LRRC8 complexes are ATP release channels that regulate platelet activation and arterial thrombosis

Figures

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  Figure 1.. LRRC8 proteins are highly expressed in platelets, functionally encode VRAC in MKs, and regulate platelet volume. (A,D) LRRC8 mRNA transcript expression in human (A) and mouse (D) platelets. (B,E) LRRC8 mRNA transcript expression compared with transcripts for ANO6 (TMEM16F), TRPV4, Piezo1, and Piezo2 in human (B) and mouse (E) platelets (data in panels A-B,D-E are from Rowley et al48; supplemental Table 4). (C,F) Western blot showing LRRC8 subunit protein expression of human (n = 3) (C) and WT C57BL/6N mouse (n = 3) (F) platelets. (G-H) Western blot detecting P-selectin, LRRC8A, integrin β3, and GAPDH in platelets isolated from Lrrc8afl/fl (WT, n = 4) and Pf4-Cre;Lrrc8afl/fl (cKO, n = 4) mice (G), with densitometric quantification (H). (I-J) MPV in whole blood from WT (n = 8), cKO (n = 8), and cHets (Pf4-Cre;Lrrc8afl/+, n = 3) (I), with corresponding platelet counts (J). (K) MPVs of washed platelets isolated from WT (n = 35) and cKO (n = 31) mice. (L) Representative images of perforated patch-clamped freshly isolated MKs from WT or cKO mice, under ISO and HYPO conditions. (M) Cell capacitances of MKs isolated from WT (n = 14) and cKO mice (n = 10). (N) VRAC current-time…

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  Figure 2.. 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…

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  Figure 3.. MK LRRC8 channels permeate ATP. (A-C) Current-time relationship of inward IATP (ATP efflux) and outward VRAC in MKs isolated from Lrrc8afl/fl (WT) mice induced by HYPO (210 mOsm) swelling with an intracellular ATP concentration of 1 mM (A) or 50 mM (B) followed by application of 10 μM SN-401 (DCPIB), and in MKs isolated from Pf4-Cre;Lrrc8afl/fl (cKO) mice (C) with an intracellular ATP concentration of 50 mM. The inward component of the current represents IATP generated from ATP efflux. (D-F) Respective current-voltage relationships of inward IATP (ATP efflux) and outward VRAC elicited from voltage ramps from −140 mV to +80 mV. (G) Mean current densities of inward IATP (ATP efflux) at −140 mV in MKs isolated from WT mice after HYPO swelling, with an intracellular concentration of 1 mM ATP (n = 7) or 50 mM ATP (n = 6), and inhibition of ATP efflux by 10 μM SN-401 (n = 7 for 1 mM ATP + SN-401; n = 6 for 50 mM ATP + SN-401), and mean current densities of inward IATP in MKs isolated from cKO mice after HYPO swelling, with an intracellular concentration of 50 mM ATP (n = 7). (H-I) Current-voltage relationship of inward IATP (ATP efflux) and outward VRAC in MEG-01 cells elicited from…

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  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…

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  Figure 5.. Platelet-targeted LRRC8A deletion impairs injury-induced arterial thrombosis, without prolonging tail bleeding. (A-C) Thrombosis in the cremasteric arterioles of Lrrc8afl/fl (WT) and Pf4-Cre;Lrrc8afl/fl (cKO) mice after laser-induced injury as observed by real-time confocal intravital microscopy (red, DyLight 649 anti-CD42C) (A) with median time course (B) and quantification of the fluorescent reporter at 60, 120, and 180 seconds after injury (n = 37 for both WT and cKO) (C). (D) Representative traces of arterial blood flow through the carotid artery of WT (top) and cKO (bottom) mice after FeCl3-induced injury. (E) Median time taken for the carotid artery of WT (n = 8) and cKO (n = 7) mice to occlude after FeCl3-induced injury. (F-G) Total bleeding time after tail tip amputation of WT (n = 10) and cKO (n = 10) mice (F), with corresponding Hb concentrations of red blood cell lysates collected during the assay (G). Data in panel C are represented as mean only, whereas data in panels B,E are represented as median. Data in panels F,G are represented as mean ± SEM. Statistical significance was determined by the Mann-Whitney U test for panels C,E, and by unpaired t test for panels F-G.…

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  Figure 6.. LRRC8 channel small-molecule inhibitors suppress agonist-induced aggregation, ATP release, and calcium influx. (A) Molecular structure of the small-molecule LRRC8 channel complex modulator SN-401 (DCPIB) and SN-406, a derivative of SN-401. (B) Current-time relationship of inward IATP (ATP efflux) and outward VRAC induced by HYPO swelling in MEG-01 cells and subsequent inhibition by application of 10 μM SN-406. (C-D) 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-406 (C), with mean current densities of inward IATP at −140 mV (n = 7-15) (D). (E-H) Aggregometry of platelets isolated from WT C57BL/6J mice stimulated with Thr (0.02 U/mL) in the presence of vehicle (0.02% dimethyl sulfoxide [DMSO]; n = 37), SN-401 (10 μM; n = 7), or SN-406 (10 μM; n = 5) (E), with concurrent ATP release (n = 5-30) (F-H). (I) Cytosolic calcium measured using the ratiometric dye Fura-2 in platelets isolated from WT C57BL/6J mice stimulated with PAR4-AP (400 μM; n = 8) in the presence of vehicle only (0.02% DMSO) or SN-406 (10 μM). (J-K) Aggregometry of human…

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  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…