To the editor:
Zhang and colleagues stated in their recent article1 that it was unknown until their study whether NO donors inhibit platelet function by cGMP-dependent and cGMP-independent mechanisms. Yet we2,3 and others4 reported several years ago that S-nitrosocysteine (CSNO) and sodium nitroprusside (SNP; Na2[Fe(CN)6NO]) inhibit platelet function by cGMP-dependent and cGMP-independent mechanisms. CSNO is a potent NO and NO+ donor,3 a crucially important extra- and intra-cellular S-nitrosating species,3 and a potent inhibitor (IC50, 100nM) of collagen-induced (1 μg/mL) aggregation of washed human platelets by different cGMP-independent mechanisms.3 These mechanisms include inhibition of thromboxane A2 (TxA2) synthesis by platelet cyclooxygenase-1 (COX-1), blockade of the platelet TxA2 receptor, and S-nitrosation of cysteine moieties on the platelet surface.3 SNP is a potent inhibitor of ADP-induced (2μM) human platelet aggregation in platelet-rich plasma (PRP) by cGMP-dependent and cGMP-independent mechanisms.3 These effects were seen at an SNP concentration of only 1μM, which is 500 times lower than the SNP concentration used by Zhang et al1 in mice. At very high concentrations, experimental NO/NO+ donors may exert concentration-dependent, diametrically different effects on platelet biochemistry.5 Our observations indicate that NO/NO+ donors can both decrease and increase COX-1 activity (Figure 1A-B).
![Figure 1. In vitro and ex vivo effects of intra- and extra-platelet NO on human platelet aggregation and of NO/NO+ donors on recombinant bovine cyclooxygenase-1 activity. (A) Effects of SNP, glycerol trinitrate (GTN), CSNO and S-nitrosoglutathione (GSNO) on the activity of recombinant bovine COX-1 (5 U, 1.2nM; Cayman Chemicals) measured as prostaglandin E2 (PGE2) formation rate (100% corresponds to 1.8 ng of PGE2 per ng COX-1 and per minute). Concentrations used were each 100μM for GSNO, CSNO, SNP and GTN, 1μM for the COX-1 inhibitor diclofenac (Diclo) serving as a positive control, and 10μM for arachidonic acid as the COX-1 substrate. Incubations were performed at 37°C in 100mM phosphate buffer, pH 8, containing 5mM EDTA, 2mM phenol and 1μM hematin. PGE2 was measured by GC-MS/MS. Data are shown as mean ± SD, n = 3. (B) Effect of GSNO on the activity of recombinant bovine COX-1. Data are shown as mean ± SD, n = 6 for each GSNO concentration. See panel A for more details. (C) Aggregation (y axis) and NOS activity (x axis) represented by the [15N]nitrate enrichment measured in platelet-rich plasma (PRP) from citrated blood donated by an apparently healthy female volunteer (aged 49 years). PRP was incubated with L-[guanidine-15N2]-arginine (98% at both 15N atoms; Cambridge Isotope Labs) at a final concentration of 40μM in the absence or in the presence of externally added recombinant human endothelial NOS (heNOS, 50 μg/mL; ALEXIS) and with all cofactors (5μM FAD, 5μM FMN, 800μM NADPH, 10μM tetrahydrobiopterin, 500nM calmodulin, 500μM calcium). Incubations were performed at 37°C. Data are shown as mean ± SD. Platelet aggregation was induced by ADP (2μM) 3 minutes before starting aggregometric measurements. Reaction was stopped by acetone and [15N]nitrate was measurement by GC-MS.9 Without heNOS, aggregation was 69%, 68% and 69% and [15N]nitrate enrichment was 7%, 6% and 7%.](/view-large/figure/8182771/zh89991291740001.jpeg)
In vitro and ex vivo effects of intra- and extra-platelet NO on human platelet aggregation and of NO/NO+ donors on recombinant bovine cyclooxygenase-1 activity. (A) Effects of SNP, glycerol trinitrate (GTN), CSNO and S-nitrosoglutathione (GSNO) on the activity of recombinant bovine COX-1 (5 U, 1.2nM; Cayman Chemicals) measured as prostaglandin E2 (PGE2) formation rate (100% corresponds to 1.8 ng of PGE2 per ng COX-1 and per minute). Concentrations used were each 100μM for GSNO, CSNO, SNP and GTN, 1μM for the COX-1 inhibitor diclofenac (Diclo) serving as a positive control, and 10μM for arachidonic acid as the COX-1 substrate. Incubations were performed at 37°C in 100mM phosphate buffer, pH 8, containing 5mM EDTA, 2mM phenol and 1μM hematin. PGE2 was measured by GC-MS/MS. Data are shown as mean ± SD, n = 3. (B) Effect of GSNO on the activity of recombinant bovine COX-1. Data are shown as mean ± SD, n = 6 for each GSNO concentration. See panel A for more details. (C) Aggregation (y axis) and NOS activity (x axis) represented by the [15N]nitrate enrichment measured in platelet-rich plasma (PRP) from citrated blood donated by an apparently healthy female volunteer (aged 49 years). PRP was incubated with L-[guanidine-15N2]-arginine (98% at both 15N atoms; Cambridge Isotope Labs) at a final concentration of 40μM in the absence or in the presence of externally added recombinant human endothelial NOS (heNOS, 50 μg/mL; ALEXIS) and with all cofactors (5μM FAD, 5μM FMN, 800μM NADPH, 10μM tetrahydrobiopterin, 500nM calmodulin, 500μM calcium). Incubations were performed at 37°C. Data are shown as mean ± SD. Platelet aggregation was induced by ADP (2μM) 3 minutes before starting aggregometric measurements. Reaction was stopped by acetone and [15N]nitrate was measurement by GC-MS.9 Without heNOS, aggregation was 69%, 68% and 69% and [15N]nitrate enrichment was 7%, 6% and 7%.
