Figure 7.
LRRC8 channel inhibitor SN-89B suppresses agonist-induced platelet activation, aggregation, PI3K-AKT-GSK3β signaling, and in vivo thrombosis. (A-B) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC during voltage ramps from −140 mV to +80 mV in MEG-01 cells after HYPO swelling in the absence or presence of 10 μM SN-89B (A); mean current densities of inward IATP at −140 mV (n = 4) (B). (C-D) P-selectin exposure (C) and PAC-1 binding to activated αIIbb3 integrin (D) as measured by flow cytometry of human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively) in the presence of vehicle (0.02% DMSO) or SN-89B (10 μM). Each sample was normalized to the average of unstimulated controls (n = 8 for all groups, isolated from 2 healthy volunteers). (E) Adhesion of platelets in reconstituted blood (platelets + red blood cells) to a von Willebrand factor–coated surface under shear (1800 s−1) conditions in the presence of vehicle (0.02% DMSO) or 10 μM SN-89B (n = 3 for both groups, isolated from 3 healthy volunteers), as determined by fluorescence microscopy. (F) Aggregometry of human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively) in the presence of vehicle (0.02% DMSO; n = 6; isolated from 1 healthy volunteer) or SN-89B (10 μM; n = 4; isolated from 1 healthy volunteer). Representative traces are provided in supplemental Figure 14. (G-H) Cytosolic calcium measured using the ratiometric dye Fura-2 in human platelets stimulated with Thr (0.01 U/mL) in the presence of vehicle (0.02% DMSO) or 10 μM SN-89B (n = 6 for both groups, isolated from 1 healthy volunteer) (G), with corresponding measurements of peak Ca2+ and rate of Ca2+ rise (H). (I-J) Western blots detecting pAKT1Ser473; AKT1; pAKT2Ser474; AKT2; pGSK3βSer9; GSK3β; and GAPDH in human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively), in the presence of vehicle (0.02% DMSO; n = 3; isolated from 1 healthy volunteer) or SN-89B (10 μM, n = 3, isolated from 1 healthy volunteer) (I), with densitometric quantification of pAKT1, pAKT2 and pGSK-3β normalized to their respective unphosphorylated forms and GAPDH (J). (K-M) Thrombosis in the cremasteric arterioles of WT mice treated with 50 mg/kg SN-89B (n = 5) or vehicle (n = 5) after laser-induced injury as observed by real-time confocal intravital microscopy (red, DyLight 649 anti-CD42C) (K) with median time course (L) and quantification of the fluorescent reporter at 30, 60, 120, 180, and 240 seconds after injury (M) (n = 36 for vehicle; n = 38 for SN-89B). (N) Time to occlusion of the carotid artery of WT C57BL/6J mice treated with 50 mg/kg SN-89B (n = 8) or vehicle (n = 5) after FeCl3-induced injury. (O-Q) Cumulative bleeding times (O) and times taken for bleeding to cease for the first time (before any rebleeding) (P) after tail tip amputation of WT C57BL/6J mice treated with vehicle (n = 13), 50 mg/kg SN-89B (n = 13), or 30 mg/kg ticagrelor (n = 10), with corresponding hemoglobin concentrations of red blood cell lysates collected during the assay (Q). Data in panel L are represented as median, whereas data in panel M are represented as mean only. All other data are represented as mean ± SEM. Statistical significance was determined by the unpaired t test for panels B-D,H,J,N,P-Q; the Mann-Whitney U test for panel M; and by the 2-way ANOVA for panels E-G. Statistical significance for panel O was determined by the unpaired t test (vehicle compared with SN-89B) or the Mann-Whitney U test (ticagrelor compared to vehicle and SN-89B). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Thr used in this experiment was sourced from the Chrono-Log Corporation. F platelets, platelet fluorescence; ns, not significant; ROI, region of interest.

LRRC8 channel inhibitor SN-89B suppresses agonist-induced platelet activation, aggregation, PI3K-AKT-GSK3β signaling, and in vivo thrombosis. (A-B) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC during voltage ramps from −140 mV to +80 mV in MEG-01 cells after HYPO swelling in the absence or presence of 10 μM SN-89B (A); mean current densities of inward IATP at −140 mV (n = 4) (B). (C-D) P-selectin exposure (C) and PAC-1 binding to activated αIIbb3 integrin (D) as measured by flow cytometry of human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively) in the presence of vehicle (0.02% DMSO) or SN-89B (10 μM). Each sample was normalized to the average of unstimulated controls (n = 8 for all groups, isolated from 2 healthy volunteers). (E) Adhesion of platelets in reconstituted blood (platelets + red blood cells) to a von Willebrand factor–coated surface under shear (1800 s−1) conditions in the presence of vehicle (0.02% DMSO) or 10 μM SN-89B (n = 3 for both groups, isolated from 3 healthy volunteers), as determined by fluorescence microscopy. (F) Aggregometry of human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively) in the presence of vehicle (0.02% DMSO; n = 6; isolated from 1 healthy volunteer) or SN-89B (10 μM; n = 4; isolated from 1 healthy volunteer). Representative traces are provided in supplemental Figure 14. (G-H) Cytosolic calcium measured using the ratiometric dye Fura-2 in human platelets stimulated with Thr (0.01 U/mL) in the presence of vehicle (0.02% DMSO) or 10 μM SN-89B (n = 6 for both groups, isolated from 1 healthy volunteer) (G), with corresponding measurements of peak Ca2+ and rate of Ca2+ rise (H). (I-J) Western blots detecting pAKT1Ser473; AKT1; pAKT2Ser474; AKT2; pGSK3βSer9; GSK3β; and GAPDH in human platelets stimulated with PAR1-AP and PAR4-AP (10 μM and 100 μM, respectively), in the presence of vehicle (0.02% DMSO; n = 3; isolated from 1 healthy volunteer) or SN-89B (10 μM, n = 3, isolated from 1 healthy volunteer) (I), with densitometric quantification of pAKT1, pAKT2 and pGSK-3β normalized to their respective unphosphorylated forms and GAPDH (J). (K-M) Thrombosis in the cremasteric arterioles of WT mice treated with 50 mg/kg SN-89B (n = 5) or vehicle (n = 5) after laser-induced injury as observed by real-time confocal intravital microscopy (red, DyLight 649 anti-CD42C) (K) with median time course (L) and quantification of the fluorescent reporter at 30, 60, 120, 180, and 240 seconds after injury (M) (n = 36 for vehicle; n = 38 for SN-89B). (N) Time to occlusion of the carotid artery of WT C57BL/6J mice treated with 50 mg/kg SN-89B (n = 8) or vehicle (n = 5) after FeCl3-induced injury. (O-Q) Cumulative bleeding times (O) and times taken for bleeding to cease for the first time (before any rebleeding) (P) after tail tip amputation of WT C57BL/6J mice treated with vehicle (n = 13), 50 mg/kg SN-89B (n = 13), or 30 mg/kg ticagrelor (n = 10), with corresponding hemoglobin concentrations of red blood cell lysates collected during the assay (Q). Data in panel L are represented as median, whereas data in panel M are represented as mean only. All other data are represented as mean ± SEM. Statistical significance was determined by the unpaired t test for panels B-D,H,J,N,P-Q; the Mann-Whitney U test for panel M; and by the 2-way ANOVA for panels E-G. Statistical significance for panel O was determined by the unpaired t test (vehicle compared with SN-89B) or the Mann-Whitney U test (ticagrelor compared to vehicle and SN-89B). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Thr used in this experiment was sourced from the Chrono-Log Corporation. F platelets, platelet fluorescence; ns, not significant; ROI, region of interest.

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