Peroxidation of arachidonic acid by human platelets

Abstract

Peroxidation of arachidonic acid by human platelets was studied by malonaldehyde measurement and oxygen uptake. Platelets were prepared by collecting blood into 3.8% trisodium citrate, isolation in the presence of EDTA, washing in 0.154M NaCl (saline) and finally suspending in Tris-buffered saline (pH 7.4) for malonaldehyde determination or in saline for measurement of oxygen uptake. Malonaledhyde was measured by reaction with thiobarbituric acid and oxygen uptake by the platinum electrode method. Peroxidation of arachidonic acid was induced by incubation of this fatty acid with platelets and the optimal incubation condition for formation of malonaldehyde was examined. The maximum value of malonaldehyde was produced approximately 1min after initiation of the reaction at 37°C. The activity of platelets was almost completely inhibited by heating them at 100°C for 5min. The highest specific activity was found in the particulate fraction that sedimented between 12, 000g and 105, 000g. A burst in oxygen uptake occurred concomitantly with peroxidation of arachidonic acid by platelets. Aspirin inhibited both malonaldehyde production and the burst in oxygen uptake induced by platelets. These results are in accord with the concept that endoperoxide intermediates in prostaglandin biosynthesis (prostaglandin G2 and prostaglandin H2) are formed by human platelets in response to arachidonic acid. Methods used in the present investigation may be employed as convenient procedures for an assay of prostaglandin synthetase activity of platelets in the evaluation of new anti-platelet drugs as well as for elucidation of pathogenesis of platelet dysfunction.