Differentiation of cGMP-dependent and -independent nitric oxide effects on platelet apoptosis and reactive oxygen species production using platelets lacking soluble guanylyl cyclase

Summary

Platelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

Keywords

Apoptosis - cGMP - NO - PKG - platelet - sGC

• References

• 1 Geddis AE. The regulation of proplatelet production. Haematologica 2009; 94: 756-759.
• 2 Li J, Xia Y, Bertino AM. et al. The mechanism of apoptosis in human platelets during storage. Transfusion 2000; 40: 1320-1329.
• 3 Perrotta PL, Perrotta CL, Snyder EL. Apoptotic activity in stored human platelets. Transfusion 2003; 43: 526-535.
• 4 Verhoeven AJ, Verhaar R, Gouwerok EG. et al. The mitochondrial membrane potential in human platelets: a sensitive parameter for platelet quality. Transfusion 2005; 45: 82-89.
• 5 Remenyi G, Szasz R, Friese P. et al. Role of mitochondrial permeability transition pore in coated-platelet formation. Arterioscler Thromb Vasc Biol 2005; 25: 467-471.
• 6 Gwozdz AM, Leung R, Wang H. et al. Calpain inhibition by calpeptin does not prevent APLT activity reduction in PS-exposing platelets, but calpeptin has independent pro-apoptotic effects. Thromb Haemost 2010; 103: 1218-1227.
• 7 Schoenwaelder SM, Yuan Y, Josefsson EC. et al. Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function. Blood 2009; 114: 663-666.
• 8 Rukoyatkina N, Begonja AJ, Geiger J. et al. Phosphatidylserine surface expression and integrin alpha IIb beta 3 activity on thrombin/convulxin stimulated platelets/particles of different sizes. Br J Haematol 2009; 144: 591-602.
• 9 Rand ML, Wang H, Bang KW. et al. Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence. J Thromb Haemost 2004; 2: 651-659.
• 10 Leung R, Gwozdz AM, Wang H. et al. Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity. J Thromb Haemost 2007; 5: 560-570.
• 11 Garthwaite J, Boulton CL. Nitric oxide signaling in the central nervous system. Annu Rev Physiol 1995; 57: 683-706.
• 12 Friebe A, Koesling D. Regulation of nitric oxide-sensitive guanylyl cyclase. Circ Res 2003; 93: 96-105.
• 13 Ozuyaman B, Godecke A, Kusters S. et al. Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation. Thromb Haemost 2005; 93: 1161-1167.
• 14 Gambaryan S, Kobsar A, Hartmann S. et al. NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity. J Thromb Haemost 2008; 6: 1376-1384.
• 15 Tymvios C, Moore C, Jones S. et al. Platelet aggregation responses are critically regulated in vivo by endogenous nitric oxide but not by endothelial nitric oxide synthase. Br J Pharmacol 2009; 158: 1735-1742.
• 16 Moore C, Tymvios C, Emerson M. Functional regulation of vascular and platelet activity during thrombosis by nitric oxide and endothelial nitric oxide synthase. Thromb Haemost 2010; 104: 342-349.
• 17 Cooper CE, Giulivi C. Nitric oxide regulation of mitochondrial oxygen consumption II: Molecular mechanism and tissue physiology. Am J Physiol Cell Physiol 2007; 292: C1993-2003.
• 18 Brown GC, Cooper CE. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett 1994; 356: 295-298.
• 19 Hall CN, Garthwaite J. What is the real physiological NO concentration in vivo?. Nitric Oxide 2009; 21: 92-103.
• 20 Beltran B, Mathur A, Duchen MR. et al. The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. Proc Natl Acad Sci USA 2000; 97: 14602-14607.
• 21 Tsikas D, Ikic M, Tewes KS. et al. Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets. FEBS Lett 1999; 442: 162-166.
• 22 Sogo N, Magid KS, Shaw CA. et al. Inhibition of human platelet aggregation by nitric oxide donor drugs: relative contribution of cGMP-independent mechanisms. Biochem Biophys Res Commun 2000; 279: 412-419.
• 23 Crane MS, Rossi AG, Megson IL. A potential role for extracellular nitric oxide generation in cGMP-independent inhibition of human platelet aggregation: biochemical and pharmacological considerations. B J Pharmacol 2005; 144: 849-859.
• 24 Marcondes S, Cardoso MH, Morganti RP. et al. Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: a role for alpha-actinin nitration. Proc Natl Acad Sci USA 2006; 103: 3434-3439.
