Oxidative inactivation of human and sheep platelet membrane-associated phosphotyrosine phosphatase activity

Incubation of human or sheep platelet crude membranes with xanthine oxidase /hypoxanthine in the presence of Fe2+/ADP inactivated phosphotyrosine phosp hatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) a ctivity in a time-dependent manner, this inhibition being significant withi n 5 min of treatment. The dynamics of protein thiols differed depending on the platelet species, but in any case decreases in protein thiols were only visible 20-45 min after the start of the treatment. The inhibition of PTPa se activity in general showed good a correlation with the production of thi obarbituric acid-reactive substances (TBARS). The results with several anti oxidants suggest that the inhibition of PTPase activity is related to the g eneration of alkoxyl and/or peroxyl radicals, Furthermore, the formation of fluorescent products and changes in amino groups were observed only after long incubation times with the oxidizing agents, these fluorescent products and the residual enzyme activity remaining in the membrane fraction. Treat ment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not with acetaldehyde, also inhibited membrane-associated PTPase activity. How ever, the amount of protein thiols was reduced only by treatment with trans -2-nonenal. Fluorescence product formation was always higher with trans-2-n onenal, these products being mainly located in the protein fraction. The re sults with aldehydes suggest that secondary degraded products of lipid hydr operoxides affect PTPase activity. Kinetic studies of PTPase activity indic ated that with all treatments enzyme inhibition is mainly due to a decrease in the V-max value. The results of fluorescence anisotropy measurements of labeled platelet membranes did not support the notion of a contribution of the lipid organization to peroxidation-mediated PTPase inhibition. All the above results indicate that platelet membrane-associated PTPase inhibition due to treatment with xanthine oxidase/hypoxanthine in the presence of Fe2 +/ADP is a very complex, time-dependent process, and that it is probably re lated, at least after long periods of peroxidation, to changes in protein t hiols and amino groups, We predict that the sensitivity of PTPase to lipid peroxidation must be physiologically relevant because of the increasing imp ortance of tyrosine phosphorylation in signal transduction, in general, and in platelet activation and aggregation in particular. (C) 1999 Elsevier Sc ience Inc.