Initiation of RVD response in human platelets: Mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A2

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A₃. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A₃ formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca²⁺-insensitive phospholipase A₂, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A₂ inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 m, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca²⁺ depletion by EGTA (up to 10 mm) does not affect RVD; (iii) intracellular Ca²⁺ depletion by BAPTAAM (100 m) inhibits RVD but not hepoxilin A₃ formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP S (1 m) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A₃; (v) NaF, 1 mm, induces hepoxilin A₃ formation, tested by the RVD reconstitution assay; and (vii) GDP S inhibits hepoxilin A₃ formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A₃ formation in human platelets.DeceasedThis work is dedicated to the memory of Prof. A.A. Livne. It was carried out at the Amelia (Mimi) Rose Laboratory for Cellular Signal Transduction at the Department of Life Sciences, Ben-Gurion University of the Negev. We thank A. Dannon for helpful discussion.