Trpc6 gain-of-function disease mutation enhances phosphatidylserine exposure in murine platelets

Platelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6(E896K/E896K) (GOF) and Trpc6(DN/DN) mutations were subject to in vitro analysis. Trpc6(E896K/E896K) and Trpc6(DN/DN) mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin alpha IIb beta 3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6(E896K/E896K), but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6(DN/DN) platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6(DN/DN), and enhanced in Trpc6(E896K/E896K), platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6(E896K/E896K) platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, the results suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype.