Figure 5

Thrombin-mediated  TNT induction is independent of CD31 but dependent on PKCα. (A) Mean data and analysis show that CD31 siRNA had no significant effect on the number of TNTs evoked by 2 U/ml thrombin (n/N = 3/17 for each group) as compared to siRNA control. (B) Mean data and analysis (n/N = 3/20 minimum for each group) from HUVECs transfected with siRNA specific to Src or a control siRNA +/− 10 μg/ml WGA showing WGA evoked significant increases in TNT formation in the presence or absence of Src kinase. (C) Example trace and mean data of HUVEC intracellular Ca²⁺ measurement in the presence of extracellular 1.5 mM Ca²⁺ showing responses to WGA (5 μg/ml) in Fura-2-loaded cells (n/N = 3/8 for each point). WGA failed to induce mobilisation of intracellular Ca²⁺. (D) Mean data and analysis (n/N = 3/23 minimum for each group) of HUVECs transfected with Src siRNA or siRNA control +/− 2 U/ml thrombin (thr). Thrombin significantly increased TNT formation in the presence or absence of Src kinase. (E) Quantification of TNT formation in cells pre-treated with the PKC inhibitor BIM, PKCδ blocker rottlerin or the PKCα selective inhibitor Go6976 prior to the addition of 2 U/ml thrombin (thr: n/N = 3/15–20 for each group). Significant formation of TNTs only occurred in those cells treated with thrombin alone or those pre-treated with rottlerin prior to thrombin treatment. (F) Mean data and analysis show that PKCα siRNA had a significant effect on the number of TNTs evoked by 2 U/ml thrombin (n = 3/N = 19 minimum for each group) as compared to siRNA control. All data are represented as mean ± SD. *P < 0.05.