Dexamethasone (Dex) Inhibits Anti-GPIIIa49-66 Ab Induced Oxidative Fragmenation of Platelets: Inhibition of Platelet Phospholipase A₂ (PLA₂) and 12-Lipoxygenase (12-LO) MembraneTranslocation and Activity.

Patients with HIV related thrombocytopenia (HIV-ITP) have a unique Ab against GPIIIa49-66 (Ab) which induces complement-independent, Fc-independent, peroxide-induced lysis of platelets (Cell, 106:551, 2001). This Ab correlates inversely with platelet count and induces oxidative-fragmentation of human and mouse platelets, and thrombocytopenia in mice. Oxidation is induced by H₂O₂ generated from platelet NADPH oxidase activated with the platelet 12-LO product, 12(S)-HETE (JCI 113: 973, 2004). The substrate for 12-LO is arachidonic acid (AA), produced by PLA₂. Similar oxidative fragmentation is also induced by the Ca⁺⁺ionophore A23187 as well as phorbol myristate acetate (PMA) (Blood 102:126A, 2003). Most patients with classic autoimmune thrombocytopenia (AITP) as well as HIV-ITP respond dramatically to treatment with steroids. The effect of Dex in AITP requires opsonized platelets interacting with Fc macrophage receptors, while Ab-induced oxidative fragmentation of platelets does not. We therefore investigated possible mechanisms of Dex inhibition of oxidative platelet fragmentation in gel-filtered human platelets (measured by flow cytometry and DCFH oxidation) induced by Ab, A23187 or PMA over 4 hr at 37 C. Dex inhibited both oxidation and platelet fragmentation ( IC ₅₀ 0.8uM, range 3.2uM to 0.2uM), n=5. Regardless of the stimulus, 12(S)-HETE production was completely inhibited during the first 60 min of Dex incubation with 2–3 fold inhibition at 60–240 minutes. Similar Dex inhibition was noted when exogenous AA was added confirming a reduction of 12-LO activity, n=4, p<0.01. The glucocorticoid receptor antagonist RU 3846 (5–50uM) did not prevent the effect of Dex, indicating independence of the known platelet steroid receptor. Dex also inhibited Ab-induced PLA₂ activity (measured by the release of ³H-AA from labeled membrane of intact platelets), as effectively as the the PLA₂ inhibitor, bromophenacyl bromide (85 ± 2% vs 84 ± 1%, n=3, p<0.001). NADPH oxidase requires transfer of several cytosol components including p67phox and activated Rac-2 to membrane gp22phox and gp91phox to initiate activity. We then examined the effect of Dex on Ab-induced assembly of NADPH oxidase by measuring p67phox translocation by immunoblot, as well as the possible translocation of PLA₂ and 12-LO. Regardless of stimulant, Dex inhibited translocations of all 3 proteins, p67phox, PLA₂ and 12-LO by 50–80%. Indeed agonist-induced membrane loading of PLA₂, 12-LO and p67phox was actually reversed with Dex, i.e. ~50% translocated to cytosol compared to buffer, after an additional 4hr of incubation. Since Dex inhibits phagocytosis by macrophages, we reasoned that the inhibitory effect of Dex on macrophage oxidative damage or killing of bacteria could also be related to its inhibition of cytosol to membrane translocation. Such proved to be the case for PMA-induced granulocyte cytosol membrane translocation of p67phox and PLA₂, n=3. Thus Ca⁺⁺ionophore, PMA and Ab-induced platelet oxidative fragmentation are inhibited by therapeutic Dex concentrations. Inhibition results in severely impaired PLA₂ and 12-LO activity as well as inhibition of translocation of these components and p67phox from cytosol to membrane, thus disrupting NADPH oxidase activity (confirmed by inhibition of Rac-2 activation, as measured by Rac-2 binding to PAK-1). This occurs in the presence of steroid receptor blockade, suggesting a direct effect of Dex on the membrane.