents Thrombin from human plasma (T7009), bovine AZD5582 research buy serum albumin (BSA), dimethyl sulfoxide
(DMSO) and polyphenol compounds, such as 4-hydroxyphenylacetic acid gallic acid, ferulic acid, caffeic acid, chlorogenic acid, coumaric acid, resveratrol, cyanin, cyanidin, (+)-catechin, (−)-epicatechin, procyanidin B2, naringenin, naringin, hesperetin, hesperidin, quercetin, rutin, genistein and silybin, were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Frozen human plasma obtained from whole blood collected into 0.32 % sodium citrate was purchased from the Regional Center for Transfusion Medicine in Lodz (Poland). Chromogenic substrate S-2238 was purchased from Chromogenix
(Italy). Sensor chips CM5, amine coupling kit containing N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ADAMTS5 (EDC) and ethanolamine hydrochloride were from BIAcore (Uppsala, Sweden). All other chemicals were of analytical grade or highest quality available products. Isolation of fibrinogen Fibrinogen (fg) was isolated from citrated human plasma by the cold ethanol precipitation technique selleck chemicals described by Doolittle et al. (1967). Its concentration was determined spectrophotometrically at 280 nm using an extinction coefficient (A = 1.55 OD for 1 mg/ml concentration of fibrinogen). Fibrinogen obtained by this method always contains a small amount of factor XIII (fibrin stabilizing factor). Blood platelet isolation Blood samples in 0.