CD39 and CD73 expression levels and enzymatic activities are significantly increased on the surface of kindlin-2^+/−ECs. (A) Representative FACS analysis of WT and kindlin-2^+/− ECs stained with PE-conjugated Abs to CD39 and CD73. Gray-filled histograms represent isotype controls. (B) FACS analyses of WT (□) and kindlin-2^+/− (▪) ECs stained with Abs to CD39, CD73, P-selectin, and ICAM-1. The data are mean ± SEM of triplicate samples and are representative of 4 independent experiments. (C) CD39 activity on the surface of WT and kindlin-2^+/− ECs. ECs (8 × 10⁵) were incubated in the absence or presence of substrate ADP (100 μM) for 0 to 60 minutes at 37°C in the presence of adenosine 5′-(α,β-methylene)diphosphate, the CD73 inhibitor. Concentration of inorganic P_i released from ADP in EC supernatants was measured using malachite green assay. (D) Platelet aggregation induced by remaining (not hydrolyzed by CD39) ADP present in the supernatants of WT (□) or kindlin-2^+/− (▪) ECs (0-8 × 10⁵) treated with ADP (100 μM) for 5 or 15 minutes; in controls, no ADP was added. (E) Platelet aggregation induced by ADP present in the supernatants of WT (□) or kindlin-2^+/− (▪) ECs (2 × 10⁵) treated with ADP (100 μM) for 5 minutes. Adenosine (0-50 ng/mL) was added to WT EC-conditioned medium, while ADP (200 μM) was added to kindlin-2^+/− EC-conditioned medium. (F) CD73 activity on the surface of WT or kindlin-2^+/− ECs. ECs (8 × 10⁵) were incubated in the absence or presence of substrate AMP (100 μM) for 0 to 60 minutes in the presence of the alkaline phosphatase inhibitor levamisole. P_i formed from AMP in EC supernatants was measured using a malachite green assay kit. Data are mean ± SEM of triplicate samples from 3 independent experiments. ADP (G) and adenosine (H) concentrations in plasma of WT and kindlin-2^+/− mice were measured as described in “Methods.” Results are expressed as mean ± SEM from 7 to 10 mice per group. MFI, mean fluorescence intensity.