Fig. 2.
Deletion of TRPC6 alters agonist-induced platelet aggregation, dense granule, alpha granules release and integrin αIIbβ3 activation.Trpc6+/+ and Trpc6−/− mouse PRP containing 3 × 108 platelets/ml, were stimulated with either U46619 (1.5 μM and 5 μM; (A)), ADP (5 μM and 10 μM; (B)); or TRAP4 (40 μM and 80 μΜ; (C)) before the aggregation response was examined. (D) Trpc6+/+ and Trpc6−/− mouse PRP containing 3 × 108 platelets/ml was stimulated with ADP (10 μM) in the absence of indomethacin, before the aggregation response was examined. Each experiment was repeated 3 times, with blood pooled from three groups of 8–10 mice. (E–G) 12.5 μL of luciferase luciferin was added to Trpc6+/+ and Trpc6−/− platelets before they were stimulated with either U46619 (1.5 μM and 5 μM; (E)), ADP (5 μM and 10 μM; (F)); TRAP (40 μM and 80 μΜ; (G)). (H) Trpc6+/+ and Trpc6−/− Platelets were stimulated with ADP (10 μM) in the absence of indomethacin. Release of ATP (for dense granule release) as a luminescence was measured by aggregometer. Each experiment was repeated 3 times, with blood pooled from three groups of 8–10 mice. Trpc6+/+ and Trpc6−/− washed platelets were stimulated with either U46619 (5 μM), ADP (10 μM; in the presence or absence of indomethacin); or TRAP4 (80 μΜ) for 5 min. The reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at room temperature. (I) Platelets were incubated with FITC-conjugated anti-P-selectin antibody (for alpha granule), or (J) with FITC-conjugated anti-JON/A antibody (for integrin αIIbβ3). The fluorescent intensities were measured by flow cytometry, and the data were plotted as bar diagram; ***P < 0.001; NS: Non-significant, t-test. (K) Quantification of U46619-induced aggregation in the Trpc6+/+ and Trpc6−/− platelets; **P < 0.01. Each experiment was repeated 3 times, with blood pooled from three groups of 8–10 mice that were 8–10 weeks old. Data was analyzed using t-test.