http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2014.974024
SHORT COMMUNICATION
Human blood platelets lack nitric oxide synthase activity Anke Bo¨hmer1, Stepan Gambaryan2,3, & Dimitrios Tsikas1 Hannover Medical School, Institute of Clinical Pharmacology, Hannover, Germany, 2Institute of Clinical Biochemistry, University of Wuerzburg, Wuerzburg, Germany, and 3Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia
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1
Abstract
Keywords
Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography–mass spectrometry (GC–MS) method to detect NOS activity in human platelets. The method measures simultaneously [15N]nitrite and [15N]nitrate formed from oxidized 15N-labeled nitric oxide (15NO) upon its NOS-catalyzed formation from the substrate L-[guanidino-15N2]-arginine. Using this GC–MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. L-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.
Enzyme activity assays, mass spectrometry, nitric oxide, stable isotopes
Introduction Nitric oxide (NO) is one of the smallest endogenous signalling molecules in humans. NO and L-citrulline are produced from L-arginine by the catalytic action of NO synthase (NOS) isoforms in various types of cells [1, 2]. In addition to endothelial and neuronal cells, human blood platelets [3–8] and red blood cells [9, 10] are also considered to express functional NOS. However, there are doubts about the presence and the functionality of NOS in human platelets [11–17] and erythrocytes [18, 19]. A recent comprehensive proteomic study revealed the absence of NOS isoforms in human platelets [16]. Extra-platelet NO is a potent inhibitor of in vivo and ex vivo platelet aggregation [20] induced by various agonists including adenosine diphosphate (ADP), collagen, thrombin, von Willebrand factor (vWF), or arachidonic acid [21, 22]. Inhibition of platelet aggregation by NO and NOcontaining species occurs via cyclic guanosine monophosphate (cGMP)-dependent and cGMP-independent mechanisms [22, 23]. A major reason for the contradictory results is the use of nonspecific methods to detect NOS proteins and RNA and to measure NOS activity [13]. A frequently used NOS activity assay is based on measuring radio-labeled L-citrulline formed upon incubation of radio-labeled L-arginine with intact or lysed cells [2]. An absolute prerequisite for the use of L-citrulline-based assays to measure NOS activity in cells is that no other pathways contribute to L-citrulline formation. However, almost all types of cells can utilize L-arginine in various pathways, including the urea cycle. For example, erythrocytes, endothelial cells, and platelets [24] express functional arginase. In this context, it is worth noting that the L-arginine/NO/L-citrulline pathway is only a very minor contributor to L-citrulline (50.1%), even in conditions of
Correspondence: Prof. Dimitrios Tsikas, PhD, Pharmacology & Toxicology Centre, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Tel: +49 511 532 3984. Fax: +49 511 532 2750. E-mail: [email protected]
History Received 26 August 2014 Revised 30 September 2014 Accepted 4 October 2014 Published online 28 October 2014
stimulated NOS expression and activity [2]. Thus, even though the urea cycle and other pathways that convert L-arginine to L-citrulline might not be abundant in human blood platelets and red blood cells, their contribution to L-citrulline is likely to approach or even exceed that of NOS. Another potential pitfall in assaying NOS activity by the L-citrulline assay is that radiolabeled L-arginine metabolites, including L-citrulline, are not entirely separated from high excess radio-labeled L-arginine and from other possible L-arginine metabolites prior to counting. These metabolites include radio-labeled L-ornithine, proline, glutamate, and polyamines that behave in a chromatographically similar manner to radio-labeled L-citrulline [25]. We have developed a specific gas chromatography–mass spectrometry (GC–MS) assay for NOS activity which is based on the simultaneous measurement of [15N]nitrite and [15N]nitrate formed from oxidized 15NO upon its NOS-catalyzed formation from L-[guanidino-15N2]-arginine [11, 26, 27]. The specificity of this GC–MS assay is based on the use of L-[guanidino-15N2]arginine as the NOS substrate and the measurement of [15N]nitrite 15 15 (15 NO 2 ) and [ N]nitrate ( NO3 ), the exclusive reaction prod15 ucts of NO produced by NOS from L-[guanidino-15N2]-arginine with L-[ureido-15N]-citrulline being the second reaction product (Reaction 1). [15N]Nitrite is likely formed via autoxidation of 15 15 NO to 15 NO 2 (Reaction 2), whereas [ N]nitrate formation 15 presumably results from oxidation of NO to 15 NO 3 by NOS (i.e., NOS[Fe2+O2]) (Reaction 3). We have recently validated this GC–MS NOS activity assay by a liquid-chromatography tandem mass spectrometry (LC–MS/MS) NOS activity assay which measures L-[ureido-15N]-citrulline formed from 15 L-[guanidino- N2]–arginine [17]. L- guanidino-15 N2 -arginine ! 15 NO þ L- ureido-15 N -citrulline þ 415 NO þ O2 þ 2H2 O ! 415 NO 2 þ 4H
ð1Þ ð2Þ
2
A. Bo¨hmer et al. 15
3þ NO þ NOS Fe2þ O2 !15 NO 3 þ NOS Fe
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ð3Þ
In the GC–MS assay, derivatization of [15N]nitrite and [ N]nitrate is carried out after stopping the reaction with acetone, and addition of the derivatization reagent 2,3,4,5,6-pentafluorobenzyl bromide to the reaction mixture and incubation for 60 minutes at 50 C. Subsequently, selected-ion monitoring (SIM) is used to separate and detect nitrite and nitrate species with massto-charge ratios (m/z) of m/z 47 for [15N]nitrite, m/z 46 for [14N]nitrite, m/z 63 for [15N]nitrate, and m/z 62 for [14N]nitrate. Peak area ratios (PAR) of 15N to 14N are calculated and used to measure 15N-enrichment for [15N]nitrite (PAR m/z 47 to m/z 46) and for [15N]nitrate (PAR m/z 63 to m/z 62). The PAR values of the 15N-to-14N-species are a measure of NOS activity. Furthermore, the concentration of [15N]nitrite and [15N]nitrate can be determined by using unlabelled nitrite and nitrate, i.e., [14N]nitrite and [14N]nitrate, as internal standards. By using this assay, we detected NOS activity in various cells including cultured endothelial cells [28] and hepatocytes [29, 30], as well as in vivo [11]. By this method, however, we did not detect any NOS activity in red blood cells of healthy subjects [19]. In the present study, we demonstrate by a specific GC–MS assay absence of NOS activity in human platelets under basal conditions as well as in platelets stimulated with ADP, collagen, thrombin, or vWF. Routinely used anticoagulants were found not to affect activity of recombinant NOS isoforms. Our results exclude the possibility that the absence of NOS activity in human blood platelets and red blood cells is due to inhibitory effects of anticoagulants.
