ABSTRACT
In previous studies, a strong synergism between low concentrations of hydrogen peroxide and nitric oxide in the inhibition of agonist-induced platelet aggregation has been established and may be due to enhanced formation of cyclic GMP. In this investigation, hydrogen peroxide and NO had no effect on the activity of pure soluble guanylyl cyclase or its activity in platelet lysates and cytosol. H(2)O(2) was found to increase the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), increasing the amount of the 50-kDa form that results from phosphorylation at serine(157). This occurs both in the presence and in the absence of low concentrations of NO, even at submicromolar concentrations of the peroxide, which alone was not inhibitory to platelets. These actions of H(2)O(2) were inhibited to a large extent by an inhibitor of cyclic AMP-dependent protein kinase, even though H(2)O(2) did not increase cyclic AMP. This inhibitor reversed the inhibition of platelets induced by combinations of NO and H(2)O(2) at low concentrations. The results suggest that the action on VASP may be one site of action of H(2)O(2) but that this event alone does not lead to inhibition of platelets; another unspecified action of NO is required to complete the events required for inhibition.
Subject(s)
Blood Platelets/physiology , Carbazoles , Cell Adhesion Molecules/metabolism , Cyclic GMP/blood , Guanylate Cyclase/blood , Hydrogen Peroxide/pharmacology , Indoles , Nitric Oxide/pharmacology , Phosphoproteins/metabolism , Platelet Aggregation/drug effects , 1-Methyl-3-isobutylxanthine/pharmacology , Alkaloids/pharmacology , Blood Platelets/drug effects , Blood Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/blood , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/blood , Cytosol/enzymology , Drug Synergism , Enzyme Inhibitors/pharmacology , Humans , Hydrazines/pharmacology , In Vitro Techniques , Indazoles/pharmacology , Kinetics , Microfilament Proteins , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Donors/pharmacology , Nitrogen Oxides , Phosphoserine/blood , Platelet Aggregation/physiology , Platelet Aggregation Inhibitors/pharmacology , Subcellular Fractions/metabolism , Thrombin/pharmacologyABSTRACT
The nitration of protein tyrosine residues by peroxynitrous acid has been associated with pathological conditions. Here it is shown, using a sensitive competitive enzyme-linked immunosorbent assay and immunoblotting for nitrotyrosine, that spontaneous nitration of specific proteins occurs during a physiological process, the activation of platelets by collagen. One of the main proteins nitrated is vasodilator-stimulated phosphoprotein. Endogenous synthesis of nitric oxide and activity of cyclo-oxygenase were required for the nitration of tyrosine. The nitration was mimicked by addition of peroxynitrite to unstimulated platelets, although the level of nitrotyrosine formation was greater and its distribution among the proteins was less specific.
