Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC130

Poster Communications

Acidic Ca2+ store refilling by SERCA3 is regulated by STIM1 in human platelets

I. Jardin1, J. J. Lopez1, R. Bobe2, J. A. Pariente1, J. Enouf2, G. M. Salido1, J. A. Rosado1

1. Department of Physiology, University of Extremadura, Caceres, Spain. 2. Hôpital Lariboisière, INSERM, CRCIL, Paris, France.

Ca2+ mobilization regulates a wide variety of cellular functions. Platelets possess agonist-releasable Ca2+ stores in acidic organelles where SERCA3 pump is involved in store refilling. STIM1, which has been presented as a central regulator of platelet function, is a Ca2+ sensor of the intracellular Ca2+ stores (Roos et al., 2005). The aim of the present study was to explore the possible involvement of STIM1 in acidic Ca2+ store refilling in human platelets. Blood was drawn from volunteers with local ethical committee approval and in accordance with the Declaration of Helsinki. Cytosolic Ca2+ concentration ([Ca2+]i) measurement, immunoprecipitation and Western blotting were performed as previously described (Redondo et al., 2004). Electroporation and incorporation (electrotransjection) of anti-STIM1 antibody (or a mouse IgG of the same nature of the anti-STIM1 antibody) into cells was performed following a previously reported procedure (Lopez et al., 2006). To compare the rate of decay of [Ca2+]i to resting values after stimulation with thrombin we used the constant of the exponential decay as previously shown (Rosado & Sage, 2000). Acidic store refilling was investigated by remobilizing Ca2+ from the acidic stores using 2,5-di-(t-butyl)-1,4-hydroquinone, a specific SERCA3 inhibitor, after a brief refilling period that followed thrombin stimulation. Electrotransjection of cells with anti-STIM1 (Y231-K243) antibody, directed towards a cytoplasmic sequence of STIM1, significantly reduced acidic store refilling by 70%. In addition, the anti-STIM1 antibody reduced the rate of decay of the [Ca2+]i to resting levels after stimulation with 1 U/mL thrombin (the decay constants were 0.0094 ± 0.0004 and 0.0078 ± 0.0004 in electroporated cells incubated with mouse IgG or anti-STIM1, respectively; P<0.05 Students t-test; n=7). Platelet treatment with thrombin or with 1 µM thapsigargin in combination with 50 nM ionomycin, to induce extensive Ca2+ store depletion, resulted in a transient increase in the interaction between STIM1 and SERCA3, reaching a maximum 30 s after stimulation and then decreased. The coupling between STIM1 and SERCA3 was abolished by electrotransjection with anti-STIM1 antibody. The interaction between STIM1 and SERCA3 induced by thrombin or by treatment with thapsigargin plus ionomycin is reduced in platelets from type 2 diabetic patients, as well as Ca2+ reuptake into the acidic Ca2+ stores. These findings provide evidence for a role of STIM1 in acidic store refilling in platelets probably acting as a Ca2+ sensor and regulating the activity of SERCA3. This action is impaired in platelets from type 2 diabetics, which might lead to the enhanced cytosolic Ca2+ concentration observed (Saavedra et al., 2004) and, therefore, in platelet hyperactivity.

Where applicable, experiments conform with Society ethical requirements