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Materials and methods Chemicals, reagents, materials, and enzymes Tetrahydrobiopterin (H4B), the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, L-[guanidino-nitro]-arginine (L-NNA), ADP, and CaCl2 were obtained from Sigma-Aldrich (Steinheim, Germany). 15 15 L-[guanidino- N2]-Arginine (declared 98 atom% N) was obtained from Cambridge Isotope Laboratories (Andover, MA). Thrombin was from Roche (Mannheim, Germany), collagen from Nycomed (Linz, Austria), and ristocetin was from Biopool (Wicklow, Ireland). The von Willebrand factor (vWF) Haemate HS 1000 (vWF activity of 2200IE), which is clinically used as a vWF substitute in vWF deficiencies, was from CLS Behring (Marburg, Germany). Recombinant human endothelial NOS (heNOS) were from ALEXIS (Gru¨nberg, Germany). The heNOS preparation was declared to contain triethanolamine (50 mM, pH 7.5), ethylenediaminetetraacetate (EDTA, 0.5 mM), glutathione (GSH, 7 mM), 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS, 20 mM), E64 (1 mM), leupeptin (1 mM), pepstatin (1 mM), trypsin inhibitor (1 mg/ml), and glycerol (10%). heNOS concentration and activity in this preparation were declared as 15 mg/ml and 100 pmol/minmg heNOS, respectively. Recombinant murine inducible NOS (iNOS) was obtained from Sigma-Aldrich (Steinheim, Germany). For blood sampling, the following Sarstedt (Nu¨mbrecht, Germany) monovettes (declared concentrations with respect to the monovette volume) were used: K-EDTA (3.94 mM), sodium citrate (10.6 mM), and lithium heparin (16 IU/ml blood). Blood platelet isolation and NOS activity assays Approval from the local Ethics Committee of the Hannover Medical School was obtained. Blood was drawn from the antecubital vein of two healthy females and males volunteers
using citrate monovettes (Sarstedt, Germany) and processed immediately. Platelets were prepared as described elsewhere [13, 16]. Residual leucocytes and erythrocytes in the platelet preparations used in the present study were counted by BD LeucocountÔ Kit (BD Biosciences, Germany). Washed human platelets contained less than 102 leucocytes per 108 platelets. This extent of contamination is comparable with that measured by us in previous studies [13, 16]. Washed human platelets (3.5 108/ml) were incubated with L[guanidino-15N2]-arginine (5 mM). Non-stimulated platelets and platelets stimulated with ADP (10 mM), thrombin (0.01 U/ml), collagen (10 mg/ml), or vWF/ristocetin (15 mg/ml and 1 mg/ml) were incubated for 30 minutes at 37 C. Data are shown as mean ± standard deviation (SD) from three independent experiments for intact platelets. NOS activity was also measured in lysed platelets spiked with a cocktail of protease inhibitors in the absence and in the presence of the NOS inhibitor L-NNA (100 mM) before addition of heNOS together with all NOS cofactors. Enzyme, substrate, and cofactors concentration in experiments with lysed platelets were as follows: heNOS, 75 mg/ml; L-[guanidino-15N2]-arginine, 5 mM; H4B, 10 mM; NADPH, 800 mM; FAD, 5 mM; FMN, 5 mM; calmodulin, 500 nM; and CaCl2, 500 mM. As a positive control, we measured NOS activity in adult rat hepatocytes proliferating in vitro in the absence and in the presence of LiCl, as described elsewhere [29]. In all experiments, reactions (100 ml aliquots) were terminated by adding ice-cold acetone (400 ml aliquots) and samples were processed for GC–MS analysis [27]. Effects of anticoagulants on recombinant NOS activity Monovettes were filled with sodium/potassium phosphate buffer (67 mM, pH 7.0) for heNOS or Tris buffer (10 mM, pH 7.4) for iNOS. Aliquots (50 ml) of these solutions or buffer as a control were mixed with a solution containing all NOS cofactors to reach final added concentrations of 500 nM for calmodulin, 500 mM for Ca2+, 5 mM for FAD, 5 mM for FMN, 10 mM for H4B, and 800 mM for NADPH. Recombinant heNOS or recombinant murine iNOS was added at final concentrations of 75 and 50 mg/ml, respectively. Reaction was started by adding 15 L-[guanidino- N2]-arginine at a final concentration of 5 mM and samples were incubated at 37 C for 30 minutes. Reaction was stopped by adding acetone.
Results The results of the present study are in part illustrated in Figure 1 and in part summarized in Table I. NOS activity in human platelets The PAR values for [15N]nitrite and [15N]nitrate measured in nonstimulated washed platelets and in platelets stimulated with ADP, collagen, thrombin, or vWF/ristocetin were of the order of the background ratios (Figure 1A and B). Indeed, the mean PAR values for m/z 47 to m/z 46 and m/z 63 to m/z 62 at the beginning of the reaction (i.e. at time zero) were 0.0075 (relative standard deviation (RSD), 7.1%; n ¼ 8) and 0.00925 (RSD, 7.6%; n ¼ 8), respectively. These findings suggest that NOS activity is not detectable in intact human platelets incubated with L[guanidino-15N2]-arginine at the exceeding concentration of 5 mM. PAR values of this order of magnitude are commonly measured for endogenous [15N]nitrite and [15N]nitrate in biological samples by the GC–MS assay [11] used in the present study. These small PAR values result from the natural abundance of the 15N isotope of the element N, which is about 0.37%. PAR values clearly higher than these values indicate the formation of
No NOS in platelets
DOI: 10.3109/09537104.2014.974024
(B)
15N/14N-Nitrite 15N/14N-Nitrate
0.012
0.10 0.08 0.06 0.04
0.008
0.004
0.02 0.000
0.00 NaCl
Thrombin (C)
Collagen
Peak area rao
heNOS
NaCl
ADP
Thrombin Collagen
vWF
0.10 15N/14N-Nitrite
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0.016
0.12 Peak area rao
PAR of m/z 47 to m/z 46
(A)
3
15N/14N-Nitrate
0.06 0.04 0.02 0.00 NaCl
NaCl+ heNOS+ NO2Arg
NaCl+ heNOS
NaCl+ NO2Arg
Figure 1. (A) Peak area ratio (PAR) of m/z 47 for [15N]nitrite to m/z 46 for [14N]nitrite upon incubation of washed human blood platelets (3.5 108/ml) with L-[15N2]-arginine (5 mM) for 30 minutes at 37 C. After addition of L-[15N2]-arginine platelets were stimulated by thrombin (0.01 U/ml) or collagen (1 mg/ml) for 5 minutes. Unstimulated platelets were incubated with physiological saline (NaCl) serving as the control in the absence and in the presence of externally added recombinant heNOS at a final concentration of 75 mg/ml. In the heNOS sample (100 ml), all NOS cofactors (10 mM H4B, 800 mM NADPH, 5 mM FAD, 5 mM for FMN, 500 nM calmodulin, and 500 mM CaCl2) were added. Data are shown as mean ± SD from three independent experiments. (B) Peak area ratio for [15N]nitrite to [14N]nitrite and [15N]nitrate to [14N]nitrate upon incubation of washed human blood platelets (3.5 108/ml) with L-[15N2]-arginine (5 mM) for 30 minutes at 37 C. After addition of L-[15N2]-arginine platelets were stimulated by ADP (10 mM), collagen (1 mg/ml), thrombin (0.01 U/ml), or vWF/ristocetin (15 mg/ml and 1 mg/ml) for 5 minutes each. Data are shown as mean ± SD from three independent experiments. (C) All platelet lysate samples were incubated with L-[15N2]-arginine (5 mM) and all NOS cofactors (10 mM H4B, 800 mM NADPH, 5 mM FAD, 5 mM for FMN, 500 nM calmodulin, and 500 mM CaCl2) for 30 minutes at 37 C. To the ‘‘NaCl’’ sample, only physiological saline was added to the platelet lysate. To the sample ‘‘NaCl+NO2Arg’’, NaCl and L-[guanidino-nitro]-arginine (NO2Arg, at a final concentration of 100 mM) was added. All NOS cofactors, heNOS, and NO2Arg (+ NO2Arg samples), were added for 5 minutes prior to start the reaction with L-[15N2]-arginine. The samples ‘‘NaCl+heNOS’’ and ‘‘NaCl+heNOS+NO2Arg’’ contained recombinant heNOS at a final concentration of 75 mg/ml. Reactions were terminated by the addition of 400-ml aliquots of ice-cold acetone and samples were processed for GC–MS analysis.
