and purpose: The modulation by flavonoids of platelet responses induced by

and purpose: The modulation by flavonoids of platelet responses induced by thrombin has been little investigated FRP-2 and the antiplatelet activity as well as possible inhibitory mechanisms E-7050 (Golvatinib) of these compounds on thrombin signalling has not yet been elucidated. secretion and aggregation for quercetin and apigenin inhibition of kinase activation may also be involved in the impairment of platelet responses. 2008 but their signalling pathways are not completely understood. Thus how differential signalling through the thrombin receptors is regulated and the potential physiological consequences of selective inactivation of PAR1 and PAR4 remain unclear. It has been shown that stimulation of platelets E-7050 (Golvatinib) with thrombin results in activation of phospholipase C β (PLCβ) and subsequent calcium mobilization and protein kinase C (PKC) activation which in turn mediate granule secretion. Recent reports establish a key role of TxA2 generation and ADP secretion in the irreversible aggregation observed in response to low or medium concentrations of thrombin through signalling amplification mechanisms involving Src kinases and the mitogen-activated protein kinase E-7050 (Golvatinib) ERK1/2 among other molecules (Falker for 15 min. Platelets were washed in the presence of 0.1 μg·mL?1 prostaglandin E1 with modified HEPES/Tyrode’s buffer pH 6.5 (137 mM NaCl 2.9 mM KCl 0.42 mM NaH2PO4 11.9 mM NaHCO3 2 mM MgCl2 5.5 mM d-glucose 10 mM HEPES) resuspended in the specific assay buffer adjusted to an appropriate count and left undisturbed for 30 min at room temperature prior to experiments. Platelet aggregation studies Washed platelets were resuspended in HEPES/Tyrode’s buffer pH 7.35 containing 2 mM CaCl2 (300 × 109 L?1) and incubated for at least 15 min with different inhibitors flavonoids or with ≤0.2% dimethylsulphoxide (DMSO) as control. They were then stimulated during 5 min with thrombin (0.2 U·mL?1) PAR1-AP (25 μM) or PAR4-AP (150 μM) in an Aggrecorder II aggregometer (Menarini Diagnostics Florence Italy) and the percentage of maximum aggregation was recorded as the percentage of light transmission with washed platelets as the baseline and HEPES/Tyrode’s buffer as 100% of light transmission. The concentration of flavonoids necessary to obtain half-maximal inhibition of platelet aggregation (IC50) was determined for each agonist by incubating washed platelets with increasing concentrations of flavonoids (0–100 μM) or DMSO (final concentration ≤0.2%). Dose-dependent inhibition curves were generated with a E-7050 (Golvatinib) non-linear curve-fitting software (GraphPad Prism for Windows version 4.0 GraphPad Inc. San Diego CA USA). 5 release Washed platelets in HEPES/Tyrode’s buffer pH 7.35 containing 2 mM CaCl2 (300 × 109 L?1) were radiolabeled with 1 μM [14C]-5-HT for 45 min at 37°C and then treated with 50 μM flavonoids or LJ-CP8 antibody (1 μM) plus 5 μM imipramine to prevent re-uptake of secreted 5-HT. The secretion of [14C]-5-HT was induced with 0.2 U·mL?1 thrombin 25 μM PAR1-AP or 150 μM PAR4-AP for 5 min at 37°C with stirring (1000 rpm) and release was analysed as described previously (Guerrero kinase assays Using a commercial kinase activity test (SelectScreen Kinase Profiling Service Invitrogen Paisley UK) several intraplatelet kinases were assayed at their approximate ATP < 0.05. Materials All the polyphenolic compounds tested except genistein were kindly provided by Nutrafur-Furfural Espa?ol S.A. (Murcia Spain) (see Supporting Information Figure S1). Genistein the protease and phosphatase inhibitor cocktails and PAR1-AP SFLLRN-OH were from Sigma-Aldrich Química (Madrid Spain). PAR4-AP (AYPGKF-amide) was obtained from Bachem (Weil am Rhein Germany). Both PAR1- and PAR4-APs had a purity of >95%. Human thrombin PLC inhibitor {“type”:”entrez-nucleotide” attrs :{“text”:”U73122″.