CHARACTERIZATION OF PHOSPHOLIPASE A(2) SECRETION FROM HUMAN PLATELETS

Human platelets secreted phospholipase A(2) in a dose- and time-depend ent manner when challenged with thrombin, 12-O-tetradecanoyl-phorbol-l 3-acetate (TPA), or collagen. Enzyme release was maximal at concentrat ions of 0.1 units/ml of thrombin, 100 nM TPA, or 2 mu g/ml of collagen ; and complete by 2 min in platelets treated with thrombin or TPA. Cel ls challenged with collagen required up to 5 min for maximal secretion . Besides dose and time functions, phospholipase A(2) secretion was al so dependent on platelet concentration and the levels of bovine serum albumin in the incubation medium. The secreted enzyme was soluble and exhibited substrate and Ca2+ requirements similar to a detergent-solub ilized, partially purified phospholipase A(2) from whole platelets [Kr amer et al., Biochim. Biophys. Actawhole platelets [Kramer et al., Bio chim. Biophys. Acta (1988) 959, 269-279]. The pH optimum of the secret ed enzyme, however, was 1-2 units lower than the pH optimum of the pho spholipase A(2) from whole cells. Secreted phospholipase A(2) hydrolyz ed phosphatidylethanolamine at 5-12 times the rate of phosphatidylchol ine when the substrates were present in pure form. These apparent diff erences in activity were greatly diminished, though, when 1:1 molar mi xtures of the two substrates were employed. Because phospholipase A(2) catalyzes a key reaction during the formation of bioactive arachidona te metabolites, the secretion of this enzyme from platelets may be imp ortant in the regulation of thrombosis.