Daniela Salvemini

Superoxide anions enhance platelet adhesion and aggregation

1 Superoxide dismutase (SOD, 60 u ml−1) or ferricytochrome c (70 μm) significantly inhibited thrombin‐stimulated platelet adhesion to gelatin‐coated plastic, whereas catalase (1000 u ml−1) or mannitol (1 mm) had no effect. 2 The platelet aggregation induced by low concentrations of thrombin (causing less than 45% maximal change in light transmission) was inhibited by SOD. Catalase or mannitol had no effect on platelet aggregation. 3 Pyrogallol (an O2− generator) enhanced both platelet adhesion to gelatin‐coated plastic and platelet aggregation induced by thrombin; this enhancement was neutralized by SOD. 4 These results indicate that O2− increase both platelet adhesion and aggregation, whereas other free radicals such as hydrogen peroxide or hydroxyl radicals are not involved.

Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase.

1 Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 μm) promoted a cell number‐related inhibition of platelet aggregation induced by thrombin (40 mu ml−1). This inhibition was not attributable to products of the cyclooxygenase pathway for the SMC were also treated with indomethacin (10 μm). 2 The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 μg ml−1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 μg ml−1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non‐treated cells. 3 The inhibition of platelet aggregation obtained with non‐treated or LPS‐treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml−1) and ablated by oxyhaemoglobin (OxyHb, 10 μm). Preincubation of the SMC with NG‐monomethyl‐l‐arginine (l‐NMMA, 30–300 μm) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with l‐arginine (l‐Arg, 100 μm) but not with d‐arginine (d‐Arg, 100 μm). 4 Exposure of the non‐treated SMC (5 × 105 cells) to stirring (1000 r.p.m., 37°C) for 10 min led to a significant increase in their levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) but not adenosine 3′:5′‐cyclic monophosphate (cyclic AMP). l‐NMMA (300 μm) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells. The inhibitory activity of l‐NMMA was reversed by co‐incubation with l‐Arg (100 μm) but not d‐Arg (100 μm). l‐Arg alone had no effect on the levels of cyclic GMP. In the absence of stirring, a 10 min stimulation of the non‐treated SMC with glyceryl trinitrate (GTN, 200 μm) or atrial natriuretic factor (ANF, 10−7 m) led to an increase in the levels of cyclic GMP but not cyclic AMP. The increase in cyclic GMP promoted by GTN or ANF was not affected by l‐NMMA. The levels of cyclic GMP were higher in the LPS (0.5 μg ml−1, 18 h)‐treated cells (5 × 10−5) and stirring was more effective in increasing the levels of cyclic GMP in these cells. 5 These findings support the idea that non‐treated or LPS‐treated cultured SMC can produce an NO‐like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO‐like factor may play a role in the auto‐regulation of smooth muscle cell reactivity through a cyclic GMP‐dependent mechanism.

Modulation of platelet function by free radicals and free-radical scavengers

Platelets have the capacity to generate oxygen-derived free radicals and are often present at inflammatory foci with other free-radical-generating cells such as white blood cells. Free radicals can modify platelet adhesion and aggregation directly or through effects on the vascular endothelium, which generates prostacyclin and nitric oxide. To defend against the overproduction of free radicals the body manufactures endogenous scavengers, which can be of enzymic or non-enzymic origin. Daniela Salvemini and Regina Botting describe how free-radical scavengers may be used therapeutically to regulate the platelet reactivity involved in many pathological phenomena.

Rat mast cells synthesize a nitric oxide like-factor which modulates the release of histamine

Rat serosal mast cells were evaluated for their capacity to generate a nitric oxide-like factor by two bioassays: inhibition of platelet aggregation and stimulation of mast cell guanylate cyclase. Incubation of mast cells with human washed platelets, both treated with indomethacin, inhibited thrombin-induced platelet aggregation which was potentiated by superoxide dismutase and reversed by oxyhaemoglobin. When mast cells alone were stirred at 1000 rpm, a time dependent increase in the levels of their cGMP but not cAMP was observed. Preincubation of mast cells with NG-monomethyl-l-arginine significantly enhanced E. coli lipopolysaccharide-evoked histamine release. Our results show that mast cell histamine release can be modulated by an intrinsically generated nitric oxide-like factor.

