Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C22
Pericellular Ca2+ recycling potentiates thrombin-evoked Ca2+ signals in human platelets.
S. O. Sage2, N. Pugh3, A. Harper3, R. W. Farndale1,2
1. Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom. 2. Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom. 3. Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Previous work has demonstrated a key role for the Na+/Ca2+ exchanger (NCX) in modulating store-operated calcium entry, as well as collagen- and P2X1-evoked Ca2+ signals in human platelets [1-3]. Initial studies examining the effects of the NCX inhibitors SN-6 and KB-R7943 on thrombin-evoked Ca2+ signals in the absence of extracellular Ca2+ demonstrated a seemingly paradoxical inhibition of the thrombin-evoked rise in cytosolic [Ca2+]. We have now investigated this further by examining the effects of NCX inhibitors on Ca2+ fluxes in different compartments in and around human platelets. Thrombin-evoked Ca2+ signals in the cytosol or pericellular space were monitored in Fura-2- or FFP-18-loaded cells respectively. Changes in the extracellular Ca2+ concentration were monitored using Fluo-4 K+ salt. Platelets were stimulated with 0.5 U/ml thrombin in the presence of 1 mM EGTA. Data are presented as mean ± SEM. Statistical significance was assessed using Student's t-test. KB-R7943 (50 µM) or SN-6 (50 µM) reduced thrombin-evoked rises in cytosolic (48.9 ± 8.9% and 65.0 ± 5.3% of control respectively; both p < 0.05; n = 5), extracellular (14.9 ± 2.8% and 77.4 ± 9.7% of control respectively; p < 0.05; n = 6), and pericellular (signal abolished and 41.8 ± 6.6% of control respectively; p < 0.05; n = 5) Ca2+ concentration. To investigate whether the pericellular Ca2+ signal influenced the cytosolic Ca2+ signal we replaced in the extracellular medium the slow Ca2+ chelator, EGTA, with same concentration of the faster Ca2+ chelator, BAPTA. This reduced the thrombin-evoked rises in pericellular (54.5 ± 9.8% of control; p < 0.05; n = 7) and cytosolic (72.8 ± 1.9% of control; p < 0.05; n = 6) Ca2+ concentration. Furthermore, blocking plasma membrane Ca2+ channels using MRS-1845 (30 µM) and 5’-Iodo-resiniferatoxin (20 µM) also reduced the cytosolic Ca2+ signal evoked by thrombin in the absence of extracellular Ca2+ (54.4 ± 3.0% of control; p < 0.05; n = 7). These results suggested that the pericellular Ca2+ signal might influence Ca2+ dynamics in the cytosol through Ca2+ recycling back into the cell across the plasma membrane. We therefore attempted to visualize the pericellular Ca2+ signal by imaging thrombin-activated single platelets immobilized on fibrinogen-coated slides in a medium containing Fluo-4 K+ salt. We observed pericellular Ca2+ signals contained within the boundary of the cells that spread in a slow, directionally-restricted manner. These results suggest that Ca2+ is specifically removed from the cytosol into invaginations of the platelet plasma membrane known as the open canalicular system (OCS), that NCXs create a rise in [Ca2+] in the OCS of thrombin-stimulated platelets and that Ca2+ can recycle from the OCS back into the cytosol so helping to maintain the rise in cytosolic Ca2+ concentration.
Where applicable, experiments conform with Society ethical requirements