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...
Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin  3 subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. IntroductionIn classical terms, reduction/oxidation systems within a cell have been represented very simply. The cytoplasmic environment is hypoxic and reducing, whereas the extracellular environment is normoxic and oxidizing. The generation of a disulfide bond from 2 cysteine residues is an oxidation reaction. To correctly generate these bonds inside the cell, there are, therefore, a group of enzymes known as the thiol isomerases. These are capable of the formation, reduction, and rearrangement of the disulfide-bonding patterns of proteins, often as part of folding of nascent proteins. The thiol isomerase enzymes are anchored to the endoplasmic reticulum via KDEL-receptor proteins. [1][2][3] Recent studies have suggested additional functions for thiol isomerase enzymes: on the surface of cells, where they participate in receptor activation and remodeling, and substrate processing. [4][5][6] Protein disulfide isomerase (PDI) is the best-characterized thiol isomerase to demonstrate this dual functionality. A number of cell types, including bovine aortic endothelial cells, 7 rat hepatocytes, 8,9 and human B cells, 5,10 have been shown to secrete PDI, which associates with the cell surface. Cell-surface PDI has been implicated in the reduction of the disulfide-linked diptheria toxin heterodimer 11 and events triggering the entry of HIV into lymphoid cells. 6,12 On the basis of a series of investigations, initially by Detweiller and coworkers, a role for PDI in platelet physiology is now established. 4,[13][14][15][16] Early studies demonstrated PDI was present on the external membrane of activated and resting platelets, and proteins with thiol isomerase activity were secreted f...
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