S-nitrosothiols cause prolonged, nitric oxide-mediated relaxation in human saphenous vein and internal mammary artery: therapeutic potential in bypass surgery

1. Reduced endothelial nitric oxide (NO) production in conduit vessels for coronary artery bypass grafting (CABG) has been implicated in post-operative complications, including spasm. 2. The brief effects of existing NO donors limits their applicability to improving patency of graft vessels. RIG200 is a novel S-nitrosothiol that might have advantages over conventional drugs because it has sustained effects in areas of endothelial damage. 3. Here we tested the hypothesis that RIG200 and S-nitrosoglutathione (GSNO) have prolonged, NO-mediated effects in human saphenous vein (SV) and internal mammary artery (IMA), compared with glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). 4. 84 SV and 80 IMA rings from 64 patients undergoing CABG were studied in vitro. Rings were precontracted with phenylephrine (EC(80) concentration) and the functional integrity of the endothelium tested with acetylcholine (10 microM). 5. Relaxation of precontracted SV and IMA rings to GTN and SNP (0.01 - 10 microM) generally recovered fully on washout. In contrast, responses to RIG200 and GSNO were sustained during washout (30 min). Sustained relaxation was reversed by the NO scavenger, ferrohaemoglobin (10 microM) but not by the NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 and 250 microM in SV and IMA respectively). 6. Pretreatment (30 min) of SV with both S-nitrosothiols (10 microM) inhibited phenylephrine-induced contraction for >180 min, compared with <90 min for GTN. In IMA, contractility was suppressed to 49+/-4% (GSNO) and 26+/-4% (RIG200) of baseline after 240 min washout. 7. Pretreatment of bypass conduits with S-nitrosothiols might improve their patency in the early post-operative period.     

Role of von Willebrand factor in tumour cell-induced platelet aggregation: differential regulation by NO and prostacyclin.

1. We have studied the effects of a novel agonist, solid-phase von Willebrand Factor (sVWF), on tumour cell-induced platelet aggregation (TCIPA). 2. Washed platelet suspensions were obtained from human blood and the effects of HT-1080 human fibrosarcoma cells and sVWF on platelets were studied using aggregometry, phase-contrast microscopy, and flow cytometry. 3. Incubation of platelets with sVWF (1.2 microg ml(-1)) and HT-1080 cells (5 x 10(3) ml(-1)) resulted in a two-phased reaction characterized first by the adhesion of platelets to sVWF, then by aggregation. 4. TCIPA in the presence of sVWF was inhibited by S-nitroso-glutathione (GSNO, 100 microM) and prostacyclin (PGI(2), 30 nM). 5. Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up-regulation of GPIIb/IIIa receptors. 6. Pre-incubation of platelets with PGI(2) (30 nM) resulted in inhibition of sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa as measured by flow cytometry using antibodies directed against both non-activated and activated receptor. In contrast, GSNO (100 microM) did not affect sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa. 7. Flow cytometry performed with PAC-1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 microM) caused inhibition of activation of GPIIb/IIIa. 8. The inhibitors exerted no significant effects on TCIPA-mediated changes in GPIb. 9. Thus, sVWF potentiates the platelet-aggregatory activity of HT-1080 cells and these effects appear to be mediated via up-regulation of platelet GPIIb/IIIa. 10. Prostacyclin and NO inhibit TCIPA-sVWF-mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI(2) differ from those of NO.     

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.     

Inhibition of platelet aggregation by carbon monoxide-releasing molecules (CO-RMs): comparison with NO donors

