Objectives: 

Altered calcium (Ca2+) signaling significantly impair platelet function and, therefore, thrombosis and hemostasis. Mechanisms of Ca2+ entry operated by previous depletion of intracellular Ca2+ stores (SOCE) and by receptor-agonist interactions (ROCE) have been described in these cells as essential for Ca2+ homeostasis. Members of the TRPC family of Ca2+ channels are expressed in platelets. However, their function in Ca2+ homeostasis is still unclear in these cells. We studied the impact of TRPC6 removal in murine platelet Ca2+ homeostasis.

Materials: 

Washed blood platelets were isolated in absence of Ca2+ from TRPC6-deficient mice, and incubated in Tyrode's buffer in the presence 50 mM Ca2+ to equilibrate possible loss of intracellular Ca2+ during the isolation process. Intracellular Ca2+ measurements were evaluated by fluorometry using the fluorescent Ca2+ probe Fura-2-AM. Molecular interactions were assessed by immunoprecipitation assays. Statistical significance assessed by Student t test.

Results: 

TRPC6-deficient platelets exhibited a mild but significant decrease (18%, p<0.05) of basal cytoplasmic free Ca2+ ([Ca2+]c) in resting conditions as compared to controls, but normal Ca2+ content in intracellular stores. TRPC6-deficient platelets also displayed reduced (40%, p<0.05), but not abrogated, OAG-induced ROCE as compared to wild-type controls, suggesting the participation of TRPC6 in diacylglycerol (DAG)-dependent ROCE. Co-immunoprecipitation assays using an anti-STIM1 antibody revealed absence of direct or indirect interactions between TRPC6 and STIM1 in murine resting platelets, indicating a redundant role of STIM1 in TRPC6-mediated regulation of basal [Ca2+]c. The involvement of other signaling pathways in TRPC6-mediated control of basal [Ca2+]c is currently under study.

Conclusions: 

Previous studies demonstrated that neither STIM1, STIM2 nor Orai1 are involved in basal [Ca2+]c regulation in resting platelets. Our results suggest that TRPC6 participates in basal [Ca2+]c and DAG-dependent ROCE together with other unidentified Ca2+ channels in murine platelets.