LRRC8A deletion impairs agonist-induced... | American Society of Hematology

LRRC8A deletion impairs agonist-induced platelet adhesion, activation, aggregation, Ca2+ signaling, and PI3K-AKT-GSK3β signaling. (A-B) Adhesion of platelets to a fibrillar collagen-coated surface under shear (A, 650 s−1; B, 2600 s−1) conditions in whole blood isolated from Lrrc8afl/fl (WT, n = 4-6) and Pf4-Cre;Lrrc8afl/fl (cKO, n = 4-5) mice. (C-D) P-selectin exposure (C) and JonA binding to activated αIIbb3 integrin (D) as measured by flow cytometry of platelets isolated from WT and cKO mice and stimulated with thrombin (Thr; 0.03 U/mL; n = 6 per group), CRP (0.2 μg/mL; n = 3), or the calcium ionophore A23187 (0.5 μM; n = 6 per group). (E-I) Aggregation of platelets isolated from WT and cKO mice stimulated with Thr (0.05 U/mL, n = 7 per group) (E), ADP (ADP, 10-20 μM; fibrinogen, 30 μg/mL; n = 6 per group) (F), CRP (0.05-0.15 μg/mL; n = 7 per group) (G), U46619 (0.5-0.8 μM; n = 6 per group) (H), and calcium ionophore A23187 (1 μM; n = 3 per group) (I). Concurrent ATP release from platelets treated with Thr (J), CRP (K), and U46619 (L). AUC of ATP release (M). Representative traces for aggregation and ATP release are provided in supplemental Figure 5. (N) Intracellular ATP content in platelets isolated from WT (n = 7) and cKO (n = 8) mice. (O-Q) Cytosolic Ca2+ measured using the ratiometric dye Fura-2 in platelets isolated from WT (n = 11) and cKO (n = 9) mice stimulated with Thr (0.02 U/mL) for 10 minutes (O), with corresponding measurements of peak Ca2+ (P) and rate of Ca2+ rise (Q). (R-S) Western blots detecting LRRC8A; pPI3KTyr458; pAKT2Ser474; AKT2; pGSK3βSer9; GSK3β; and GAPDH in platelets from WT and cKO mice after stimulation with PAR4-AP (400 μM) for 5 minutes during aggregometry (R), with densitometric quantification (S). Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels A-L,N,P,Q,S; and by 2-way ANOVA for panel O. For panel M, statistical significance was determined by unpaired t test (Thr) or Mann-Whitney U test (CRP and U46619). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Thr used in panels C-E,J,M was sourced from Sigma-Aldrich (St. Louis, MO), whereas Thr used in panels O-Q was sourced from the Chrono-Log Corporation (Havertown, PA). a.u., arbitrary units; AUC, area under curve; GSK3β, glycogen synthase kinase-3β; pAKT2, phosphorylated AKT2; pGSK3β, phosphorylated glycogen synthase kinase-3β; pPI3K, phosphorylated PI3K; ROI, region of interest.

LRRC8A deletion impairs agonist-induced platelet adhesion, activation, aggregation, Ca²⁺ signaling, and PI3K-AKT-GSK3β signaling. (A-B) Adhesion of platelets to a fibrillar collagen-coated surface under shear (A, 650 s⁻¹; B, 2600 s⁻¹) conditions in whole blood isolated from Lrrc8a^fl/fl (WT, n = 4-6) and Pf4-Cre;Lrrc8a^fl/fl (cKO, n = 4-5) mice. (C-D) P-selectin exposure (C) and JonA binding to activated αIIbb3 integrin (D) as measured by flow cytometry of platelets isolated from WT and cKO mice and stimulated with thrombin (Thr; 0.03 U/mL; n = 6 per group), CRP (0.2 μg/mL; n = 3), or the calcium ionophore A23187 (0.5 μM; n = 6 per group). (E-I) Aggregation of platelets isolated from WT and cKO mice stimulated with Thr (0.05 U/mL, n = 7 per group) (E), ADP (ADP, 10-20 μM; fibrinogen, 30 μg/mL; n = 6 per group) (F), CRP (0.05-0.15 μg/mL; n = 7 per group) (G), U46619 (0.5-0.8 μM; n = 6 per group) (H), and calcium ionophore A23187 (1 μM; n = 3 per group) (I). Concurrent ATP release from platelets treated with Thr (J), CRP (K), and U46619 (L). AUC of ATP release (M). Representative traces for aggregation and ATP release are provided in supplemental Figure 5. (N) Intracellular ATP content in platelets isolated from WT (n = 7) and cKO (n = 8) mice. (O-Q) Cytosolic Ca²⁺ measured using the ratiometric dye Fura-2 in platelets isolated from WT (n = 11) and cKO (n = 9) mice stimulated with Thr (0.02 U/mL) for 10 minutes (O), with corresponding measurements of peak Ca²⁺ (P) and rate of Ca²⁺ rise (Q). (R-S) Western blots detecting LRRC8A; pPI3K^Tyr458; pAKT2^Ser474; AKT2; pGSK3β^Ser9; GSK3β; and GAPDH in platelets from WT and cKO mice after stimulation with PAR4-AP (400 μM) for 5 minutes during aggregometry (R), with densitometric quantification (S). Data are represented as mean ± SEM. Statistical significance was determined by unpaired t test for panels A-L,N,P,Q,S; and by 2-way ANOVA for panel O. For panel M, statistical significance was determined by unpaired t test (Thr) or Mann-Whitney U test (CRP and U46619). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Thr used in panels C-E,J,M was sourced from Sigma-Aldrich (St. Louis, MO), whereas Thr used in panels O-Q was sourced from the Chrono-Log Corporation (Havertown, PA). a.u., arbitrary units; AUC, area under curve; GSK3β, glycogen synthase kinase-3β; pAKT2, phosphorylated AKT2; pGSK3β, phosphorylated glycogen synthase kinase-3β; pPI3K, phosphorylated PI3K; ROI, region of interest.

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