Table I. Recombinant human endothelial nitric oxide synthase (heNOS) and inducible nitric oxide synthase (iNOS) activity measured in the absence (control) and in the presence of the anticoagulants ethylenediaminetetraacetate (EDTA), citrate, and heparin. heNOS activity (pmol/minmg) 15
15
iNOS activity (nmol/minmg)
Anticoagulant
[ N]nitrite
[ N]nitrate
Total (%)
Control EDTA Citrate Heparin
3.20 ± 0.15 3.11 ± 0.46 2.30 ± 0.15 2.84 ± 0.09
2.21 ± 0.38 1.13 ± 0.05 0.97 ± 0.02 0.98 ± 0.02
100 78.4 60.4 70.8
a
15
[ N]nitrite
[15N]nitrate
Total (%)a
3.13 ± 0.06 2.94 ± 0.03 3.83 ± 0.07 2.65 ± 0.08
2.84 ± 0.06 2.41 ± 0.04 2.84 ± 0.06 2.14 ± 0.06
100 89.6 112 80.2
Data are shown as mean ± SD of two independent analyses. a Total activity was calculated by using the sum of [15N]nitrite and [15N]nitrate concentrations. The mean total activity is expressed as percent of control (no anticoagulation agent).
[15N]nitrite and [15N]nitrate from 15NO formed from 15 L-[guanidino- N2]-arginine [11]. In order to eliminate potential problems associated with the supply of intra-platelet NOS with its substrate, we lysed washed human blood platelets and incubated the lysate with the same
concentration of L-[guanidino-15N2]-arginine together with all cofactors required for functional endothelial NOS. In the presence of the NOS inhibitor L-NNA, the PAR values measured for [15N]nitrite and [15N]nitrate were the same as in the lysed platelets in the absence of L-NNA (Figure 1C). In contrast, the
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PAR values of [15N]nitrite and [15N]nitrate were about eight and seven times higher when the platelet lysate was spiked with heNOS, respectively. Using various concentrations of heNOS and an incubation time of 30 minutes, we found that 1 mg/ml heNOS was the lowest enzyme concentration that yielded PAR values being significantly different from those obtained in the absence of heNOS. In addition, L-NNA inhibited remarkably heNOS activity suggesting that the recombinant heNOS added to the platelet lysate was functional (Figure 1C). By means of the same GC–MS assay, we found that adult rat hepatocytes proliferating in vitro express functional NOS isoforms confirming previous results [29].
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Effects of anticoagulants on recombinant NOS activity Anticoagulants routinely used in blood sampling may affect the activity of NOS isoforms, for example, by complexing Ca2+ ions which are required for enzyme activity of eNOS. We investigated this issue using recombinant heNOS and iNOS. The latter was used as a positive control, as the iNOS activity is independent of Ca2+ ions. Expectedly, in the absence of any anticoagulant (Control), the iNOS activity was almost three orders of magnitude higher than the heNOS activity (Table I). The low-molecularmass, Ca2+ ions-complexing anticoagulants EDTA and citrate did not affect the activity of both NOS isozymes. The high-molecularmass anticoagulant heparin (used as its lithium salt) also did not affect heNOS and iNOS activities. Our results indicate that the anticoagulants tested in the study did influence the bioavailability of NO produced by the recombinant heNOS but not iNOS. EDTA, citrate, and heparin almost doubled the molar ratio of [15N]nitrite to [15N]nitrate in heNOS incubation mixtures, while this ratio did not change in the iNOS samples upon addition of the anticoagulants.
Discussion Occurrence and functionality of NOS in platelets of humans and other species are still contradictory topics in the literature [11– 17]. Differences in the analytical methods used are the most likely explanation for diametrically opposite results (see below). Analytical shortcomings may also include involvement of additional pathways which contribute to NO or L-citrulline from L-arginine in platelets independently of NOS. The very recent proof of lacking expression of NOS isoforms in human blood platelets by means of MS-based proteomics [16] suggests that the most likely explanation of our findings is not due to the presence of non-functioning NOS in stimulated and non-stimulated platelets of healthy subjects, but is due to the entire absence of NOS isozymes in human platelets. These issues are discussed below, in more detail. Demonstration of expression and enzymatic activity of NOS isoforms in certain cells such as human platelets requires the use of reliable analytical methods in terms of specificity and detection sensitivity. We have previously observed that detection of platelet eNOS by Western blot or immunoprecipitation may provide falsepositive results because of the variable specificity of commercially available eNOS antibodies [13]. Indeed, our recent proteomic studies on human blood platelets provide unequivocal evidence of lacking expression of NOS isoforms in these cells [16]. Vane’s group demonstrated in 1989 that human neutrophils and mononuclear cells inhibit platelet aggregation by L-argininederived NO in a manner depending on the number of cells added to washed human platelets [31]. Thus, addition of 2 106 neutrophils to about 2 108 washed platelets (‘‘contamination’’ degree, 1%) inhibited thrombin-stimulated platelet aggregation by 50%. Therefore, contamination of platelets with leucocytes and/or other blood cells that express iNOS is likely to be a major source
Platelets, Early Online: 1–6
of extra-platelet NO. Because iNOS produces NO at several orders of magnitude higher rates than eNOS (Table I), even a low-extent contamination of platelets with circulating iNOSexpressing cells may simulate NO production in human platelets. In our study, the leucocytes contamination degree was about 104% and, therefore, negligible. With respect to the measurement of NOS activity, the most useful method is, in our opinion, the use of L-arginine labeled with the stable isotope 15N being on the guanidine group in combination with the measurement of stable 15N-labeled metabolites of NO, notably [15N]nitrite in platelets [11, 15, 17] and [15N]nitrate in erythrocytes [19]. Other spectrophotometric and fluorescence-based assays including the widely used diaminofluorescein (DAF) reagents, as well as chemiluminescence-based assays are likely to fail because they lack specificity. For example, such assays cannot discriminate between NO coming from NOScatalyzed oxidation of one of the guanidine N atoms of L-arginine and NO coming from other sources. In the case of NOSindependency, measurement of NO, nitrite, or nitrate released by oxidation of the a-NH2 group of L-arginine [32] or other amino acids and amines would falsely contribute to NOS activity. The L-citrulline assay is the most widely used NOS activity assay. However, the L-citrulline assay lacks specificity because of the involvement of additional pathways in the production and utilization of L-citrulline. Another serious shortcoming of the L-citrulline assay is often omitting additional chromatographic steps for the sake of simplicity and labor and time saving. As platelets and erythrocytes possess arginase activity [24] and are capable of synthesizing proteins and metabolizing enzymes, omission of chromatographic steps beyond removal of exceeding L-arginine holds the potential for counting radioactivity other than radio-labeled L-citrulline [25], thus simulating NOS activity. Interestingly, human blood platelets express arginineproteinmethyltransferases (PRMTs), various proteases, and dimethylarginine dimethylaminohydrolase (DDAH) [16]. DDAH hydrolyses asymmetric dimethylarginine (NG,NG-dimethylarginine) to L-citrulline and dimethylamine. In such a scenario, the interplay of these enzymes is likely to finally yield (radio-)labeled L-citrulline from externally added (radio-)labeled L-arginine in the L-citrulline assay. Another critical point in measuring NOS activity in thrombocytes, erythrocytes, and other intact cells concerns the biochemistry of NOS, i.e., the substrate L-arginine and L-arginine analogues which act as NOS inhibitors. The latter include the endogenous guanidine-methylated L-monomethylarginine (L-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) [33], as well as the exogenous L-NNA and its methyl ester (L-NAME). L-NMMA, ADMA and SDMA, and L-arginine are transported through the same cationic amino acid transporter in various cells. L-NAME requires preceding activation by esterases which release the free acid L-NNA. L-NMMA, ADMA, and SDMA may occur in different molar ratios with respect to L-arginine, in health and disease, so that the activity of intra-cellular NOS isoforms is basally inhibited to a different extent [33]. The use of L-arginine at quasi-physiological concentrations in NOS assays, including the L-citrulline assay, in intact cells may change the extent of NOS activity inhibition, thus providing misleading information. In contrast, the use of L-arginine at high concentrations in NOS assays in intact cells is likely to overcome problems potentially arising from amino acid transport and basal NOS inhibition phenomena. In dengue hemorrhagic fever, NO synthesis [34, 35], presumably in cells other than platelets such as macrophages, and prostacyclin synthesis [36], presumably in endothelial cells, are elevated. As extra-platelet NO and extra-platelet prostacyclin are both potent inhibitors of platelet aggregation, reduced aggregation
No NOS in platelets
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DOI: 10.3109/09537104.2014.974024
seen in dengue hemorrhagic fever is difficult to attribute to NO as suggested [35] or to prostacyclin alone. The contribution of platelet-derived NO to dengue hemorrhagic fever and the involvement of platelet-NOS need to be demonstrated by reliable approaches. In the present study, EDTA, citrate, and heparin (as lithium salt), commonly used anticoagulants in blood sampling, were found not to severely compromise the activity of recombinant heNOS and iNOS activities at concentrations similar to those present in commercially available vacutainers when filled with blood. Assessment of NOS activity in blood cells can be performed by drawing blood with routinely used anticoagulants or vacutainers/monovettes without the risk of considerable NOS activity inhibition by removing Ca2+ ions which are essential for the activity of constitutive NOS isozymes. The discrepancies seen in the literature regarding functional NOS in human platelets and erythrocytes are likely to be due to measurement of NO and Lcitrulline produced independently of NOS. EDTA, citrate, and lithium heparin double the molar ratio of [15N]nitrite to [15N]nitrate in heNOS incubation mixtures, suggesting that these substances may partially inhibit NOS-catalyzed oxidation of 15NO to [15N]nitrate. In conclusion, specific assessment of NOS activity in cells is possible by GC–MS by using L-[guanidino-15N2]-arginine as a substrate and by measuring 15N-labeled nitrite and/or nitrate. By means of this highly specific and sensitive (the limit of detection, 3 amol [15N]nitrite; the limit of quantitation, 4 nM [15N]nitrite) GC–MS assay [26], we did not detect NOS activity in nonstimulated and stimulated human blood platelets. Current controversy on the expression and functionality of NOS in platelets arises in part from the use of non-specific methods to detect NOS proteins and to measure NOS activity and in part from contaminating iNOS-expressing cells in platelet preparations. It is worth mentioning that the doubts prevailing in humans and animals may also apply to plant NOS [37].
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Declaration of interest
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Financial support was granted to DT (TS 60/4-1) and SFB 688 (TP A2) to S. G. from the Deutsche Forschungsgemeinschaft (Germany).
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cells express a functional endothelial nitric oxide synthase. Blood 2006;107:2943–2951. Cortese-Krott MM, Rodriguez-Mateos A, Sansone R, Kuhnle GGC, Thasian-Sivarajah S, Krenz T, Horn P, Krisp C, Wolters D, Heib C, et al. Human red blood cells at work: Identification and visualization of erythrocytic eNOS activity in health and disease. Blood 2012; 120:4229–4237. Tsikas D. Measurement of nitric oxide synthase activity in vivo and in vitro by gas chromatography-mass spectrometry. Methods Mol Biol 2004;279:81–103. Ozuyaman B, Gudecke A, Kusters S, Kirchhoff E, Scharf RE, Schrader J. Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation. Thromb Haemost 2005; 93:1161–1167. Gambaryan S, Kobsar A, Hartmann S, Birschmann I, Kuhlencordt PJ, Mu¨ller-Esterl W, Lohmann SM, Walter U. NO-synthase-/ NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity. J Thromb Haemost 2008;6:1376–1384. Tymvios C, Moore C, Jones S, Solomon A, Sanz-Rosa D, Emerson M. 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. Tsikas D, Flentje M, Niemann J, Bo¨hmer A, Stichtenoth DO. Extraplatelet NO and NO+-containing drugs are potent inhibitors of platelet aggregation in humans by cGMP-dependent and cGMPindependent mechanisms. Blood 2012;119:5337–5339. Burkhart JM, Vaudel M, Gambaryan S, Radau S, Walter U, Martens L, Geiger J, Sickmann A, Zahedi RP. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012;120:e73–e82. Bo¨hmer A, Gambaryan S, Flentje M, Jordan J, Tsikas D. [ureido-15N]Citrulline UPLC–MS/MS nitric oxide synthase (NOS) activity assay: Development, validation, and applications to assess NOS uncoupling and human platelets NOS activity. J Chromatogr B 2014;965:173–182. Kang ES, Ford K, Grokulsky G, Wang YB, Chiang TM, Acchiardo SR. Normal circulating adult human red blood cells contain inactive NOS proteins. J Lab Clin Med 2000;135:444–451. Bo¨hmer A, Beckmann B, Sandmann J, Tsikas D. Doubts concerning functional endothelial nitric oxide synthase in human erythrocytes. Blood 2012;119:1322–1323. Bode-Bo¨ger SM, Bo¨ger RH, Galland A, Fro¨lich JC. Differential inhibition of human platelet aggregation and thromboxane A2 formation by L-arginine in vivo and in vitro. Naunyn Schmiedebergs Arch Pharmacol 1998;357:143–150. Zhang G, Xiang B, Dong A, Skoda RC, Daugherty A, Smyth SS, Du X, Li Z. Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation. Blood 2011;118:3670–3678. Tsikas D, Ikic M, Tewes KS, Raida M, Fro¨lich JC. 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. Gordge MP, Hothersall JS, Noronha-Dutra AA. Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione. Br J Pharmacol 1998;124:141–148. Villanueva VR, Giret M. Human platelet arginase. Mol Cell Biochem 1980;33:97–100. Barilli A, Visigalli R, Rotoli BM, Bussolati O, Gazzola GC, Parolari A, Dall’Asta V. Radiochemical high-performance liquid chromatography detection of arginine metabolism in human endothelial cells. Anal Biochem 2012;424:156–161. Tsikas D, Sandmann J, Savva A, Luessen P, Bo¨ger RH, Gutzki FM, Mayer B, Fro¨lich JC. Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography-mass spectrometry. J Chromatogr B 2000;742:143–153. Tsikas D. Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography/mass spectrometry. Anal Chem 2000;72: 4064–4072. Thum T, Tsikas D, Fro¨lich JC, Borlak J. Growth hormone induces eNOS expression and nitric oxide release in a cultured human endothelial cell line. FEBS Lett 2003;555:567–571. Krause P, Wa¨tzig E, Acil H, Ko¨nig S, Unthan-Fechner Kb, Tsikas D, Probst I. Role of carbon monoxide and nitric oxide in adult rat
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hepatocytes proliferating in vitro: Effects of CAS 1609. Nitric Oxide 2010;23:220–226. 30. Trettin A, Bo¨hmer A, Suchy MT, Probst I, Staerk U, Stichtenoth DO, Fro¨lich JC, Tsikas D. Effects of paracetamol on NOS, COX, and CYP activity and on oxidative stress in healthy male subjects, rat hepatocytes, and recombinant NOS. Oxid Med Cell Longev 2014; 2014:212576. 31. Salvemini D, de Nucci G, Gryglewski RJ, Vane JR. Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor. Proc Natl Acad Sci USA 1989; 86:6328–6332. 32. Marescau B, Pintens J, Lowenthal A, Terheggen HG. Excretion of a-keto-d-guanidinovaleric acid and its cyclic form in patients with hyperarginemia. Clin Chim Acta 1979;98:35–38.