Carbon monoxide (CO) and CO-releasing molecules (CO-RMs) inhibit platelet aggregation in vitro. Herein, we compare the anti-platelet action of CORM-3, which releases CO rapidly (t (?) 1?min), and CORM-A1, which slowly releases CO (t(?)?=?21?min). The anti-platelet effects of NO donors with various kinetics of NO release were studied for comparison. The effects of CO-RMs and NO donors were analyzed in washed human platelets (WP), platelets rich plasma (PRP), or whole blood (WB) using aggregometry technique. CORM-3 and CORM-A1 inhibited platelet aggregation in human PRP, WP, or WB, in a concentration-dependent manner. In all three preparations, CORM-A1 was more potent than CORM-3. Inhibition of platelets aggregation by CORM-A1 was not significantly affected by a guanylate cyclase inhibitor (ODQ) and a phosphodiesterase-5 inhibitor, sildenafil. In contrast, inhibition of platelet aggregation by NO donors was more potent with a fast NO releaser (DEA-NO, t (?)?=?2?min) than slow NO releasers such as PAPA-NO (t (?)?=?15?min) or other slow NO donors. Predictably, the anti-platelet effect of DEA-NO and other NO donors was reversed by ODQ while potentiated by sildenafil. In contrast to NO donors which inhibit platelets proportionally to the kinetics of NO released via activation of soluble guanylate cyclase (sGC), the slow CO-releaser CORM-A1 is a superior anti-platelet agent as compared to CORM-3 which releases CO instantly. The anti-platelet action of CO-RMs does not involve sGC activation. Importantly, CORM-A1 or its derivatives representing the class of slow CO releasers display promising pharmacological profile as anti-platelet agents.     

Antiplatelet effect of the anaesthetic drug propofol: influence of red blood cells and leucocytes

1. The present study was designed to investigate the mechanism of the antiplatelet action of the anaesthetic propofol in vitro. 2. Human whole blood was incubated with different concentrations of propofol and its solvent Intralipid(R). We determined, platelet aggregometry in whole blood, platelet-enriched plasma (PRP), PRP plus red blood cells (RBC), and PRP plus leucocytes (LC); platelet production of thromboxane B2 (TxB2), ATP release by platelet dense granules, adenosine uptake by RBC, intraplatelet levels of cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP), and LC production of nitric oxide (NO). 3. Propofol-induced inhibition of platelet aggregation was greater in whole blood (IC50 80 - 136 microM) than in PRP (IC50>600 microM), except when aggregation was induced by arachidonic acid, in which case the antiaggregatory effect of the anaesthetic was similar in both media (IC50 72 - 85 microM). Inhibition of platelet aggregation correlated significantly with inhibition of TxB2 synthesis (r2=0.83). Propofol also inhibited granular ATP release; this effect was greatest in whole blood. 4. The presence of RBC or LC increased the antiaggregatory effect of propofol, mainly when collagen was used as aggregating agent. Intralipid inhibited the uptake of adenosine by RBC, however this effect probably does not contribute significantly to its antiaggregatory effect. 5. The anaesthetic potentiated the NO-cyclic GMP pathway, mainly by increasing the synthesis of NO by LC. Intralipid had no effect on the NO-cyclic GMP pathway in the LC-platelet interaction. 6. Propofol inhibited platelet aggregation in human whole blood, possibly through the sum of the effects of Intralipid on the platelet-RBC interaction and the increased synthesis of NO by LC in the platelet-LC interaction.     

Differences in the influence of the interaction between acetylsalicylic acid and salicylic acid on platelet function in whole blood and isolated platelets: influence of neutrophils.

The aim of this study was to characterize the influence of the interaction between acetylsalicylic acid (ASA) and salicylic acid (SA) on the inhibition by ASA of platelet aggregation in platelets isolated from whole blood, and to determine whether leukocytes influence this pharmacological interaction. This in vitro study was done in human blood from which we prepared samples of whole blood, platelet-rich plasma (PRP), PRP plus mononuclear leukocytes, and PRP plus neutrophils. The variables recorded were maximum platelet aggregation intensity, thromboxane B2 (TxB2) production, and nitric oxide (NO) production (N=10 different samples in each type of experiment). Different concentrations of ASA and SA were incubated with all samples. In PRP, the concentration of ASA that inhibited maximum aggregation by 50% (IC50) (281+/-16microM) increased with increasing SA concentration to a maximum of more than 2mM when 500microM SA was used. In whole blood, the IC50 for ASA (24.9+/-1.2microM) decreased with decreasing SA concentrations to 7.9+/-0.8microM with 50microM SA and 15.6+/-0.9microM with 125microM SA, and increased to 46.2+/-2.6microM with 250microM SA and 96.3+/-7.2microM with 500microM SA. In experiments with PRP+neutrophils the IC50 of ASA increased in the presence of all concentrations of SA. The antagonistic interactions were also reflected in the changes in TxB2 production in all samples. In samples of neutrophils incubated with ASA, the curve for NO production was shifted to the right, a finding that paralleled the changes in platelet aggregation. In conclusion, the influence of the interaction between ASA and its metabolite SA on platelet aggregation difference depending on the type of sample, and was antagonistic in PRP but partially agonistic in whole blood. Nitric oxide synthesis showed an additive effect of the two compounds.     