• 25 Begonja AJ, Gambaryan S, Geiger J. et al. Platelet NAD(P)H-oxidase-generated ROS production regulates alphaIIbbeta3-integrin activation independent of the NO/cGMP pathway. Blood 2005; 106: 2757-2760.
• 26 Amer J, Fibach E. Oxidative status of platelets in normal and thalassemic blood. Thromb Haemost 2004; 92: 1052-1059.
• 27 Liaudet L, Vassalli G, Pacher P. Role of peroxynitrite in the redox regulation of cell signal transduction pathways. Front Biosci 2009; 14: 4809-4814.
• 28 Mergia E, Friebe A, Dangel O. et al. Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system. J Clin Invest 2006; 116: 1731-1737.
• 29 Friebe A, Mergia E, Dangel O. et al. Fatal gastrointestinal obstruction and hypertension in mice lacking nitric oxide-sensitive guanylyl cyclase. Proc Natl Acad Sci USA 2007; 104: 7699-7704.
• 30 Dangel O, Mergia E, Karlisch K. et al. Nitric oxide-sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition. J Thromb Haemost 2010; 8: 1343-1352.
• 31 Almeida A, Almeida J, Bolanos JP. et al. Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection. Proc Natl Acad Sci USA 2001; 98: 15294-15299.
• 32 Mitchell DA, Morton SU, Fernhoff NB. et al. Thioredoxin is required for S-nitrosation of procaspase-3 and the inhibition of apoptosis in Jurkat cells. Proc Natl Acad Sci USA 2007; 104: 11609-11614.
• 33 Groneberg D, Konig P, Wirth A. et al. Smooth muscle-specific deletion of nitric oxide-sensitive guanylyl cyclase is sufficient to induce hypertension in mice. Circulation 2010; 121: 401-409.
• 34 Tiedt R, Schomber T, Hao-Shen H. et al. Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood 2007; 109: 1503-1506.
• 35 Gambaryan S, Kobsar A, Rukoyatkina N. et al. Thrombin and collagen induce a feedback inhibitory signaling pathway in platelets involving dissociation of the catalytic subunit of protein kinase A from an NFkappaB-IkappaB complex. J Biol Chem 2010; 285: 18352-18363.
• 36 Gruner S, Prostredna M, Aktas B. et al. Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy. Circulation 2004; 110: 2946-2951.
• 37 Gambaryan S, Geiger J, Schwarz UR. et al. Potent inhibition of human platelets by cGMP analogs independent of cGMP-dependent protein kinase. Blood 2004; 103: 2593-2600.
• 38 Mergia E, Koesling D, Friebe A. Genetic mouse models of the NO receptor 'soluble' guanylyl cyclases. Handb Exp Pharmacol 2009: 33-46.
• 39 Haslam RJ, Dickinson NT, Jang EK. Cyclic nucleotides and phosphodiesterases in platelets. Thromb Haemost 1999; 82: 412-423.
• 40 Garthwaite J. New insight into the functioning of nitric oxide-receptive guanylyl cyclase: physiological and pharmacological implications. Mol Cell Biochem 2010; 334: 221-232.
• 41 Feelisch M. The use of nitric oxide donors in pharmacological studies. Naunyn-Schmiedeberg Arch Pharmacol 1998; 358: 113-122.
• 42 Thompson AJ, Mander PK, Brown GC. The NO donor DETA-NONOate reversibly activates an inward current in neurones and is not mediated by the released nitric oxide. Br J Pharmacol 2009; 158: 1338-1343.
• 43 Bellamy TC, Griffiths C, Garthwaite J. Differential sensitivity of guanylyl cyclase and mitochondrial respiration to nitric oxide measured using clamped concentrations. J Biol Chem 2002; 277: 31801-31807.
• 44 Rodriguez-Juarez F, Aguirre E, Cadenas S. Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration. Biochem J 2007; 405: 223-231.
• 45 Echtay KS, Roussel D, St-Pierre J. et al. Superoxide activates mitochondrial uncoupling proteins. Nature 2002; 415: 96-99.
• 46 Begonja AJ, Teichmann L, Geiger J. et al. Platelet regulation by NO/cGMP signaling and NAD(P)H oxidase-generated ROS. Blood Cells Mol Dis 2006; 36: 166-170.
• 47 Brookes PS, Salinas EP, Darley-Usmar K. et al. Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release. J Biol Chem 2000; 275: 20474-20479.
• 48 Piantadosi CA. Regulation of mitochondrial processes by protein S-nitrosylation. Biochimica et biophysica acta. 2011. in print
• 49 Schrammel A, Behrends S, Schmidt K. et al. Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol Pharmacol 1996; 50: 1-5.