Figure 3.
P2YR antagonists but not COX inhibitors reduce oxLDL potentiation of platelet GPVI responses. (A) Replicate samples (n ≥ 3) of washed human platelets (2 × 108/mL; in modified HEPES/Tyrode buffer without addition of calcium) were treated with P2YR inhibitors ticagrelor (750 nM) and ARC/MRS (10 μM), and COX inhibitors aspirin (1 mM) and indomethacin (10 μM) for 10 minutes and stimulated with oxLDL (20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0.5 μg/mL). Platelet samples were monitored using the luciferase enzyme CHRONO-LUME to detect the luminescence of ATP released as a measure of platelet dense granule secretion. (B) Platelets were stained with APC-CD62P to monitor for platelet surface expression of P-selectin and (C) stained with FITC-PAC-1 to monitor for platelet surface integrin activation. Statistical analysis was performed using a one-way ANOVA test and the corresponding P values of significance are as indicated: ∗∗∗P < .001. (D) Replicate samples (n = 4) of washed human platelets (2 × 108/mL) were treated with P2YR inhibitors ticagrelor (750 nM) and ARC/MRS (10 μM), and COX inhibitors aspirin (1 mM) and indomethacin (10 μM) for 10 minutes and stimulated with oxLDL (20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0.5 μg/mL). Platelet samples were monitored at 37°C under continuous stirring at 1200 rpm and the changes in light transmission were measured. (E) Representative traces of platelet samples treated with each select inhibitor are shown and quantified. (F) Platelets were stained with FITC-lactadherin to monitor for platelet surface PS exposure. (G) Platelet samples were incubated with CaCl2 (8.3 mM) and citrated PPP (33% final). Fibrin formation was measured as a change in turbidity at an absorbance of 405 nm. (H-I) Representative traces are shown and the lag time and time to half the maximum absorbance were calculated. Statistical analysis was performed using a one-way ANOVA test and the corresponding P values of significance are as indicated: ∗P < .05; ns, not significant.

P2YR antagonists but not COX inhibitors reduce oxLDL potentiation of platelet GPVI responses. (A) Replicate samples (n ≥ 3) of washed human platelets (2 × 108/mL; in modified HEPES/Tyrode buffer without addition of calcium) were treated with P2YR inhibitors ticagrelor (750 nM) and ARC/MRS (10 μM), and COX inhibitors aspirin (1 mM) and indomethacin (10 μM) for 10 minutes and stimulated with oxLDL (20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0.5 μg/mL). Platelet samples were monitored using the luciferase enzyme CHRONO-LUME to detect the luminescence of ATP released as a measure of platelet dense granule secretion. (B) Platelets were stained with APC-CD62P to monitor for platelet surface expression of P-selectin and (C) stained with FITC-PAC-1 to monitor for platelet surface integrin activation. Statistical analysis was performed using a one-way ANOVA test and the corresponding P values of significance are as indicated: ∗∗∗P < .001. (D) Replicate samples (n = 4) of washed human platelets (2 × 108/mL) were treated with P2YR inhibitors ticagrelor (750 nM) and ARC/MRS (10 μM), and COX inhibitors aspirin (1 mM) and indomethacin (10 μM) for 10 minutes and stimulated with oxLDL (20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0.5 μg/mL). Platelet samples were monitored at 37°C under continuous stirring at 1200 rpm and the changes in light transmission were measured. (E) Representative traces of platelet samples treated with each select inhibitor are shown and quantified. (F) Platelets were stained with FITC-lactadherin to monitor for platelet surface PS exposure. (G) Platelet samples were incubated with CaCl2 (8.3 mM) and citrated PPP (33% final). Fibrin formation was measured as a change in turbidity at an absorbance of 405 nm. (H-I) Representative traces are shown and the lag time and time to half the maximum absorbance were calculated. Statistical analysis was performed using a one-way ANOVA test and the corresponding P values of significance are as indicated: ∗P < .05; ns, not significant.

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