Figure 3. Tumor microvesicles induce platelet activation in a tissue factor and thrombin-dependent manner.

(A) BxPc-3 (black bars) and L3.6pl (grey bars) microvesicles (MVs) (50 μg protein/mL) were incubated with washed human platelets in the presence and absence of diluted human plasma (1:100). Platelet activation was monitored by flow cytometry for Pac-1 binding to activated αIIb/β3 and P-selectin expression. Data are presented as the mean fluorescence intensity (MFI) of total CD41a-positive events (+/- SEM). Data are representative of 3 independent experiments. *P<0.01, n=3. Data were analyzed by one-way ANOVA with Bonferroni posttests comparing indicated pairs of data on Graphpad Prism software v5.01. (B) BxPc-3 and L3.6pl MVs (50 μg protein/mL) were pre-incubated with HTF-1 (50 μg/ml) or IgG control (50 μg/ml) for 30 minutes at 4°C. TMVs were then incubated with human platelets in the presence of diluted human plasma (1:100). Data are presented as the MFI of total CD41a-positive events (+/- SEM). Data are representative of 3 independent experiments. *P<0.0001, n=3. (C) Platelets were pre-incubated with inhibitors as indicated for 5 minutes at room temperature. BxPc-3 and L3.6pl MVs (50 μg/mL) were then incubated with human platelets in the presence of diluted plasma for 15 minutes at 37°C. Recombinant TF (Innovin^TM) (0.5 pg) and alpha-thrombin (5 nM) were used as controls. After the activation reaction was complete, the reactions were diluted in Tyrode’s buffer + 1 mM CaCl₂ + 0.5% formalin and P-selectin expression evaluated by flow cytometry. Data are presented as the MFI of total CD41a-positive events (± SEM). Data are representative of 3 independent experiments. *P<0.0001, n=3. Data were analyzed by one-way ANOVA with Bonferroni posttests comparing all data to the no MV control.