Pericellular Ca²⁺recycling potentiates thrombin-evoked Ca²⁺signals in human platelets

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+].

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…Fluo-4 fluorescence was recorded with excitation at 480 nm and emission at 515 nm, and calibrated as previously described [3]. A validation and quantitative analysis of this technique is presented in [3].…”

“…Yet these studies are limited by their ability to only monitor the net effect of any experimental manipulation on each of the component processes involved in controlling [Ca 2+ ] cyt (Ca 2+ buffering, sequestration, release, entry and removal), and lack the ability to resolve the source of the Ca 2+ or the transporters or channels affected. Recent work in human platelets has highlighted the many pitfalls of drawing conclusions as to the molecular pathways involved in eliciting Ca 2+ signals based solely on measurements of [Ca 2+ ] cyt [1][2][3].…”

“…We have recently developed and validated methods to measure agonist-evoked changes in extracellular and intracellular store Ca 2+ concentrations ([Ca 2+ ] ext and [Ca 2+ ] st , respectively) in platelet cell suspensions [3,4], which can be utilized alongside the measurement of agonist-evoked changes in [Ca 2+ ] cyt to improve the interpretation of results compared with simple cytosolic Ca 2+ measurements alone. This methodology has been successfully used to demonstrate how the Na + /Ca 2+ exchanger acts to create a pericellular source for Ca 2+ recycling into the platelet, as well as allowing estimation of the Ca 2+ buffering capacity of the platelet cytosol [3].…”

“…The P2Y 1 receptor couples via G q to phospholipase C-β (PLC-β) leading to the formation of IP 3 , which releases Ca 2+ from the dense tubular system (DTS) [15,16]. The P2Y 12 receptor (formerly known as P2 T or P2 AC ) couples via G i to adenylyl cyclase [17] and phosphoinositide 3-kinase [18].…”

“…Thrombin activates human platelets via the protease-activated receptors (PARs) 1 and 4 [19]. PAR-1 and PAR-4 couple via Gq to PLC-β resulting in Ca 2+ release from the DTS via IP 3 formation.…”