Fig 1.
Effect of TRPC6 inhibitor on human platelet aggregation.
PRP was incubated without or with the TRPC6 inhibitor (5 μM and/or 10 μM) for 5 min followed by stimulation with (A) U46619 (0.5 μM), (B) ADP (5 μM), (C) TRAP4 (60 μM), or (D) U46619 (0.5 μM) with or without BAPTA (10 μM). Each experiment was repeated three times, with three separate donors.
Fig 2.
Effect of TRPC6 inhibitor on glycoprotein IIb-IIIa activation in vitro.
Washed platelets were incubated in the presence or absence of TRPC6 inhibitor (10 μM) for 5 minutes and then stimulated with U46619 (0.5 μM) and ADP (5 μM) for 3 minutes. The reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at room temperature. Platelets were incubated with FITC-conjugated PAC-1 antibody, the fluorescent intensities were measured by flow cytometry, and the data were plotted as histogram. Each experiment was repeated at least four times, with blood obtained from four separate donors (n = 4; P < 0.01, Mann-Whitney test).
Fig 3.
Effect of TRPC6 inhibitor on human platelet ATP secretion stimulated by U46619.
PRP was incubated without or with TRPC6 inhibitor (10 μM) for 5 min followed by stimulation of PRP with (A) U46619 (0.5 μM), or (B) ADP (5 μM). Each experiment was repeated three times, with three separate donors.
Fig 4.
Effect of TRPC6 inhibitor on alpha granule secretion (P-selectin expression).
Washed platelets were incubated in the presence or absence of TRPC6 inhibitor (10 μM) for 5 minutes and then stimulated with U46619 (0.5 μM) and ADP (5 μM) for 3 minutes. The reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at room temperature. Platelets were incubated with FITC-conjugated anti–P-selectin antibody, the fluorescent intensities were measured by flow cytometry, and the data were plotted as histogram. Each experiment was repeated at least four times, with blood obtained from four separate donors (P < 0.01, Mann-Whitney test).
Fig 5.
Effect of TRPC6 inhibitor on intracellular calcium in human platelets in vitro and on ERK and Akt phosphorylation.
(A) Human platelets were loaded with Fura-2/AM to measure intracellular [Ca2+]i, in the presence or absence of TRPC6 inhibitor (10 μM), and activated with U46619 (0.5 μM), ADPβS (100 μM) and OAG (150 μM). Each experiment was repeated at least three times, with blood obtained from three separate donors. (B) Human platelets were incubated in the presence or absence of TRPC6 inhibitor (10 μM) for 5 minutes and then stimulated with U46619 (0.5 μM), ADPβS (100 μM) for 3 minutes, and subjected to immunoblotting with ERK, pERK, Akt and pAkt antibodies.
Fig 6.
Effect of the TRPC6 inhibitor on binding of the radiolabelled TPR antagonist [3H]SQ29,548.
Binding displacement of 1 nM [3H]SQ29,548 with increasing concentrations of the TRPC6 inhibitor (10–50 μM), in human platelets (n = 3; P < 0.01, Mann-Whitney test).
Fig 7.
Effect of TRPC6 inhibitor on clot retraction.
Clot retraction of washed platelets was intiated by adding thrombin (0.1 U/ml) in the presence or absence of TRPC6 inhibitor (10 μM) and photographed for every 10 minutes up to one hour (n = 4).
Fig 8.
Effect of TRPC6 inhibitor tail vein injections on mouse hemostasis following tail tip excision and on the time for occlusion in a carotid artery injury thrombosis model.
Mice were injected with TRPC6 inhibitor (10 μM) or vehicle 1 hour before their tail bleeding times and time for occlusion are measured. TRPC6 inhibitor treatment resulted in a significant increase in bleeding time (n = 3; P < 0.01, Mann-Whitney test) (A) and significantly prolonged time for occlusion (n = 3; P < 0.01, Mann-Whitney test) (B). Each point represents a single animal.