The nature of the mechanism underlying store-mediated Ca 2؉ entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca 2؉ entry, showing an initial potentiation followed by inhibition of Ca 2؉ entry. Moreover, addition of these agents after induction of store-mediated Ca 2؉ entry inhibited the Ca 2؉ influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca 2؉ entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca 2؉ entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca 2؉ entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca 2؉ entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca 2؉ entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.In many cell types, including human platelets, depletion of the intracellular Ca 2ϩ stores induces entry of Ca 2ϩ across the plasma membrane (PM) 1 (1). However, the mechanism by which the filling state of stores is communicated to the PM remains unclear. Hypotheses have considered both indirect and direct coupling mechanisms. Indirect coupling assumes the existence of a diffusible messenger generated by the storage organelles such as cyclic GMP (2), cytochrome P-450 metabolites (3), tyrosine kinases (4, 5), or release of a calcium influx factor from depleted stores (6); however, no messenger molecule has been identified. Alternatively, direct coupling (conformational coupling) proposes a physical interaction between the endoplasmic reticulum (ER) and the PM and considers that some calcium sensitivity may reside on the InsP 3 receptor, which is thought to be responsible for transmitting information from the ER to the PM (7). Consistent with this model, some studies indicate that Ca 2ϩ entry is closely localized to sites where InsP 3 evokes emptying of the Ca 2ϩ stores and that Ca 2ϩ entry is unlikely to be activated by a diffusible molecule (8 -10).A different model suggests that Ca 2ϩ channels or membranebound activator molecules are exocytotically incorporated into the PM upon store depletion (10). This hypothesis is compatible with the conformational coupling model only if it is assumed that the link between the ER and the PM is mechanically weak before store depletion and strong afterward.Recently, a secretion-like coupling model has been proposed by Patterson et al. (11). This mechanism involves a phys...

To investigate the possible involvement of tyrosine phosphorylation in the process of store-regulated Ca2+ entry, ionomycin (in the presence of EGTA) was used to deplete the intracellular Ca2+ stores of fura-2-loaded human platelets, and the effect of refilling with Ca2+, Ba2+ or Sr2+ evaluated. Depletion of the intracellular Ca2+ stores resulted in an increase in protein tyrosine phosporylation. This increase is reversed when the stores were refilled in Ca2+ or Sr2+, but not Ba2+. Refilling of the stores with Ca2+ or Sr2+, but not Ba2+, suppressed Mn2+ entry. These findings support the hypothesis that tyrosine phosphorylation plays a role in mediating store-regulated Ca2+ entry in human platelets and provides evidence for tyrosine phosphatase activity regulated by the Ca2+ content of the intracellular stores.

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The effects of the WT on store-mediated Ca 2+ entry and protein tyrosine phosphorylation were investigated in fura-2-loaded human platelets. Wortmannin (2 laM) attenuated the rise in [Ca2+]i caused by Ca 2÷ entry while having no effect on the mobilisation of Ca 2+ from internal stores. It also reduced storedepletion-evoked protein tyrosine phosphorylation. These findings demonstrate that WT is an inhibitor of tyrosine phosphorylation and store-mediated calcium entry and provide further evidence for the involvement of a tyrosine phosphorylation step in the link between Ca 2÷ store depletion and Ca 2+ influx in human platelets.