Agonist-induced Ca²⁺ mobilization in HUVECs and effect of calcium chelation on thrombin-, PAR1-AP–, and PAR2-AP–mediated release of VWF and P-selectin. Cells were plated into 96-well plates and stimulated with indicated concentrations of agonists, and Ca²⁺ was measured fluorometrically. Stimulation with indicated doses of histamine and the calcium ionophore A23187 (10 μM) (A) and stimulation with indicated concentrations of PAR2-AP and calcium ionophore A23187 (10 μM) (B) are represented. The y-axis represents the relative fluorescence units (RFUs). Data represent the mean ± SEM of 2 independent experiments for histamine and 3 independent experiments for PAR2-AP. (C-D) Dose-response curves were generated using the area under the curve function in SOFTmax PRO (Molecular Devices) and then normalized to fraction of maximal response and plotted with the data from Figure 2 to graphically represent the different potencies between agonists with respect to Ca²⁺ mobilization and release of VWF and P-selectin. (E) The effect of BAPTA-AM on agonist-mediated stimulation of Ca²⁺ mobilization is represented. HUVECs were pretreated with BAPTA-AM at indicated concentrations for 1 hour and stimulated with thrombin (10 nM). Figure represents the mean ± SEM of 2 independent experiments conducted in triplicate. (F) HUVECs that were pretreated either with 10 μM BAPTA-AM for 1 hour and stimulated with thrombin (10 nM), PAR1-AP (100 μM), or PAR2-AP (200 μM), are represented. The y-axis represents percentage of inhibition and was normalized by dividing the agonist stimulation by the basal levels in the presence of BAPTA-AM. Data represent the mean ± SEM of 3 independent experiments conducted in triplicate. *Statistical difference (P < .05 as determined by 2-tailed t test) between VWF and P-selectin in the presence of BAPTA-AM. Figure represents the mean ± SEM of 3 independent experiments conducted in triplicate.