The use of oxyhaemoglobin to explore the events underlying inhibition of platelet aggregation induced by NO or NO‐donors

1 Full inhibition of thrombin‐induced platelet aggregation was elicited by the least maximal platelet inhibitory concentrations of nitric oxide (NO; 7 ± 1 μm) or NO‐donors which included sodium nitroprusside (NaNp; 80 ± 13 μm), 3‐morpholinosydnonimine (SIN‐1; 3 ± 0.1 μm) or endothelial cells (EC; 2.36 ± 0.12 × 105) added 1 min before thrombin. Oxyhaemoglobin (oxyHb; 10 μm) administered 30s to 10 min after stimulation with thrombin caused a time‐dependent reversal of the inhibition induced by these agents. OxyHb was ineffective when these agents were co‐incubated with the non‐selective phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX, 0.05 mm). 2 OxyHb did not reverse the platelet inhibition with IBMX (0.2 mm) or that caused by a selective guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP) phosphodiesterase inhibitor 2‐O‐propoxyphenyl‐8‐azapurin‐6‐one, (M & B 22948; 20 μm). In addition, oxyHb did not reverse the inhibition with iloprost (1 nm) which inhibits platelet aggregation through stimulation of adenylate cyclase. 3 The inhibition of platelet aggregation by NO (7 ± 1 μm) or NaNp (80 ± 13 μm) was accompanied by a 13 fold increase in cyclic GMP levels occurring within 15 s of addition of these agents. In the continued presence of NO or NaNp, the reversing effect of oxyHb given 1 min after thrombin was associated with a pronounced decrease in cyclic GMP levels. 4 We conclude that the inhibition of platelet aggregation by activators of guanylate cyclase depends in the first few minutes on continuous stimulation of the enzyme in order to maintain intracellular concentrations of cyclic GMP, except when its breakdown is inhibited. 5 The addition of agents such as oxyHb after the inhibition of platelet aggregation offers another way of investigating the biochemical changes involved in maintaining platelets in an inactive state.

Conversion of glyceryl trinitrate to nitric oxide in tolerant and non-tolerant smooth muscle and endothelial cells.

1 Exposure of smooth muscle cells (SMC) to glyceryl trinitrate (GTN, 75–600 μm) for 30 min led to a concentration‐dependent increase in nitrite (NO2−), one of the breakdown products of nitric oxide (NO). This was not affected by 30 min pretreatment of the cells with 0.5 mm of sulphobromophthalein (SBP) an inhibitor of glutathione‐S‐transferase (GST), by metyrapone or SKF‐525A inhibitors of cytochrome P450. These experiments were confirmed by organ bath studies using rabbit aortic strips denuded of endothelium and contracted with phenylephrine. Thus, a 30 min incubation of the strips with 0.5 mm SPB, metyrapone or SKF‐525A did not affect the relaxations in response to GTN (10−10−10−6 m). 2 Potentiation of the anti‐platelet effect of GTN (44 μm) by endothelial cells (EC, 40 × 103 cells) was not affected by prior incubation of EC with SBP, metyrapone or SKF‐525A (all at 0.5 mm). 3 Potentiation of the antiplatelet activity of GTN (11–352 μm) by small numbers of SMC (24 × 103 cells) or EC (40 × 103 cells) treated with indomethacin (10 μm) was attenuated when the SMC or EC were treated in culture with a high concentration of GTN (600 μm) for 18 h beforehand (referred to as ‘tolerant’ cells). In addition, tolerant SMC produced far less NO2− than non‐tolerant SMC. 4 Exposure of non‐tolerant SMC or EC (105 cells) to GTN (200 μm) for 3 min increased (3–4 fold) the levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP). This increase was much less (≤ 1 fold) in the tolerant SMC or EC (105 cells). The basal levels of cyclic GMP were similar in normal or tolerant SMC or EC. Sodium nitroprusside (80 μm) or atrial natriuretic factor (ANF, 10−7 m) increased the levels of cyclic GMP in normal or tolerant SMC or EC to the same extent. 5 The anti‐platelet effects of GTN (44 μm) were potentiated by the sulphydryl donor N‐acetylcysteine (NAC, 0.5 mm). Incubation of GTN (150–1200 μm) for 30 min with NAC (0.1–1 mm) led to a concentration‐dependent increase in NO2− formation. The reduced ability of tolerant SMC or EC to potentiate the anti‐platelet activity of GTN was restored by NAC (0.5 mm). These anti‐aggregatory effects were abolished by concurrent co‐incubation with oxyhaemoglobin (10 μm) indicating that they were due to NO release. 6 Thus, in SMC or EC, metabolism of GTN to NO does not depend on glutathione‐S‐transferase or the cytochrome P450 system. Furthermore, when compared to normal cells, tolerant SMC or EC metabolize GTN to NO less effectively as assessed by the reduced capacity to potentiate the antiplatelet effects of GTN, to release NO2− and to increase the level of cyclic GMP. This decrease in NO formation shows that tolerance to GTN is mainly due to impaired biotransformation of GTN to NO. NAC, by directly forming NO from GTN, compensates for this failing mechanism.