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33. Tsikas D, Bo¨ger RH, Sandmann J, Bode-Bo¨ger SM, Fro¨lich JC. Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox. FEBS Lett 2000;478:1–3. 34. Valero N, Espina LM, Anˇez G, Torres E, Mosquera JA. Short report: Increased levels of serum nitric oxide in patients with dengue. Am J Trop Med Hyg 2002;66:762–764. 35. Mendes-Ribeiro AC, Moss MB, Siqueira MA, Moraes TL, Ellory JC, Mann GE, Brunini TM. Dengue fever activates L-arginine-nitric oxide pathway: An explanation for reduced aggregation of human platelets. Clin Exp Pharmacol Physiol 2008;35:1143–1146. 36. Leithner C, Sinzinger H, Silberbauer K. Prostacyclin as a protective factor for blood vessels. Fortschr Med 1980;98:1845–1849. 37. Fro¨hlich A, Durner J. The hunt for plant nitric oxide synthase (NOS): Is one really needed? Plant Sci 2011;181:401–404.
SHORT COMMUNICATION
Human blood platelets lack nitric oxide synthase activity Anke Bo¨hmer1, Stepan Gambaryan2,3, & Dimitrios Tsikas1 Hannover Medical School, Institute of Clinical Pharmacology, Hannover, Germany, 2Institute of Clinical Biochemistry, University of Wuerzburg, Wuerzburg, Germany, and 3Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia
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Abstract
Keywords
Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography–mass spectrometry (GC–MS) method to detect NOS activity in human platelets. The method measures simultaneously [15N]nitrite and [15N]nitrate formed from oxidized 15N-labeled nitric oxide (15NO) upon its NOS-catalyzed formation from the substrate L-[guanidino-15N2]-arginine. Using this GC–MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. L-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.
Enzyme activity assays, mass spectrometry, nitric oxide, stable isotopes
Introduction Nitric oxide (NO) is one of the smallest endogenous signalling molecules in humans. NO and L-citrulline are produced from L-arginine by the catalytic action of NO synthase (NOS) isoforms in various types of cells [1, 2]. In addition to endothelial and neuronal cells, human blood platelets [3–8] and red blood cells [9, 10] are also considered to express functional NOS. However, there are doubts about the presence and the functionality of NOS in human platelets [11–17] and erythrocytes [18, 19]. A recent comprehensive proteomic study revealed the absence of NOS isoforms in human platelets [16]. Extra-platelet NO is a potent inhibitor of in vivo and ex vivo platelet aggregation [20] induced by various agonists including adenosine diphosphate (ADP), collagen, thrombin, von Willebrand factor (vWF), or arachidonic acid [21, 22]. Inhibition of platelet aggregation by NO and NOcontaining species occurs via cyclic guanosine monophosphate (cGMP)-dependent and cGMP-independent mechanisms [22, 23]. A major reason for the contradictory results is the use of nonspecific methods to detect NOS proteins and RNA and to measure NOS activity [13]. A frequently used NOS activity assay is based on measuring radio-labeled L-citrulline formed upon incubation of radio-labeled L-arginine with intact or lysed cells [2]. An absolute prerequisite for the use of L-citrulline-based assays to measure NOS activity in cells is that no other pathways contribute to L-citrulline formation. However, almost all types of cells can utilize L-arginine in various pathways, including the urea cycle. For example, erythrocytes, endothelial cells, and platelets [24] express functional arginase. In this context, it is worth noting that the L-arginine/NO/L-citrulline pathway is only a very minor contributor to L-citrulline (50.1%), even in conditions of
Correspondence: Prof. Dimitrios Tsikas, PhD, Pharmacology & Toxicology Centre, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Tel: +49 511 532 3984. Fax: +49 511 532 2750. E-mail: [email protected]
History Received 26 August 2014 Revised 30 September 2014 Accepted 4 October 2014 Published online 28 October 2014
stimulated NOS expression and activity [2]. Thus, even though the urea cycle and other pathways that convert L-arginine to L-citrulline might not be abundant in human blood platelets and red blood cells, their contribution to L-citrulline is likely to approach or even exceed that of NOS. Another potential pitfall in assaying NOS activity by the L-citrulline assay is that radiolabeled L-arginine metabolites, including L-citrulline, are not entirely separated from high excess radio-labeled L-arginine and from other possible L-arginine metabolites prior to counting. These metabolites include radio-labeled L-ornithine, proline, glutamate, and polyamines that behave in a chromatographically similar manner to radio-labeled L-citrulline [25]. We have developed a specific gas chromatography–mass spectrometry (GC–MS) assay for NOS activity which is based on the simultaneous measurement of [15N]nitrite and [15N]nitrate formed from oxidized 15NO upon its NOS-catalyzed formation from L-[guanidino-15N2]-arginine [11, 26, 27]. The specificity of this GC–MS assay is based on the use of L-[guanidino-15N2]arginine as the NOS substrate and the measurement of [15N]nitrite 15 15 (15 NO 2 ) and [ N]nitrate ( NO3 ), the exclusive reaction prod15 ucts of NO produced by NOS from L-[guanidino-15N2]-arginine with L-[ureido-15N]-citrulline being the second reaction product (Reaction 1). [15N]Nitrite is likely formed via autoxidation of 15 15 NO to 15 NO 2 (Reaction 2), whereas [ N]nitrate formation 15 presumably results from oxidation of NO to 15 NO 3 by NOS (i.e., NOS[Fe2+O2]) (Reaction 3). We have recently validated this GC–MS NOS activity assay by a liquid-chromatography tandem mass spectrometry (LC–MS/MS) NOS activity assay which measures L-[ureido-15N]-citrulline formed from 15 L-[guanidino- N2]–arginine [17]. L- guanidino-15 N2 -arginine ! 15 NO þ L- ureido-15 N -citrulline þ 415 NO þ O2 þ 2H2 O ! 415 NO 2 þ 4H
ð1Þ ð2Þ
