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Previous studies have shown that platelets exhibit a H2O2producing, NADH-dependent system that is activated by interaction with particulate material. Current evidence suggests that this system could be critically involved in the generation of chemotactic factor(s). In the present studies, chemotactic activity for polymorphonuclear leukocytes of supernatants derived from zymosan-stimulated human platelets has been evaluated using an agarose gel technique. Supernatants of opsonized zymosan-stimulated platelets showed significant chemotactic activity (migration index = 300 ± 50), in comparison with supernatants prepared from platelet suspensions stimulated with nonopsonized-zymosan (migration index = 15 ± 15) or resting platelet supernatants (migration index = 15 ± 15). Furthermore, a marked increase in chemotactic activity of the opsonized zymosan-treated platelet supernatants was demonstrated after the additon of NADH (migration index = 525 ± 100). The inclusion of specific inhibitors of the cycloxygenase and lipoxygenase pathways resulted in a marked reduction of chemotactic activity, which was restored in the presence of NADH. Further, the addition of superoxide dismutase completely abolished the chemotactic response induced by NADH. These data suggest that platelets are the source of chemotactic factor(s) derived from the activation of a superoxide generating system.

The oxidative metabolic burst of mononuclear and polymorphonuclear phagocytes can be stimulated to produce free oxygen radicals. Several substances can enhance this respiratory burst activity by a priming action: recently growth hormone (rat and porcine) was demonstrated to act as a priming agent on rat peritoneal and on porcine alveolar macrophages. In our study we wanted to verify whether also human GH had a similar priming action on homologous cells, in particular on polymorphonuclear leukocytes. To determine the oxidative activity of polymorphonuclear leukocytes, after stimulation with phorbol myristate acetate, a flow-cytometric assay was employed which registered the intracellular formation of highly fluorescent products as indicators for the intracellular formation of hydrogen peroxide. The incubation of phorbol myristate acetate-stimulated polymorphonuclear leukocytes with GH resulted in a time-dependent and dose-dependent increase in fluorescence, thus demonstrating that human GH enhances in vitro the oxidative metabolic burst of these cells. The action of GH appeared to be significant after 30 min of incubation, was maximal at 60 min, and decreased after 90 min. After one hour of incubation, the first significant variation of fluorescence appeared with GH at a concentration of 50 μg/l. The maximum effect was seen at 100 μg/l with no further increase. Specificity of GH action was demonstrated by the inhibition of its effect by the addition of monoclonal antibodies to GH.

Summary: Previous studies have shown that platelets exhibit a H2O2producing, NADH-dependent system that is activated by interaction with particulate material. Current evidence suggests that this system could be critically involved in the generation of chemotactic factor(s). In the present studies, chemotactic activity for polymorphonuclear leukocytes of supernatants derived from zymosan-stimulated human platelets has been evaluated using an agarose gel technique. Supernatants of opsonized zymosan-stimulated platelets showed significant chemotactic activity (migration index = 300 ± 50), in comparison with supernatants prepared from platelet suspensions stimulated with nonopsonized-zymosan (migration index = 15 ± 15) or resting platelet supernatants (migration index = 15 ± 15). Furthermore, a marked increase in chemotactic activity of the opsonized zymosan-treated platelet supernatants was demonstrated after the additon of NADH (migration index = 525 ± 100). The inclusion of specific inhibitors of the cycloxygenase and lipoxygenase pathways resulted in a marked reduction of chemotactic activity, which was restored in the presence of NADH. Further, the addition of superoxide dismutase completely abolished the chemotactic response induced by NADH. These data suggest that platelets are the source of chemotactic factor(s) derived from the activation of a superoxide generating system.Speculation: Platelets play an important role in the nonspecific immune mechanisms by participating in the recruitment of inflammatory cells to the site of inflammation and in the cell-to-cell interaction. This function appears to be related to the production of chemotactic factors some of which are associated with a superoxide generating system.

Human platelets have been shown to release H2O2when challenged with particulate stimuli. Here we report that platelets stimulated by thrombin also generate H2O2inside, as detected by the peroxidation of the fluorogenic 2, 7-dichlorofluorescein. The increase of the fluorescence is proportional to the amount of H2O2generated by platelets. Under our experimental conditions, the fluorescence of intra-platelet 2, 7-dichlorofluorescein is increased by 2-5 folds by stimulation with 1 U/ml of thrombin. Intracellular catalase reduces the extent of H2O2production or its steady-state concentration. The ability of platelets to produce reduced, reactive oxygen radical species is noteworthy, since they have been found to play a role in pathological process, like the Kawasaki disease (Del Principe et al., FEBS Lett.185:142, 1985).