Extra-platelet NO and NO⁺-containing drugs are potent inhibitors of platelet aggregation in humans by cGMP-dependent and cGMP-independent mechanisms

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 we^2,3  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).

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 platelet^4,9  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.