A potential role for extracellular nitric oxide generation in cGMP-independent inhibition of human platelet aggregation: biochemical and pharmacological considerations

1. Nitric oxide (NO) is a potent inhibitor of platelet activation, that inhibits the agonist-induced increase in cytosolic Ca2+ concentration through both cGMP-dependent and independent pathways. However, the NO-related (NOx) species responsible for cGMP-independent signalling in platelets is unclear. We tested the hypothesis that extracellular NO, but not NO+ or peroxynitrite, generated in the extracellular compartment is responsible for cGMP-independent inhibition of platelet activation via inhibition of Ca2+ signalling. 2. Concentration-response curves for diethylamine diazeniumdiolate (DEA/NO; a spontaneous NO generator), S-nitroso-N-valerylpenicillamine (SNVP; an S-nitrosothiol) and 3-morpholinosydnonomine (SIN-1; a peroxynitrite generator) were generated in platelet-rich plasma (PRP) and washed platelets (WP) in the presence and absence of a supramaximal concentration of the soluble guanylate cyclase inhibitor, ODQ (20 microM). All three NOx donors displayed cGMP-independent inhibition of platelet aggregation in PRP, but only DEA/NO exhibited cGMP-independent inhibition of aggregation in WP. 3. Analysis of NO generation using an isolated NO-electrode revealed that cGMP-independent effects coincided with the generation of substantial levels of extracellular NO (>40 nM) from the NOx donors. 4. Reconstitution of WP with plasma factors indicated that the copper-containing plasma protein, caeruloplasmin (CP), catalysed the release of NO from SNVP, while Cu/Zn superoxide dismutase (SOD) unmasked NO generated from SIN-1. The increased generation of extracellular NO correlated with a switch to cGMP-independent effects with both NOx donors. 5. Analysis of Fura-2 loaded WP revealed that only DEA/NO inhibited Ca2+ signalling in platelets via a cGMP-independent mechanism. However, preincubation of SNVP and SIN-1 with CP and SOD, respectively, induced cGMP-independent inhibition of intraplatelet Ca2+ trafficking by the NOx donors. 6. Taken together, our data suggest that extracellular NO (>40 nM) is required for cGMP-independent inhibition of platelet activation. Plasma constituents may play an important pharmacological role in activating cGMP-independent signalling by S-nitrosothiols or peroxynitrite generators.     

Differential inhibition of human platelet aggregation and thromboxane A2 formation by L-arginine in vivo and in vitro

We compared the effects of L-arginine (L-ARG), the precursor of endogenous NO, on platelet aggregation and thromboxane A₂ formation in vivo and in vitro. Human platelet-rich plasma (PRP) was anticoagulated with citrate (which decreases extracellular Ca²⁺) or with recombinant hirudin (which does not affect extracellular Ca²⁺). Two groups of 10 healthy male volunteers received intravenous infusions of L-ARG (30 g or 6 g, 30 min) or placebo. Blood was collected immediately before and at the end of the infusions for aggregation by ADP or collagen. Infusion of L-ARG inhibited ADP-induced aggregation in PRP anticoagulated with citrate by 37.5 ± 6.3% (P < 0.05). In PRP anticoagulated with hirudin, aggregation was inhibited by 33.6 ± 16.0% (P < 0.05). L-ARG infusion also inhibited platelet TXB₂ formation and slightly, but not significantly decreased the urinary excretion rate of 2,3-dinor-TXB₂; cGMP concentrations in PRP were significantly elevated during L-arginine infusion. In vitro preincubation with L-ARG (10 μM–2.5 mM) inhibited platelet aggregation in PRP anticoagulated with r-hirudin, but not citrate. This effect was stereospecific for L-arginine, as D-arginine had no effect. It was dependent upon NO synthase activity, as indicated by increased cGMP levels in PRP. Moreover, both the NOS inhibitor L-NMMA and the inhibitor of soluble guanylyl cyclase ODQ antagonized the effects of L-ARG. Haemoglobin, an extracellular scavenger of NO, partly antagonized the antiplatelet effects of L-ARG. 8-Br-cyclic GMP and the exogenous NO donor linsidomine inhibited aggregation in PRP anticoagulated with citrate or r-hirudin. The inhibitory effects of L-ARG on platelet aggregation in vitro were paralleled by increased cyclic GMP levels; L-ARG also inhibited platelet TXB₂ formation in PRP anticoagulated with r-hirudin, but not citrate. We conclude that the L-arginine/NO pathway is present in human platelets as a Ca²⁺-dependent anti-aggregatory pathway. In vivo the formation of NO from L-ARG by endothelial cells may contribute to the platelet-inhibitory effects of L-ARG. NO-releasing compounds like linsidomine inhibit platelet aggregation in vitro independent of extracellular Ca²⁺. Received: 17 April 1997 / Accepted: 17 October 1997     

