Mouse transient receptor potential channel type 6 selectively regulates agonist-induced platelet function

Abstract

While changes in intracellular calcium levels is a central step in platelet activation and thrombus formation, the contribution and mechanism of receptor-operated calcium entry (ROCE) via transient receptor potential channels (TRPCs) in platelets remains poorly defined. In previous studies, we have shown that TRPC6 regulates hemostasis and thrombosis, in mice. In the present studies, we employed a knockout mouse model system to characterize the role of TRPC6 in ROCE and platelet activation. It was observed that the TRPC6 deletion (Trpc6 ^-/- ) platelets displayed impaired elevation of intracellular calcium, i.e., defective ROCE. Moreover, these platelets also exhibited defects in a host of functional responses, namely aggregation, granule secretion, and integrin αIIbβ3. Interestingly, the aforementioned defects were specific to the thromboxane receptor (TPR), as no impaired responses were observed in response to ADP or the thrombin receptor-activating peptide 4 (TRAP4). The defect in ROCE in the Trpc6 ^-/- was also observed with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Finally, our studies also revealed that TRPC6 regulates clot retraction. Taken together, our findings demonstrate that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Thus, TRPC6 may serve as a novel target for the therapeutic management of thrombotic diseases.

Keywords: Calcium channel; Platelets; Receptor-operated calcium entry; TRPC6.

Fig. 1

TRPC6 deficiency does not affect platelet count but results in defective receptor-operated calcium entry is defective in Trpc6^−/−platelets. (A) Platelet extracts (2 × 10⁸/ml) were prepared from Trpc6^+/+ and the Trpc6^−/− mice, and proteins (including β-Actin as loading control) were probed using Western Blot. (B) Platelet count was performed for the Trpc6^+/+ and the Trpc6^−/− mice. Trpc6^+/+ and Trpc6^−/− washed platelets were loaded with Fura-2/AM and activated with either U46619 (5 μM; C; inset shows quantification of data expressed as mean [Ca²⁺]_i ± SEM (n = 3)); ADP (10 μM; D; inset shows quantification of data expressed as mean [Ca²⁺]_i ± SEM (n = 3)); TRAP4 (80 μM; E; inset shows quantification of data expressed as mean [Ca²⁺]_i ± SEM (n = 3)), or ADP (10 μM; F inset shows quantification of data expressed as mean [Ca²⁺]_i ± SEM (n = 3))) in the absence of indomethacin, before the maximum intracellular calcium levels were measured at the end of the measurement. (G) Trpc6^+/+ and Trpc6^−/− washed platelets were loaded with Fura-2/AM and activated with OAG (150 μM). NS: Non-significant; *P < 0.05; ***P < 0.001; ****P < 0.0001, t-test. 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.

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.

Fig. 3

Clot retraction is impaired in Trpc6^−/−platelets. (A) Trpc6^+/+ and Trpc6^−/− platelets were washed and resuspended at 1 × 10⁸/mL in buffer (see “Methods”) in the presence of 500 μg/mL fibrinogen, with erythrocytes included to enhance visualization. Fibrin clot formation was initiated by thrombin (0.4 U/mL) at 37 °C. Images represent time-frame of a retracting clot at the indicated time points. (B) Clot retraction quantification by digital processing and plotted as percentage of clot retraction; *P < 0.05; ***P < 0.001; ****P < 0.0001, t-test. This experiment was repeated 3 times, with blood pooled from three groups of 8–10 mice that were 8–10 weeks old.

Fig. 4

TRPC6 expression increases with age. Platelet extracts (2 × 10⁸/ml) from Trpc6^+/+ mice that are 4, 6 or 10 weeks-old were prepared, and the TRPC6 protein (and β-Actin as loading control) was probed using Western Blot. (A) Quantification of protein expression determined via Western blot; ***P < 0.001; ****P < 0.001, t-test. (B) Western blot data showing expression of TRPC6 in an age-dependent manner, along with β-Actin as loading control. This experiment was repeated 3 times, with blood pooled from three groups of 5–6 mice, at the specified age.