Figure 4.
LRRC8A deletion impairs ATP release despite preserved dense granule secretion. (A-L) Aggregation (A,E,I), P-selectin exposure (B,F,J), ATP release (C,G,K), and CD63 exposure (D,H,L) in platelets isolated from WT (n = 4) and cKO (n = 4) mice stimulated with 0.02 U/mL (A-D), 0.06 U/mL (E-H) and 0.08 U/mL (I-L) Thr. (M) Serotonin secretion from platelets isolated from WT (n = 4) and cKO (n = 4) mice stimulated with 0.08 U/mL Thr. (N) Summary of ATP release from platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (O) Summary of P-selectin exposure in platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (P) Summary of CD63 expression in platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (Q) Summary of serotonin secretion from platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (R) ATP-to-serotonin ratios for platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (S) Inhibition of ATP and serotonin secretion from platelets isolated from cKO mice stimulated with 0.08 U/mL Thr (n = 4 for all groups) relative to ATP and serotonin secretion from platelets isolated from WT mice also treated with 0.08 U/mL Thr. (T) Aggregometry of platelets isolated from WT and cKO mice stimulated with 400 μM PAR4-AP only (n = 5 for WT; n = 5 for cKO), or with 400 μM PAR4-AP in the presence of 40 μM ATP (n = 4 for WT; n = 4 for cKO). Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels B,D,F,H,J,L-Q,S; and by 2-way ANOVA for panels A,C,E,G,I,K,R,T. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Thr used in this experiment was sourced from the Chrono-Log Corporation. Data for panels G,K were calculated based on extrapolation as described in supplemental Methods and shown in supplemental Figure 7. Representative aggregometry traces for panels A,E,I, and ATP traces for panels C,G,K are also provided in supplemental Figure 7, and for panel T is provided in supplemental Figure 8. A.U., arbitrary units; ctrl, control; MFI, mean fluorescence intensity; ns, not significant.

LRRC8A deletion impairs ATP release despite preserved dense granule secretion. (A-L) Aggregation (A,E,I), P-selectin exposure (B,F,J), ATP release (C,G,K), and CD63 exposure (D,H,L) in platelets isolated from WT (n = 4) and cKO (n = 4) mice stimulated with 0.02 U/mL (A-D), 0.06 U/mL (E-H) and 0.08 U/mL (I-L) Thr. (M) Serotonin secretion from platelets isolated from WT (n = 4) and cKO (n = 4) mice stimulated with 0.08 U/mL Thr. (N) Summary of ATP release from platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (O) Summary of P-selectin exposure in platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (P) Summary of CD63 expression in platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (Q) Summary of serotonin secretion from platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (R) ATP-to-serotonin ratios for platelets isolated from WT and cKO mice stimulated with 0.02 to 0.08 U/mL Thr (n = 4 for all groups). (S) Inhibition of ATP and serotonin secretion from platelets isolated from cKO mice stimulated with 0.08 U/mL Thr (n = 4 for all groups) relative to ATP and serotonin secretion from platelets isolated from WT mice also treated with 0.08 U/mL Thr. (T) Aggregometry of platelets isolated from WT and cKO mice stimulated with 400 μM PAR4-AP only (n = 5 for WT; n = 5 for cKO), or with 400 μM PAR4-AP in the presence of 40 μM ATP (n = 4 for WT; n = 4 for cKO). Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels B,D,F,H,J,L-Q,S; and by 2-way ANOVA for panels A,C,E,G,I,K,R,T. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Thr used in this experiment was sourced from the Chrono-Log Corporation. Data for panels G,K were calculated based on extrapolation as described in supplemental Methods and shown in supplemental Figure 7. Representative aggregometry traces for panels A,E,I, and ATP traces for panels C,G,K are also provided in supplemental Figure 7, and for panel T is provided in supplemental Figure 8. A.U., arbitrary units; ctrl, control; MFI, mean fluorescence intensity; ns, not significant.

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