Platelet inhibitory effects of the nitric oxide donor drug MAHMA NONOate in vivo in rats

The platelet inhibitory effects of the nitric oxide (NO) donor drug MAHMA NONOate ((Z-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen-1-ium-1,2-diolate) were examined in anaesthetised rats and compared with those of S-nitrosoglutathione (GSNO; an S-nitrosothiol). Bolus administration of the aggregating agent ADP dose-dependently reduced the number of circulating free platelets. Intravenous infusions of MAHMA NONOate (3-30 nmol/kg/min) dose-dependently inhibited the effect of 0.3 micromol/kg ADP. MAHMA NONOate was approximately 10-fold more potent than GSNO. MAHMA NONOate (0.3-10 nmol/kg/min) also reduced systemic artery pressure and was again 10-fold more potent than GSNO. Thus MAHMA NONOate has both platelet inhibitory and vasodepressor effects in vivo. The dose ranges for these two effects overlapped, although blood pressure was affected at slightly lower doses. The platelet inhibitory effects compared favourably with those of GSNO, even though NONO-ates generate free radical NO which, in theory, could have been scavenged by haemoglobin. Therefore platelet inhibition may be a useful therapeutic property of NONOates.     

S-nitroso-glutathione inhibits platelet activation in vitro and in vivo.

1. The effect of S-nitroso-glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2. The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3. GSNO caused a concentration-dependent inhibition of collagen-induced platelet aggregation in vitro and decreased ADP-induced aggregation in the conscious rat. 4. Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5. The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6. The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.     

Role of a copper (I)-dependent enzyme in the anti-platelet action of S-nitrosoglutathione.

1. S-nitrosoglutathione (GSNO) is a potent and selective anti-platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti-aggregatory action of GSNO. 2. The biological action of GSNO could be mediated by NO released from S-nitrosocystylglycine, following enzymatic cleavage of GSNO by gamma-glutamyl transpeptidase. The anti-aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the gamma-glutamyl transpeptidase inhibitor acivicin (1 mM). gamma-Glutamyl transpeptidase is not, therefore, involved in mediating the action of GSNO. 3. The rate of breakdown of S-nitrosoalbumin was increased from 0.19 +/- 0.086 nmol min-1 to 1.52 +/- 0.24 nmol min-1 (mean +/- s.e.mean) in the presence of cysteine (P < 0.05, n = 4). Inhibition of platelet aggregation by S-nitrosoalbumin was also significantly increased by cysteine (P < 0.05, n = 4), suggesting that the biological activity of S-nitrosoalbumin is mediated by exchange of NO from the protein carrier to form the unstable compound cysNO. Breakdown of GSNO showed a non-significant acceleration in the presence of cysteine, from 0.56 +/- 0.22 to 1.77 +/- 0.27 nmol min-1 (mean +/- s.e.mean) (P = 0.064, n = 4), and its ability to inhibit platelet aggregation was not enhanced by cysteine. This indicates that the anti-platelet action of GSNO is not dependent upon transnitrosation to form cysNO. 4. Platelets pretreated with the copper (I)-specific chelator bathocuproine disulphonic acid (BCS), then resuspended in BCS-free buffer, showed resistance to the inhibitory effect of GSNO. These findings suggest that BCS impedes the action of GSNO by binding to structures on the platelet, rather than by chelating free copper in solution. 5. Release of NO from GSNO was catalysed enzymatically by ultrasonicated platelet suspensions. This enzyme had an apparent K(m) for GSNO of 12.4 +/- 2.64 microM and a Vmax of 0.21 +/- 0.03 nmol min-1 per 10(8) platelets (mean +/- s.e.mean, n = 5). It was inhibited by BCS, but not by the iron chelator bathophenathroline disulphonic acid, nor by acivicin. 6. We conclude that the stable S-nitrosothiol compound GSNO may exert its anti-platelet action via enzymatic, rather than spontaneous release of NO. This is mediated by a copper-dependent mechanism. The potency and platelet-selectivity of GSNO may result from targeted NO release at the platelet surface.     