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3þ NO þ NOS Fe2þ O2 !15 NO 3 þ NOS Fe
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ð3Þ
In the GC–MS assay, derivatization of [15N]nitrite and [ N]nitrate is carried out after stopping the reaction with acetone, and addition of the derivatization reagent 2,3,4,5,6-pentafluorobenzyl bromide to the reaction mixture and incubation for 60 minutes at 50 C. Subsequently, selected-ion monitoring (SIM) is used to separate and detect nitrite and nitrate species with massto-charge ratios (m/z) of m/z 47 for [15N]nitrite, m/z 46 for [14N]nitrite, m/z 63 for [15N]nitrate, and m/z 62 for [14N]nitrate. Peak area ratios (PAR) of 15N to 14N are calculated and used to measure 15N-enrichment for [15N]nitrite (PAR m/z 47 to m/z 46) and for [15N]nitrate (PAR m/z 63 to m/z 62). The PAR values of the 15N-to-14N-species are a measure of NOS activity. Furthermore, the concentration of [15N]nitrite and [15N]nitrate can be determined by using unlabelled nitrite and nitrate, i.e., [14N]nitrite and [14N]nitrate, as internal standards. By using this assay, we detected NOS activity in various cells including cultured endothelial cells [28] and hepatocytes [29, 30], as well as in vivo [11]. By this method, however, we did not detect any NOS activity in red blood cells of healthy subjects [19]. In the present study, we demonstrate by a specific GC–MS assay absence of NOS activity in human platelets under basal conditions as well as in platelets stimulated with ADP, collagen, thrombin, or vWF. Routinely used anticoagulants were found not to affect activity of recombinant NOS isoforms. Our results exclude the possibility that the absence of NOS activity in human blood platelets and red blood cells is due to inhibitory effects of anticoagulants.
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Materials and methods Chemicals, reagents, materials, and enzymes Tetrahydrobiopterin (H4B), the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, L-[guanidino-nitro]-arginine (L-NNA), ADP, and CaCl2 were obtained from Sigma-Aldrich (Steinheim, Germany). 15 15 L-[guanidino- N2]-Arginine (declared 98 atom% N) was obtained from Cambridge Isotope Laboratories (Andover, MA). Thrombin was from Roche (Mannheim, Germany), collagen from Nycomed (Linz, Austria), and ristocetin was from Biopool (Wicklow, Ireland). The von Willebrand factor (vWF) Haemate HS 1000 (vWF activity of 2200IE), which is clinically used as a vWF substitute in vWF deficiencies, was from CLS Behring (Marburg, Germany). Recombinant human endothelial NOS (heNOS) were from ALEXIS (Gru¨nberg, Germany). The heNOS preparation was declared to contain triethanolamine (50 mM, pH 7.5), ethylenediaminetetraacetate (EDTA, 0.5 mM), glutathione (GSH, 7 mM), 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS, 20 mM), E64 (1 mM), leupeptin (1 mM), pepstatin (1 mM), trypsin inhibitor (1 mg/ml), and glycerol (10%). heNOS concentration and activity in this preparation were declared as 15 mg/ml and 100 pmol/minmg heNOS, respectively. Recombinant murine inducible NOS (iNOS) was obtained from Sigma-Aldrich (Steinheim, Germany). For blood sampling, the following Sarstedt (Nu¨mbrecht, Germany) monovettes (declared concentrations with respect to the monovette volume) were used: K-EDTA (3.94 mM), sodium citrate (10.6 mM), and lithium heparin (16 IU/ml blood). Blood platelet isolation and NOS activity assays Approval from the local Ethics Committee of the Hannover Medical School was obtained. Blood was drawn from the antecubital vein of two healthy females and males volunteers
using citrate monovettes (Sarstedt, Germany) and processed immediately. Platelets were prepared as described elsewhere [13, 16]. Residual leucocytes and erythrocytes in the platelet preparations used in the present study were counted by BD LeucocountÔ Kit (BD Biosciences, Germany). Washed human platelets contained less than 102 leucocytes per 108 platelets. This extent of contamination is comparable with that measured by us in previous studies [13, 16]. Washed human platelets (3.5 108/ml) were incubated with L[guanidino-15N2]-arginine (5 mM). Non-stimulated platelets and platelets stimulated with ADP (10 mM), thrombin (0.01 U/ml), collagen (10 mg/ml), or vWF/ristocetin (15 mg/ml and 1 mg/ml) were incubated for 30 minutes at 37 C. Data are shown as mean ± standard deviation (SD) from three independent experiments for intact platelets. NOS activity was also measured in lysed platelets spiked with a cocktail of protease inhibitors in the absence and in the presence of the NOS inhibitor L-NNA (100 mM) before addition of heNOS together with all NOS cofactors. Enzyme, substrate, and cofactors concentration in experiments with lysed platelets were as follows: heNOS, 75 mg/ml; L-[guanidino-15N2]-arginine, 5 mM; H4B, 10 mM; NADPH, 800 mM; FAD, 5 mM; FMN, 5 mM; calmodulin, 500 nM; and CaCl2, 500 mM. As a positive control, we measured NOS activity in adult rat hepatocytes proliferating in vitro in the absence and in the presence of LiCl, as described elsewhere [29]. In all experiments, reactions (100 ml aliquots) were terminated by adding ice-cold acetone (400 ml aliquots) and samples were processed for GC–MS analysis [27]. Effects of anticoagulants on recombinant NOS activity Monovettes were filled with sodium/potassium phosphate buffer (67 mM, pH 7.0) for heNOS or Tris buffer (10 mM, pH 7.4) for iNOS. Aliquots (50 ml) of these solutions or buffer as a control were mixed with a solution containing all NOS cofactors to reach final added concentrations of 500 nM for calmodulin, 500 mM for Ca2+, 5 mM for FAD, 5 mM for FMN, 10 mM for H4B, and 800 mM for NADPH. Recombinant heNOS or recombinant murine iNOS was added at final concentrations of 75 and 50 mg/ml, respectively. Reaction was started by adding 15 L-[guanidino- N2]-arginine at a final concentration of 5 mM and samples were incubated at 37 C for 30 minutes. Reaction was stopped by adding acetone.
