Abstract
We have previously demonstrated that Na+/Ca2+ exchangers (NCXs) potentiate Ca2+ signaling evoked by thapsigargin in human platelets, via their ability to modulate the secretion of autocoids from dense granules. This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca2+ removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near-membrane indicator FFP-18, thrombin stimulation was observed to elicit an NCX-dependent accumulation of Ca2+ in a pericellular region around the platelets. To test whether this pericellular Ca2+ accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca2+ chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca2+ rise reduced thrombin-evoked Ca2+ signals and dense granule secretion. Blocking Ca2+-permeable ion channels had a similar effect, suggesting that Ca2+ exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo-4 indicated that thrombin-evoked rises in extracellular [Ca2+] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP-18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca2+] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca2+].
Highlights
The importance of rises in cytosolic Ca2+ concentration ([Ca2+]cyt) in the activation of human platelets is well established (Rink and Sage 1990)
Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society
If the role of the Na+/Ca2+ exchangers (NCXs) in Ca2+ signal generation was through reverse-mode exchange, the NCX inhibitors should have no effect when platelets are activated in the absence of extracellular Ca2+
Summary
The importance of rises in cytosolic Ca2+ concentration ([Ca2+]cyt) in the activation of human platelets is well established (Rink and Sage 1990). This has led to great interest in identifying the molecular mechanisms that generate and modulate agonist-evoked rises in platelet [Ca2+]cyt, in the hope of identifying novel targets for antithrombotic drugs. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society
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