The effect of 6-oxo-prostaglandin E1 on human platelet aggregation in whole blood in-vitro

The effect of PGI2, 6-oxo-PGE1 and PGE1 on ADP-induced human platelet aggregation has been assessed in whole blood and in blood centrifuged to prepare platelet-rich plasma (PRP). PGI2 was the most potent anti-aggregatory agent in both media. The concentration of PGI2 required to produce 50% inhibition of platelet aggregation was approximately 0.3 ng ml-1 in each case. In contrast both E series prostaglandins exhibited significantly greater (400-700%) anti-aggregatory activity when tested in whole blood than when tested in PRP. Since whole blood presumably represents a truer reflection of platelet reactivity in-vivo, we believe that the potency of 6-oxo-PGE1 (and PGE1) as inhibitors of platelet aggregation has been underestimated in previous experiments using PRP. In human whole blood 6-oxo-PGE1 has approximately 40% the anti-aggregatory activity of PGI2. The reasons for the increased anti-aggregatory potency of E series prostaglandins in whole blood is not known. We suggest that 6-oxo-PGE1 and PGE1 (but not PGI2) may prevent the release of pro-aggregatory ADP from red blood cells thereby enhancing their ability to inhibit platelet aggregation.     

Mechanisms of aggregation inhibition by aspirin and nitrate-aspirin prodrugs in human platelets

Objectives Aspirin is the mainstay of anti‐platelet therapy in the secondary prevention of cardiovascular disease. However, problems with aspirin safety and resistance demand clinical strategies based on multiple pharmacological approaches. Prodrugs of aspirin may offer beneficial effects in terms of gastro‐intestinal safety and multiple pharmacological approaches. However, the pharmacological profile of aspirin prodrugs in human platelets has not been completed yet. We aimed to compare the effects of aspirin and prodrugs of aspirin ( 1 – 5 ) on human platelet aggregation stimulated by ADP and collagen and associated receptor expression (GPIIb/IIIa and P‐selectin) in platelet‐rich plasma (PRP) and washed platelets (WP). Methods As aspirin is released from prodrugs following esterase hydrolysis we studied the expression and activity of butyrylcholineterase (BuChE) and carboxyesterase (CE) in plasma and platelets. The mechanism of prodrug‐induced platelet aggregation inhibition was explored by studying the effects of plasma and purified human BuChE on aggregation. Finally, the relative contribution of nitric oxide (NO) bioactivity to nitrate‐containing prodrugs of aspirin‐induced inhibition of aggregation was determined using 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ,) a selective inhibitor of the soluble guanylyl cyclase. Key findings ST0702, 2 , a nicotinic acid‐aspirin codrug was equipotent with aspirin with respect to inhibition of collagen‐induced platelet aggregation. Compound 4 , a NO releasing aspirin was the most potent inhibitor of ADP‐induced platelet aggregation, an effect partially reversed by ODQ. The platelet inhibitory effects of aspirin prodrugs were time‐dependent as the maximal inhibitory effects against collagen‐induced aggregation were achieved by aspirin at 2 min, 1 at 5 min and ST0702 at 15 min. The aspirin prodrugs were significantly less potent in WP than in PRP and the reverse was true of aspirin. In the presence of complete BuChE inhibition in PRP, there was almost complete loss of aspirin prodrug, but not aspirin anti‐aggregatory activity. Interestingly, CE activity was observed in WP and platelet lysate with pNPA substrate. Accordingly, 1 and ST0702 retained 50% and 100% anti‐aggregatory activity at maximal concentrations in WP, which was attenuated in the presence of esterase inhibitor phenylmethylsulphonyl fluoride. Conclusions The inhibitory effect of aspirin prodrugs in PRP is due to prodrug activation by BuChE. In contrast, the platelet‐inhibitory effects of aspirin prodrugs in WP may be mediated through the activity of platelet CE. Compound 4 , a NO‐containing aspirin prodrug, may exert dual inhibitory effects in platelets. Thus, aspirin prodrugs effectively inhibit human platelet aggregation and as such may be an alternative to conventional aspirin.     