Results The results of the present study are in part illustrated in Figure 1 and in part summarized in Table I. NOS activity in human platelets The PAR values for [15N]nitrite and [15N]nitrate measured in nonstimulated washed platelets and in platelets stimulated with ADP, collagen, thrombin, or vWF/ristocetin were of the order of the background ratios (Figure 1A and B). Indeed, the mean PAR values for m/z 47 to m/z 46 and m/z 63 to m/z 62 at the beginning of the reaction (i.e. at time zero) were 0.0075 (relative standard deviation (RSD), 7.1%; n ¼ 8) and 0.00925 (RSD, 7.6%; n ¼ 8), respectively. These findings suggest that NOS activity is not detectable in intact human platelets incubated with L[guanidino-15N2]-arginine at the exceeding concentration of 5 mM. PAR values of this order of magnitude are commonly measured for endogenous [15N]nitrite and [15N]nitrate in biological samples by the GC–MS assay [11] used in the present study. These small PAR values result from the natural abundance of the 15N isotope of the element N, which is about 0.37%. PAR values clearly higher than these values indicate the formation of
No NOS in platelets
DOI: 10.3109/09537104.2014.974024
(B)
15N/14N-Nitrite 15N/14N-Nitrate
0.012
0.10 0.08 0.06 0.04
0.008
0.004
0.02 0.000
0.00 NaCl
Thrombin (C)
Collagen
Peak area rao
heNOS
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ADP
Thrombin Collagen
vWF
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0.016
0.12 Peak area rao
PAR of m/z 47 to m/z 46
(A)
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15N/14N-Nitrate
0.06 0.04 0.02 0.00 NaCl
NaCl+ heNOS+ NO2Arg
NaCl+ heNOS
NaCl+ NO2Arg
Figure 1. (A) Peak area ratio (PAR) of m/z 47 for [15N]nitrite to m/z 46 for [14N]nitrite upon incubation of washed human blood platelets (3.5 108/ml) with L-[15N2]-arginine (5 mM) for 30 minutes at 37 C. After addition of L-[15N2]-arginine platelets were stimulated by thrombin (0.01 U/ml) or collagen (1 mg/ml) for 5 minutes. Unstimulated platelets were incubated with physiological saline (NaCl) serving as the control in the absence and in the presence of externally added recombinant heNOS at a final concentration of 75 mg/ml. In the heNOS sample (100 ml), all NOS cofactors (10 mM H4B, 800 mM NADPH, 5 mM FAD, 5 mM for FMN, 500 nM calmodulin, and 500 mM CaCl2) were added. Data are shown as mean ± SD from three independent experiments. (B) Peak area ratio for [15N]nitrite to [14N]nitrite and [15N]nitrate to [14N]nitrate upon incubation of washed human blood platelets (3.5 108/ml) with L-[15N2]-arginine (5 mM) for 30 minutes at 37 C. After addition of L-[15N2]-arginine platelets were stimulated by ADP (10 mM), collagen (1 mg/ml), thrombin (0.01 U/ml), or vWF/ristocetin (15 mg/ml and 1 mg/ml) for 5 minutes each. Data are shown as mean ± SD from three independent experiments. (C) All platelet lysate samples were incubated with L-[15N2]-arginine (5 mM) and all NOS cofactors (10 mM H4B, 800 mM NADPH, 5 mM FAD, 5 mM for FMN, 500 nM calmodulin, and 500 mM CaCl2) for 30 minutes at 37 C. To the ‘‘NaCl’’ sample, only physiological saline was added to the platelet lysate. To the sample ‘‘NaCl+NO2Arg’’, NaCl and L-[guanidino-nitro]-arginine (NO2Arg, at a final concentration of 100 mM) was added. All NOS cofactors, heNOS, and NO2Arg (+ NO2Arg samples), were added for 5 minutes prior to start the reaction with L-[15N2]-arginine. The samples ‘‘NaCl+heNOS’’ and ‘‘NaCl+heNOS+NO2Arg’’ contained recombinant heNOS at a final concentration of 75 mg/ml. Reactions were terminated by the addition of 400-ml aliquots of ice-cold acetone and samples were processed for GC–MS analysis.
Table I. Recombinant human endothelial nitric oxide synthase (heNOS) and inducible nitric oxide synthase (iNOS) activity measured in the absence (control) and in the presence of the anticoagulants ethylenediaminetetraacetate (EDTA), citrate, and heparin. heNOS activity (pmol/minmg) 15
15
iNOS activity (nmol/minmg)
Anticoagulant
[ N]nitrite
[ N]nitrate
Total (%)
Control EDTA Citrate Heparin
3.20 ± 0.15 3.11 ± 0.46 2.30 ± 0.15 2.84 ± 0.09
2.21 ± 0.38 1.13 ± 0.05 0.97 ± 0.02 0.98 ± 0.02
100 78.4 60.4 70.8
a
15
[ N]nitrite
[15N]nitrate
Total (%)a
3.13 ± 0.06 2.94 ± 0.03 3.83 ± 0.07 2.65 ± 0.08
2.84 ± 0.06 2.41 ± 0.04 2.84 ± 0.06 2.14 ± 0.06
100 89.6 112 80.2
Data are shown as mean ± SD of two independent analyses. a Total activity was calculated by using the sum of [15N]nitrite and [15N]nitrate concentrations. The mean total activity is expressed as percent of control (no anticoagulation agent).
[15N]nitrite and [15N]nitrate from 15NO formed from 15 L-[guanidino- N2]-arginine [11]. In order to eliminate potential problems associated with the supply of intra-platelet NOS with its substrate, we lysed washed human blood platelets and incubated the lysate with the same
concentration of L-[guanidino-15N2]-arginine together with all cofactors required for functional endothelial NOS. In the presence of the NOS inhibitor L-NNA, the PAR values measured for [15N]nitrite and [15N]nitrate were the same as in the lysed platelets in the absence of L-NNA (Figure 1C). In contrast, the
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PAR values of [15N]nitrite and [15N]nitrate were about eight and seven times higher when the platelet lysate was spiked with heNOS, respectively. Using various concentrations of heNOS and an incubation time of 30 minutes, we found that 1 mg/ml heNOS was the lowest enzyme concentration that yielded PAR values being significantly different from those obtained in the absence of heNOS. In addition, L-NNA inhibited remarkably heNOS activity suggesting that the recombinant heNOS added to the platelet lysate was functional (Figure 1C). By means of the same GC–MS assay, we found that adult rat hepatocytes proliferating in vitro express functional NOS isoforms confirming previous results [29].
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Effects of anticoagulants on recombinant NOS activity Anticoagulants routinely used in blood sampling may affect the activity of NOS isoforms, for example, by complexing Ca2+ ions which are required for enzyme activity of eNOS. We investigated this issue using recombinant heNOS and iNOS. The latter was used as a positive control, as the iNOS activity is independent of Ca2+ ions. Expectedly, in the absence of any anticoagulant (Control), the iNOS activity was almost three orders of magnitude higher than the heNOS activity (Table I). The low-molecularmass, Ca2+ ions-complexing anticoagulants EDTA and citrate did not affect the activity of both NOS isozymes. The high-molecularmass anticoagulant heparin (used as its lithium salt) also did not affect heNOS and iNOS activities. Our results indicate that the anticoagulants tested in the study did influence the bioavailability of NO produced by the recombinant heNOS but not iNOS. EDTA, citrate, and heparin almost doubled the molar ratio of [15N]nitrite to [15N]nitrate in heNOS incubation mixtures, while this ratio did not change in the iNOS samples upon addition of the anticoagulants.
