Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) is an important factor involved in the modulation of many physiological processes. In non-excitable cells, such as platelets, agonists induce a transient release of Ca²⁺ from internal stores, which activates a sustained store-mediated Ca²⁺ entry (SMCE). In contrast, removal of Ca²⁺ from the cytosol and maintenance of a low [Ca²⁺]_i is mainly mediated by the plasma membrane Ca²⁺ ATPase (PMCA; Rosado et al. 2000). The redox state has been reported to modulate [Ca²⁺]_i in different cell types (Pariente et al. 2001; Törnquist et al. 2000) suggesting that oxygen radicals, such as H₂O₂, might act as second messengers. The aim of the present study was to investigate the effect of H₂O₂ in both SMCE and the PMCA activity in human platelets.

Fura-2-loaded human platelets, from volunteers with local ethical committee approval according to the Declaration of Helsinki, were prepared and fluorescence measured as previously described (Rosado et al. 2000). SMCE induced by treatment with thapsigargin (Tg) plus ionomycin (IONO) or thrombin was estimated as the integral of the rise in [Ca²⁺]_i for 2.5 min after addition of Ca²⁺. To study the effect of H₂O₂ in the PMCA activity, we compared the rate of decay of [Ca²⁺]_i to basal values, after treatment in a Ca²⁺-free medium with Tg plus IONO, in the absence and presence of H₂O₂, by using the constant of the exponential decay. Since H₂O₂ has been shown to release Ca²⁺ from mitochondrial as well as agonist-releasable pools (Pariente et al. 2001) a buffer supplemented with oligomycin (10 µM) and rotenone (10 µM) was used to avoid interference with the analysis of Ca²⁺ release from mitochondrial stores.

In a Ca²⁺-free medium, treatment of platelets with 1 µM Tg plus 50 nM IONO resulted in a transient increase in [Ca²⁺]_i due to release of Ca²⁺ from intracellular stores; subsequent addition of Ca²⁺ (1.5 mM) to the medium resulted in a sustained increase in [Ca²⁺]_i indicative of SMCE. Addition of 1 mM H₂O₂ significantly reduced Ca²⁺ entry by 65.1 ± 5.3 % (S.E.M.; n = 7; P < 0.01, Student’s paired t test). Similar results were found when Ca²⁺ entry was activated by the agonist thrombin (0.5 U ml^-1). Treatment of human platelets with H₂O₂ (1 mM) reduced thrombin-induced Ca²⁺ entry by 50.8 ± 8.6 % (n = 5; P < 0.01). In addition, H₂O₂ decreased the rate of decay of [Ca²⁺]_i to basal levels after activation with 1 µM Tg plus 50 nM IONO. The decay constants were 0.0006 ± 0.0003 in H₂O₂-treated platelets and 0.0069 ± 0.0011 in controls (n = 5; P < 0.001). These results indicate that H₂O₂ inhibits both SMCE and Ca²⁺ efflux across the plasma membrane in human platelets.

This work was supported by DGESIC (BFI2001-0624) and Consejer’a de Sanidad y Consumo, Junta de Extremadura (02/0002). P.C.R. is supported by a DGISIC fellowship.