Abstract Thu129: Leucine Rich Repeat Containing 8 Heterohexameric Complexes Differentially Modulate Platelet Activation

Leucine rich repeat containing 8 (LRRC8) forms the heterohexameric volume-regulated anion channel (VRAC) that conducts an anionic current in response to cell swelling. Immunoprecipitation western blotting indicates that platelet LRRC8 is composed of a combination of the LRRC8A, B, C, and D subunits with LRRC8A being the essential subunit. Previous work utilizing selective LRRC8A deletion in murine platelets (8a cKO) demonstrated that the LRRC8 channel complex releases cytoplasmic ATP in response to platelet shape change to amplify platelet activation and calcium signaling via P2X1 activation. To investigate the individual contributions of the non-essential LRRC8 subunits to platelet function, we generated global LRRC8B, C, and D knock-out mice (8BKO, 8CKO, 8DKO respectively). Like the 8AKO, mean platelet volume is increased in 8DKO mice, while platelet volumes in 8CKO and 8BKO mice are unchanged from control. Phenocopying the 8AKO platelets, 8DKO platelets exhibit a marked defect in aggregation, granule secretion, integrin activation, and ATP release in response to weak agonist-stimulation. In response to a stronger agonist-stimulation, the aggregation, granule secretion, and integrin activation defects were rescued in the 8DKO while ATP release remained impaired. These data suggest that an LRRC8A/D containing channel complex induces platelet ATP release independent of dense-granule secretion to amplify platelet function.