Discussion Occurrence and functionality of NOS in platelets of humans and other species are still contradictory topics in the literature [11– 17]. Differences in the analytical methods used are the most likely explanation for diametrically opposite results (see below). Analytical shortcomings may also include involvement of additional pathways which contribute to NO or L-citrulline from L-arginine in platelets independently of NOS. The very recent proof of lacking expression of NOS isoforms in human blood platelets by means of MS-based proteomics [16] suggests that the most likely explanation of our findings is not due to the presence of non-functioning NOS in stimulated and non-stimulated platelets of healthy subjects, but is due to the entire absence of NOS isozymes in human platelets. These issues are discussed below, in more detail. Demonstration of expression and enzymatic activity of NOS isoforms in certain cells such as human platelets requires the use of reliable analytical methods in terms of specificity and detection sensitivity. We have previously observed that detection of platelet eNOS by Western blot or immunoprecipitation may provide falsepositive results because of the variable specificity of commercially available eNOS antibodies [13]. Indeed, our recent proteomic studies on human blood platelets provide unequivocal evidence of lacking expression of NOS isoforms in these cells [16]. Vane’s group demonstrated in 1989 that human neutrophils and mononuclear cells inhibit platelet aggregation by L-argininederived NO in a manner depending on the number of cells added to washed human platelets [31]. Thus, addition of 2 106 neutrophils to about 2 108 washed platelets (‘‘contamination’’ degree, 1%) inhibited thrombin-stimulated platelet aggregation by 50%. Therefore, contamination of platelets with leucocytes and/or other blood cells that express iNOS is likely to be a major source
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of extra-platelet NO. Because iNOS produces NO at several orders of magnitude higher rates than eNOS (Table I), even a low-extent contamination of platelets with circulating iNOSexpressing cells may simulate NO production in human platelets. In our study, the leucocytes contamination degree was about 104% and, therefore, negligible. With respect to the measurement of NOS activity, the most useful method is, in our opinion, the use of L-arginine labeled with the stable isotope 15N being on the guanidine group in combination with the measurement of stable 15N-labeled metabolites of NO, notably [15N]nitrite in platelets [11, 15, 17] and [15N]nitrate in erythrocytes [19]. Other spectrophotometric and fluorescence-based assays including the widely used diaminofluorescein (DAF) reagents, as well as chemiluminescence-based assays are likely to fail because they lack specificity. For example, such assays cannot discriminate between NO coming from NOScatalyzed oxidation of one of the guanidine N atoms of L-arginine and NO coming from other sources. In the case of NOSindependency, measurement of NO, nitrite, or nitrate released by oxidation of the a-NH2 group of L-arginine [32] or other amino acids and amines would falsely contribute to NOS activity. The L-citrulline assay is the most widely used NOS activity assay. However, the L-citrulline assay lacks specificity because of the involvement of additional pathways in the production and utilization of L-citrulline. Another serious shortcoming of the L-citrulline assay is often omitting additional chromatographic steps for the sake of simplicity and labor and time saving. As platelets and erythrocytes possess arginase activity [24] and are capable of synthesizing proteins and metabolizing enzymes, omission of chromatographic steps beyond removal of exceeding L-arginine holds the potential for counting radioactivity other than radio-labeled L-citrulline [25], thus simulating NOS activity. Interestingly, human blood platelets express arginineproteinmethyltransferases (PRMTs), various proteases, and dimethylarginine dimethylaminohydrolase (DDAH) [16]. DDAH hydrolyses asymmetric dimethylarginine (NG,NG-dimethylarginine) to L-citrulline and dimethylamine. In such a scenario, the interplay of these enzymes is likely to finally yield (radio-)labeled L-citrulline from externally added (radio-)labeled L-arginine in the L-citrulline assay. Another critical point in measuring NOS activity in thrombocytes, erythrocytes, and other intact cells concerns the biochemistry of NOS, i.e., the substrate L-arginine and L-arginine analogues which act as NOS inhibitors. The latter include the endogenous guanidine-methylated L-monomethylarginine (L-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) [33], as well as the exogenous L-NNA and its methyl ester (L-NAME). L-NMMA, ADMA and SDMA, and L-arginine are transported through the same cationic amino acid transporter in various cells. L-NAME requires preceding activation by esterases which release the free acid L-NNA. L-NMMA, ADMA, and SDMA may occur in different molar ratios with respect to L-arginine, in health and disease, so that the activity of intra-cellular NOS isoforms is basally inhibited to a different extent [33]. The use of L-arginine at quasi-physiological concentrations in NOS assays, including the L-citrulline assay, in intact cells may change the extent of NOS activity inhibition, thus providing misleading information. In contrast, the use of L-arginine at high concentrations in NOS assays in intact cells is likely to overcome problems potentially arising from amino acid transport and basal NOS inhibition phenomena. In dengue hemorrhagic fever, NO synthesis [34, 35], presumably in cells other than platelets such as macrophages, and prostacyclin synthesis [36], presumably in endothelial cells, are elevated. As extra-platelet NO and extra-platelet prostacyclin are both potent inhibitors of platelet aggregation, reduced aggregation
No NOS in platelets
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DOI: 10.3109/09537104.2014.974024
seen in dengue hemorrhagic fever is difficult to attribute to NO as suggested [35] or to prostacyclin alone. The contribution of platelet-derived NO to dengue hemorrhagic fever and the involvement of platelet-NOS need to be demonstrated by reliable approaches. In the present study, EDTA, citrate, and heparin (as lithium salt), commonly used anticoagulants in blood sampling, were found not to severely compromise the activity of recombinant heNOS and iNOS activities at concentrations similar to those present in commercially available vacutainers when filled with blood. Assessment of NOS activity in blood cells can be performed by drawing blood with routinely used anticoagulants or vacutainers/monovettes without the risk of considerable NOS activity inhibition by removing Ca2+ ions which are essential for the activity of constitutive NOS isozymes. The discrepancies seen in the literature regarding functional NOS in human platelets and erythrocytes are likely to be due to measurement of NO and Lcitrulline produced independently of NOS. EDTA, citrate, and lithium heparin double the molar ratio of [15N]nitrite to [15N]nitrate in heNOS incubation mixtures, suggesting that these substances may partially inhibit NOS-catalyzed oxidation of 15NO to [15N]nitrate. In conclusion, specific assessment of NOS activity in cells is possible by GC–MS by using L-[guanidino-15N2]-arginine as a substrate and by measuring 15N-labeled nitrite and/or nitrate. By means of this highly specific and sensitive (the limit of detection, 3 amol [15N]nitrite; the limit of quantitation, 4 nM [15N]nitrite) GC–MS assay [26], we did not detect NOS activity in nonstimulated and stimulated human blood platelets. Current controversy on the expression and functionality of NOS in platelets arises in part from the use of non-specific methods to detect NOS proteins and to measure NOS activity and in part from contaminating iNOS-expressing cells in platelet preparations. It is worth mentioning that the doubts prevailing in humans and animals may also apply to plant NOS [37].
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Declaration of interest
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Financial support was granted to DT (TS 60/4-1) and SFB 688 (TP A2) to S. G. from the Deutsche Forschungsgemeinschaft (Germany).
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