Role of adenosine uptake and metabolism by blood cells in the antiplatelet actions of dipyridamole, dilazep and nitrobenzylthioinosine

Adenosine (Ado, 10 microM) did not inhibit ADP-induced human platelet aggregation in whole blood. However, if the blood was preincubated with dipyridamole (10 microM), a potent inhibitor of the erythrocytic nucleoside transport system (NTS), Ado acted as a strong inhibitor of platelet aggregation. Similarly, Ado inhibited platelet aggregation in whole blood in the presence of other potent NTS inhibitors, dilazep (1 microM) and p-nitrobenzylthioinosine (NBMPR, 1 microM). RA 233 (10 microM), an analog of dipyridamole which is a potent inhibitor of platelet cAMP phosphodiesterase (PDE), did not evoke the Ado effect in whole blood. However, in platelet-rich plasma (PRP), RA 233 potentiated strongly Ado-mediated inhibition, whereas dipyridamole, dilazep and NBMPR were without activity. 5'-Methylthioadenosine (MTA), an Ado receptor antagonist, reversed the inhibition produced by a nucleoside transport system inhibitor plus Ado in whole blood. Dipyridamole (10 microM), dilazep (1 microM) or NBMPR (1 microM) blocked [14C]Ado (10 microM) uptake by blood cells in whole blood, whereas RA 233 (10 microM) was not effective. The combination of 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of adenosine deaminase (ADA), plus 5-iodotubercidin (ITu, 10 microM), a potent inhibitor of adenosine kinase (Ado kinase), gave comparable Ado-mediated inhibition of platelet aggregation in whole blood as was obtained when the blood was pretreated with dilazep. These studies suggest that the in vivo antiplatelet actions of drugs such as dipyridamole and dilazep result from their abilities to block erythrocytic Ado uptake and subsequent metabolism, thus elevating the extracellular steady-state concentration of the physiologically occurring, antiplatelet agent, Ado.     

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.     

Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets

1 The pharmacological effects of endothelium-derived relaxing factor (EDRF), nitric oxide (NO) and prostacyclin on human and rabbit platelets were examined. 2 EDRF is released from porcine aortic endothelial cells, cultured on microcarriers and treated with indomethacin, in sufficient quantities to inhibit platelet aggregation induced by 9,11-dideoxy-9 alpha, 11 alpha-methano epoxy-prostaglandin F2 alpha (U46619) and collagen. 3 The anti-aggregating activity of EDRF was potentiated by M&B 22948, a selective inhibitor of cyclic GMP phosphodiesterase, and by superoxide dismutase (SOD) and was inhibited by haemoglobin and Fe2+. 4 Both NO and prostacyclin inhibited platelet aggregation. 5 The anti-aggregatory activity of NO, but not that of prostacyclin, was potentiated by M&B 22948 and by SOD and was inhibited by haemoglobin and Fe2+. Thus NO is a potent inhibitor of platelet aggregation whose activity on platelets mimics that of EDRF. 6 It is likely that the inhibitory effect of NO on platelets represents the action of endogenous EDRF and therefore this substance, together with prostacyclin, is a regulator of platelet-vessel wall interactions.     

Copper chelation-induced reduction of the biological activity of S-nitrosothiols.

1. The effect of copper on the activity of the S-nitrosothiol compounds S-nitrosocysteine (cysNO) and S-nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sulphonate (BCS), and human washed platelets as target cells. 2. Chelation of trace copper with BCS (10 microM) in washed platelet suspensions reduced the inhibition of thrombin-induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti-aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3. The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 x 10(9) l-1. 4. In BCS-treated platelet suspensions, the addition of Cu2+ ions (0.37-2.37 microM) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5. The anti-aggregatory activity of GSNO was reduced in a concentration-dependent manner by the copper (I)-specific chelators BCS and neocuproine, and to a smaller extent by desferal. No effect was observed with the copper (II) specific chelator, cuprizone, the iron-specific chelator, bathophenanthroline sulphonate, or the broader-specificity copper chelator, D-penicillamine. 6. In both BCS-treated and -untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS-treated platelet suspensions there was no significant difference between the anti-aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1. Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen-1-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and to investigate the possible role of activation of sarco-endoplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2-10 micro g ml(-1)) and adenosine diphosphate (ADP; 2 micro M) in platelet rich plasma. It was (i). more effective at inhibiting aggregation induced by collagen than by ADP, and (ii). less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3. ODQ (10 micro M) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4. The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; 1-100 micro M), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1-1 mM), caused minimal inhibition. 5. On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 micro M present) were abolished by thapsigargin (200 nM). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6. Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7. Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.     

