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 relationship of WT MK cell induced by HYPO (210 mOsm) swelling, and subsequent inhibition upon application of 10 μM SN-401 (DCPIB). (O-P) VRAC current-voltage relationship after HYPO swelling during voltage ramps from −100 mV to +100 mV in MKs isolated from WT ± SN-401 (O) and cKO (P) mice. (Q) Mean outward (+100 mV) and inward (−100 mV) current densities measured in WT MKs after HYPO stimulation in the presence (green, n = 6) or absence (gray, n = 6) of 10 μM SN-401, or in cKO MKs after HYPO stimulation (red, n = 9). Data are represented as mean ± standard error of the mean (SEM). Statistical significance was determined by unpaired t test for panels H,K,M, Q; and by ordinary 1-way analysis of variance (ANOVA) with the Tukey multiple comparisons test for panels I-J. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. cHet, heterozygous control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HYPO, hypotonic; ISO, isotonic; ns, not significant; RPKM, reads per kilobase million.

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 relationship of WT MK cell induced by HYPO (210 mOsm) swelling, and subsequent inhibition upon application of 10 μM SN-401 (DCPIB). (O-P) VRAC current-voltage relationship after HYPO swelling during voltage ramps from −100 mV to +100 mV in MKs isolated from WT ± SN-401 (O) and cKO (P) mice. (Q) Mean outward (+100 mV) and inward (−100 mV) current densities measured in WT MKs after HYPO stimulation in the presence (green, n = 6) or absence (gray, n = 6) of 10 μM SN-401, or in cKO MKs after HYPO stimulation (red, n = 9). Data are represented as mean ± standard error of the mean (SEM). Statistical significance was determined by unpaired t test for panels H,K,M, Q; and by ordinary 1-way analysis of variance (ANOVA) with the Tukey multiple comparisons test for panels I-J. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. cHet, heterozygous control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HYPO, hypotonic; ISO, isotonic; ns, not significant; RPKM, reads per kilobase million.

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