Store-operated Ca ؉؉ entry (SOCE) is thought to comprise the major pathway for Ca ؉؉ entry in platelets. Recently, a number of transient receptor potential (TRP) proteins, which have been divided into 3 groups (TRPC, TRPM, and TRPV), have been suggested as SOCE channels. We report the expression and function of TRPC proteins in human platelets. TRPC6 is found at high levels and TRPC1 at low levels. Using purified plasma (PM) and intracellular membranes (IM), TRPC6 is found in the PM, but TRPC1 is localized to the IM. Using Fura-2-loaded platelets, we report that, in line with TRPC6 expression, 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the entry of Ca ؉؉ and Ba 2؉ independently of protein kinase C. Thrombin also induced the entry of Ca ؉؉ and Ba 2؉ , but thapsigargin, which depletes the stores, induced the entry of only Ca ؉؉ . Thus, thrombin activated TRPC6 via a SOCE-independent mechanism. In phosphorylation studies, we report that neither TRPC6 nor TRPC1 was a substrate for tyrosine kinases. TRPC6 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK) and associated with other cAMP-PK substrates. TRPC1 was not phosphorylated by cAMP-PK but also associated with other substrates. Activation of cAMP-PK inhibited Ca ؉؉ but not Ba 2؉ entry induced by thrombin and neither Ca ؉؉ nor Ba 2؉ entry stimulated by OAG. These results suggest that TRPC6 is a SOCE-independent, nonselective cation entry channel stimulated by thrombin and OAG. TRPC6 is a substrate for cAMP-PK, although phosphorylation appears to not affect cation permeation. TRPC1 is located in IM, suggesting a role at the level of the stores. IntroductionPlatelet activation forms an integral component of hemostasis and contributes to the events leading to thrombosis. Complete activation of platelets by all stimulatory agents leads to an increase of cytosolic Ca ϩϩ levels, which triggers many intracellular signaling processes important for the expression of functional responses. 1 Conversely, the vasodilators prostacyclin (PGI 2 ) and nitric oxide (NO) inhibit platelet function, with inhibition of Ca ϩϩ elevation an identified mechanism. 2 Cytosolic Ca ϩϩ elevation occurs as a consequence of release of the cation from intracellular stores and influx from the outside medium. Whilst the mechanism for Ca ϩϩ release from the stores in nonexcitable cells is well accepted, Ca ϩϩ entry mechanisms are less understood. The key elements involved in Ca ϩϩ signaling include activated surface receptors that lead to the stimulation of phospholipase C (PLC), resulting in the hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to release inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 binds to the IP 3 receptor (IP 3 R) on intracellular stores, releasing Ca ϩϩ , and DAG is a potent activator of protein kinase C (PKC). Ca ϩϩ entry is thought to occur predominantly as a consequence of store depletion and has been referred to as store-operated Ca ϩϩ entry (SOCE) or capacitative Ca ϩϩ entry (CCE). 3 However, the d...

Changes in [Ca 2+ ] i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca 2+ stores triggers Ca 2+ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca 2+ sensor that regulates storeoperated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1.

Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.