In vitro and ex vivo effects of intra- and extra-platelet NO on human platelet aggregation and of NO/NO+ donors on recombinant bovine cyclooxygenase-1 activity. (A) Effects of SNP, glycerol trinitrate (GTN), CSNO and S-nitrosoglutathione (GSNO) on the activity of recombinant bovine COX-1 (5 U, 1.2nM; Cayman Chemicals) measured as prostaglandin E2 (PGE2) formation rate (100% corresponds to 1.8 ng of PGE2 per ng COX-1 and per minute). Concentrations used were each 100μM for GSNO, CSNO, SNP and GTN, 1μM for the COX-1 inhibitor diclofenac (Diclo) serving as a positive control, and 10μM for arachidonic acid as the COX-1 substrate. Incubations were performed at 37°C in 100mM phosphate buffer, pH 8, containing 5mM EDTA, 2mM phenol and 1μM hematin. PGE2 was measured by GC-MS/MS. Data are shown as mean ± SD, n = 3. (B) Effect of GSNO on the activity of recombinant bovine COX-1. Data are shown as mean ± SD, n = 6 for each GSNO concentration. See panel A for more details. (C) Aggregation (y axis) and NOS activity (x axis) represented by the [15N]nitrate enrichment measured in platelet-rich plasma (PRP) from citrated blood donated by an apparently healthy female volunteer (aged 49 years). PRP was incubated with L-[guanidine-15N2]-arginine (98% at both 15N atoms; Cambridge Isotope Labs) at a final concentration of 40μM in the absence or in the presence of externally added recombinant human endothelial NOS (heNOS, 50 μg/mL; ALEXIS) and with all cofactors (5μM FAD, 5μM FMN, 800μM NADPH, 10μM tetrahydrobiopterin, 500nM calmodulin, 500μM calcium). Incubations were performed at 37°C. Data are shown as mean ± SD. Platelet aggregation was induced by ADP (2μM) 3 minutes before starting aggregometric measurements. Reaction was stopped by acetone and [15N]nitrate was measurement by GC-MS.9 Without heNOS, aggregation was 69%, 68% and 69% and [15N]nitrate enrichment was 7%, 6% and 7%.
NO is endogenously produced in various types of cells by constitutive and inducible NO synthase (NOS) isoforms and plays multiple physiologic roles.6,7 Blood platelets and red blood cells are considered to express NOS isoforms, but there are doubts about functional erythrocytic8 and platelet4,9 NOS. Using a sophisticated gas chromatography–mass spectrometry method, we did not detect any NOS activity in human platelets (Figure 1C). Addition of functional recombinant human endothelial NOS to human PRP resulted in NO formation and platelet aggregation inhibition (Figure 1C; supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
In summary, in human blood platelets and erythrocytes, expression and functionality of NO synthase are in dispute. Extra-platelet NO/NO+ species including endothelium-derived NO and NO/NO+-releasing drugs are potent inhibitors of platelet aggregation. They are unlikely to modulate platelet function by influencing platelet L-arginine/NO pathway, but they may modulate platelet COX-1. Effects of NO/NO+ donors on platelet function observed experimentally at therapeutically irrelevantly high drug concentrations are rather artifactual and misleading. Extension of observations on platelet function from mice to humans should be treated with caution.
Authorship
The online version of this article contains a data supplement.
Acknowledgments: This study was supported in part by the Deutsche Forschungsgemeinschaft (TS 60/4-1). The authors thank M. T. Suchy for laboratory assistance and F. M. Gutzki for performing GC-MS analyses.
Contribution: D.T. and D.O.S. designed the study and wrote the manuscript; and M.F., J.N. and A.B. performed the experimental work and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Prof Dimitrios Tsikas, Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; e-mail: tsikas.dimitros@mh-hannover.de.
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