Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A 2 in response to thrombin in rabbit platelets

Abstract

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP 3) in [ 3H]inositol-labeled platelets, increases of [ 3H]arachidonic acid ([ 3H]AA) release, and [ 3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A 2 or C inhibitor suggested that not only phospholipase C but also phospholipase A 2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A 2 activity. Guanosine 5′-(3- O-thio)triphosphate (GTP-γS), guanyl-5′-(β,γ-iminio)triphosphate), or AlF 4 − caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5′-(2- O-thio)diphosphate) and was also inhibited by decreased Mg 2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP 3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTPγS-stimulated release from permeabilized platelets were both markedly dependent on Ca 2+. However, Ca 2+ addition could not enhance AA release without GTPγS even when Ca 2+ was increased up to 10 −4 m in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A 2 activity, not by phospholipase C activity, and that Ca 2+ is an important factor to the activation of phospholipase A 2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A 2.