Effect of Cu2+ on relaxations to the nitrergic neurotransmitter, NO and S-nitrosothiols in the rat gastric fundus.

1. The effects of addition of Cu2+ and chelation of Cu2+ were studied on relaxations in response to S-nitrosothiols and on relaxations to non-adrenergic non-cholinergic (NANC) nerve stimulation, nitric oxide (NO) and glyceryl trinitrate (GTN) in the rat gastric fundus. 2. The S-nitrosothiols S-nitroso-L-cysteine (NOCys, 1-300 nM), S-nitrosoglutathione (GSNO, 0.01-3 microM) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.01-3 microM) induced concentration-dependent relaxations of the rat gastric fundus muscle strip. The relaxant potencies of the S-nitrosothiols were NOCys > SNAP > GSNO. Relaxations to NOCys were transient and comparable to those to NANC nerve stimulation and NO whereas relaxations to GSNO and SNAP were sustained. The relaxations to NOCys, GSNO and SNAP were significantly and concentration-dependently enhanced by CuSO4 (3-30 microM). The order of relaxant potency in the presence of CuSO4 was reversed to GSNO approximately SNAP > NOCys. 3. In the presence but not in the absence of 0.1 microM GSNO, CuSO4 (1 microM) induced a rapid and transient relaxation which was inhibited by the superoxide radical generator, pyrogallol (30 microM). CuCl2 but not FeSO4 mimicked the effect of CuSO4. 4. Electrical stimulation (0.5-8 Hz) of the rat gastric fundus strips induced frequency-dependent relaxations which were previously shown to be nitrergic in nature and which were not affected by CuSO4 (3-30 microM). Relaxations to NO (3-100 nM) and GTN (0.01-1 microM) were not affected by 3 and 10 microM CuSO4 but were inhibited by 30 microM CuSO4. 5. The Cu2+ chelator, bathocuproine (3-30 microM) significantly and concentration-dependently inhibited the relaxations to NOCys (0.01-3 microM), GSNO (0.01-10 microM) and SNAP (0.01-3 microM). The inhibitory effect of 10 microM bathocuproine was reversed by 3 microM CuSO4. 6. Bathocuproine (3-30 microM) had no effect on the relaxations to NANC nerve stimulation (0.5-8 Hz) or on the concentration-response curve to NO (0.01-0.3 microM), whereas relaxations to GTN (0.01-1 microM) were significantly inhibited by 30 microM bathocuproine. 7. From these results we conclude that relaxations to S-nitrosothiols and to nitrergic stimulation of the rat gastric fundus are differentially affected by addition and chelation of Cu2+, suggesting that the nitrergic NANC neurotransmitter in the rat gastric fundus is not an S-nitrosothiol but is more likely to be free nitric oxide.     

Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP).
2. Using 10 μM of each donor compound, mean±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min⁻¹ (for SNP) to 3.15±0.29 nmol min⁻¹ (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 10⁸ platelets (for SNP) to 2.67±0.31 pmol per 10⁸ platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC).
3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34).
4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8-bromo cyclic GMP).
5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu⁺) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO.
6. Cyclic GMP accumulation in response to GSNO (10⁻⁹–10⁻⁵M) was undetectable following treatment of platelets with ODQ (100 μM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu⁺ ions were required for the cyclic GMP-independent pathway to operate.
7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP.
8. The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (CMPS) produced a concentration-dependent inhibition of aggregation of ODQ-treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP-independent inhibition of platelet aggregation.
9. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP-dependent and -independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP-independent pathway may depend upon a cellular system which involves both copper (I) (Cu⁺) ions and surface membrane thiol groups. The potent anti-platelet action of GSNO results from its ability to exploit this cyclic GMP-independent mechanism.