In non-excitable cells, such as platelets, activation of phospholipase C-coupled receptors results in a transient increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) due to the release of Ca²⁺ from agonist-sensitive stores. In addition, mitochondria, whose redox state control is important for cellular energy conversion, have been reported to be involved in the regulation of Ca²⁺ homeostasis (Rizzuto et al. 1999). Ca²⁺ mobilisation plays an important role in the regulation of several cellular events, such as secretion or platelet aggregation. The oxidative balance has been reported to play a crucial role in regulating platelet activity. Hence, we have investigated the effect of oxygen free radicals such as H₂O₂ on Ca²⁺ homeostasis in human platelets.

Blood was drawn from volunteers with local ethical committee approval according to the Declaration of Helsinki. Fura-2-loaded human platelets were prepared as previously described (Rosado et al. 2000) and fluorescence was measured using a Shimadzu spectrophotometer.

In a Ca²⁺-free medium, treatment of platelets with 1 mM H₂O₂ caused a sustained increase in [Ca²⁺]_i due to the release of Ca²⁺ from internal stores. Addition of H₂O₂ to platelet suspensions whose agonist-releasable Ca²⁺ pools had been previously depleted by thrombin (1 U ml^-1) or simultaneous treatment with thapsigargin (TG; 1 µM) and a low concentration of ionomycin (50 nM) was still able to induce a sustained increase in [Ca²⁺]_i (n = 7). Similar results were observed when human platelets were treated with the mitochondrial uncoupler FCCP (1 µM), to dissipate the membrane potential across the inner mitochondrial membrane that sustains the activity of the Ca²⁺ uniporter (Medler & Gleason, 2002), prior to the treatment with H₂O₂ (n = 8). In contrast, treatment of platelets with a combination of either FCCP plus thrombin or FCCP plus TG and ionomycin abolished the H₂O₂-induced calcium release (n = 5). Finally, addition of H₂O₂ to platelet suspensions reduced calcium mobilisation induced by the physiological agonist thrombin or with TG plus ionomycin by 78 ± 6 and 90 ± 6 %, respectively (S.E.M.; n = 4-8, P < 0.01, Student’s paired t test).

Our findings suggest that H₂O₂ releases Ca²⁺ from mitochondrial and agonist-releasable Ca²⁺ pools which might be involved in the reported effects of the oxygen free radicals in human platelets.

This work was supported by DGESIC (BFI2001-0624) and Programa Propio of UEX. P.C.R. is supported by a DGESIC fellowship.