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Nitric oxide (NO) is a diffusible, short-lived, diatomic free radical ubiquitously produced by mammalian cells. The generation of NO from L-arginine is enzymatically regulated by three different isoforms of NO synthases. The NO signaling pathway involves mainly the activation of soluble guanylyl cyclase to produce cyclic GMP (cGMP) as a second messenger and downstream mediator. In addition, the free radical activity of NO can cause cellular damage through a phenomenon known as nitrosative stress. NO is a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. In the hematopoietic system, NO is thought to be an autocrine or paracrine messenger but also an intracellular effector molecule. Megakaryopoiesis and subsequent thrombopoiesis occur through complex biologic steps that involve hematopoietic stem cell commitment to megakaryocytic lineage, megakaryocyte maturation and finally, platelet release. Here, we summarize the current knowledge regarding the role of exogenous and endogenous NO in hematopoietic stem cell biology, megakaryocyte development and platelet biogenesis as well as relevance of platelet-derived NO generation on platelet function. Dysregulation of NO synthesis has been observed in several diseases, and the evaluation of a series of pharmacological agents with the ability to modulate the NO/cGMP pathway in platelets will also be discussed.

1 We have previously demonstrated that nitric oxide (NO) triggers CD34(+)-derived megakaryocyte apoptosis. We here show that prostacyclin (PGI(2)) inhibits PAPA/NO-induced megakaryocyte death detected by fluorescent microscopy and flow cytometry. 2 The cAMP-specific phosphodiesterase inhibitor, Ro 20-1724, and the permeable analog dibutyryl-cAMP also delayed apoptosis. PGI(2) effect was fully prevented when adenylyl cyclase activity was suppressed by SQ 22536, and partially reversed by the permeable protein kinase A inhibitor PKI 14-22 amide. ELISA showed that while both PGI(2) and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels. 3 Treatment of megakaryocytes with PGI(2) abolished both basal and NO-raised cGMP levels. Addition of 8-pCPT-cGMP or activation of soluble guanylyl cyclase by BAY 41-2272 induced cell death in a concentration-dependent manner, and ODQ, an inhibitor of guanylyl cyclase, prevented both PAPA/NO- or BAY 41-2272-induced apoptosis. Specific cGMP phosphodiesterase inhibition by Zaprinast or suppression of adenylyl cyclase by SQ 22536 enhanced the PAPA/NO proapoptotic effect. 4 PGI(2) completely inhibited NO-mediated generation and the increased activity of the cleaved form of caspase-3. 5 In conclusion, our results demonstrate that contrary to their well-known direct and synergistic inhibitory effects on platelets, PGI(2) and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase-3 activity control.

The present study investigated the effect of nitric oxide (NO) on megakaryocyte (Mk) proliferation induced by thrombopoietin (TPO). Low-density mononuclear cells (MNCs) and CD34+ cells from human bone marrow (BM) were cultured in liquid medium in the presence of sodium nitroprusside (SNP) or (Z)-1-[2-(aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA/NO) and then stimulated with TPO. Mk number decreased in both NO donors, as identified by flow cytometry 11 to 13 days after TPO stimulation. Nitrite, cyanide, or the carrier molecule DETA failed to reproduce the inhibition caused by NO donors. When CD34+ cells were treated with DETA/NO, the inhibition of Mk growth was even more pronounced than that in MNCs. Failure of the guanosine 3',5'-cyclic monophosphate (cGMP) analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) to inhibit Mk proliferation suggests that cGMP is not involved in Mk suppression mediated by NO. On the other hand, DNA analysis by flow cytometry showed that apoptosis of CD34+ cells and Mks seemed to be at least one of the mechanisms associated with the cytotoxic DETA/NO effect. Stimulation of MNCs or CD34+ cells with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) increased endogenous NO levels and suppressed Mk growth. Treatment with NO synthesis inhibitors such as L -N(G)-monomethyl arginine (L -NMMA) or L -N(G)-nitroarginine methyl ester hydrochloride (L -NAME) partially reversed Mk growth inhibition induced by TNF-alpha and IFN-gamma, although increased NO levels returned to normal values. The results presented here strongly indicate that NO regulates the growth of Mks induced by TPO by a direct effect on both progenitors and mature Mks.