Store-mediated Ca²⁺ entry (SMCE), a mechanism modulated by the filling state of the Ca²⁺ stores, is an important mechanism for Ca²⁺ influx in non-excitable cells. Although the mechanism involved in the activation of SMCE is still not clearly described, the secretion-like coupling is the most likely model to explain SMCE in several cell types (Patterson et al. 1999; Redondo et al. 2003), including human platelets (Rosado et al. 2000b). This mechanism involves a physical and reversible interaction between the endoplasmic reticulum and the plasma membrane where the actin cytoskeleton and tyrosine kinases, such as pp60^src, play an important role (Rosado et al. 2000a; Redondo et al. 2003). Reactive oxygen species, including H₂O₂, have been recently presented as intracellular messengers required for a large number of cellular events, especially those mediated by tyrosine kinases. Hence, we have investigated the involvement of H₂O₂ generation in the activation of SMCE in human platelets.

Blood was drawn from volunteers with local ethical committee approval. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) measurement and Western blotting were performed as previously described (Rosado et al. 2000a; Redondo et al. 2003). Treatment of human platelets with H₂O₂ resulted in a time-and concentration-dependent activation of pp60 ^src. Incubation for 30 min with 5 µM GF 109203X, a PKC inhibitor, prevented pp60^src activation induced by 10 µM H₂O₂. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60^src by H₂O₂ does not require rises in [Ca²⁺]_i. Treatment of platelets for 40 min with 10 µM cytochalasin D, a inhibitor of actin polymerization, significantly reduced pp60^src activation induced by 10 µM H₂O₂ by 85 % (n = 6; P < 0.01, Student’s t test). We found that platelet stimulation with 1 µM thapsigargin (TG) plus a low concentration of ionomycin (Iono; 50 nM) or with the physiological agonist thrombin (0.5 U/ml) induced rapid H₂O₂ production as detected by using CM-H2DCFDA. Treatment of human platelets for 5 min with 300 U/ml catalase, an enzyme that activates the decomposition of H₂O₂ into water and oxygen, abolished TG plus Iono-and thrombin-induced activation of pp60^src. In addition, catalase inhibited TG plus Iono-and thrombin-induced SMCE by 59.3 ± 4.5 and 37.4 ± 3.9 %, respectively (S.E.M.; n = 6, P < 0.01, paired Student’s t test), without having any effect on the maintenance of SMCE. Our findings suggest that platelet activation with TG plus Iono or thrombin is associated with H₂O₂ production, which acts as a second messenger involved in the activation of SMCE by pp60^src activation in these cells.

This work was supported by DGESIC (BFI2001-0624) and Cons. Sanidad y Consumo (Junta de Extremadura) (03/51).