Phorbol ester mediated activation of inducible nitric oxide synthase results in platelet profilin nitration

Nitric oxide is an important precursor for peroxynitrite production under in vivo conditions leading to cell injury and cell death. In platelets, a number of cytosolic and actin binding proteins were shown to be nitrated [K.M. Naseem, S.Y. Low, M. Sabetkar, N.J. Bradley, J. Khan, M. Jacobs, K.R. Bruckdorfer, The nitration of platelet cytosolic proteins during agonist-induced activation of platelets. FEBS Lett. 473 (1) (2000) 199-122 and M. Sabetkar, S.Y. Low, K.M. Naseem, K.R. Bruckdorfer, The nitration of proteins in platelets: significance in platelet function, Free Radic. Biol. Med. 33 (6) (2002) 728-736]. We investigated the possible mechanism that regulates profilin (an actin binding protein) nitration in platelets. Activation of bovine platelets with arachidonic acid, thrombin, and phorbol 12,13-dibutyrate resulted in nitration of profilin on tyrosine residue. In vivo profilin nitration showed a four- and eight-fold increase in the presence of thrombin and phorbol 12,13-dibutyrate, respectively. Analysis of nitroprofilin levels in the presence of NOS inhibitors (1400W and EGTA), indicated that profilin nitration in phorbol 12,13-dibutyrate treated platelets is mediated by inducible nitric oxide synthase. Phorbol ester treated platelets exhibited higher levels by inducible nitric oxide synthase (491% over control), while total nitric oxide synthase activity increased by 5% over control. Higher levels of peroxynitrite in platelets treated with phorbol 12,13-dibutyrate indicated that profilin nitration is mediated by peroxynitrite. Increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity in platelets treated with thrombin and phorbol 12,13-dibutyrate indicates that nitration of platelet profilin could be mediated by PI 3-kinase. A decrease in the level of nitroprofilin in PDBu treated platelets in the presence of inducible nitric oxide synthase inhibitor, 1400W, was observed suggesting that profilin nitration is mediated by PI 3-kinase dependent activation of inducible nitric oxide synthase.