Modulation of the pharmacological actions of nitrovasodilators by methylene blue and pyocyanin

1 In superfused precontracted strips of rabbit aorta, methylene blue (MeB) or pyocyanin (Pyo, 1‐hydroxy‐5‐methyl phenazinum betaine) at concentrations of 1–10 μm inhibited relaxations induced by endothelium‐derived relaxing factor (EDRF), glyceryl trinitrate (GTN), S‐nitroso‐N‐acetyl‐penicillamine (SNAP) or 3‐morpholino‐sydnonimine (SIN‐1). However, the vasorelaxant actions of sodium nitroprusside (NaNP) or sodium nitrite (NaNO2) were enhanced by MeB or Pyo. Oxyhaemoglobin (HbO2, 1 μm) inhibited the activities of EDRF and all of the nitrovasodilators studied. Vascular preparations were not relaxed by Pyo unless pretreated with NaNP (0.05–10 μm). 2 In bathed, precontracted rings of rabbit aorta, Pyo (10 μm) produced a shift to the left of the cumulative concentration‐response curve for NaNP (0.01–10 μm). The rise in guanosine‐3′:5′‐cyclic monophosphate (cyclic GMP) content of aortic tissue was also enhanced. 3 The vasorelaxant potency of NaNP (30 μm) at pH 5–8 and at 37°C remained unchanged over 2.5 h while a solution of SNAP (30 μm) progressively lost its biological activity over 60 min. The in vitro degradation of the biological activity of SNAP was accelerated by MeB (150 μm) or Pyo (150 μm), whereas the vasorelaxant potency NaNP (30 μm) was doubled when incubated with MeB or Pyo. 4 In human platelet‐rich plasma, MeB or Pyo (0.3–3.0 μm) uncovered an anti‐aggregatory action of subthreshold concentrations of NaNP (4–8 μm). This was abrogated by HbO2 (10 μm). In contrast to NaNP the anti‐aggregatory effect of SNAP (2–20 μm) was antagonized by MeB (10 μm), Pyo (10 μm) or HbO2 (10 μm). 5 We conclude that MeB or Pyo differ from HbO2 in their mode of interaction with nitrovasodilators. HbO2 scavenges nitric oxide that is released from all types of nitrovasodilators. MeB and Pyo exert a similar action towards organic nitrovasodilators (e.g. SNAP, SIN‐1). However, the pharmacological actions of inorganic nitrovasodilators (e.g. NaNP or NaNO2) are potentiated by MeB and Pyo owing to facilitation of the intracellular release of nitric oxide from the inorganic nitrovasodilators.

Cultured astrocytoma cells inhibit platelet aggregation by releasing a nitric oxide-like factor

Cultured astrocytoma cells were tested for their ability to generate a nitric-oxide like factor using platelet aggregation as a bioassay. Incubation of astrocytoma cells with human washed platelets resulted in an inhibition of thrombin-induced platelet aggregation which was proportional to the number of astrocytoma cells added. The inhibition was potentiated by superoxide dismutase (SOD) and prevented by oxyhaemoglobin (oxyHb). The inhibitory activity of astrocytoma cells was also prevented by the NO biosynthesis inhibitor NG-monomethyl-L-arginine (MeArg), an effect reversed by co-incubation with L-arginine (L-Arg) but not D-arginine (D-Arg). These results demonstrate that astrocytoma cells release, independent of added agonist, a factor with the same pharmacological profile as NO, which is likely to be derived from L-arginine.

Cultured astrocytoma cells generate a nitric oxide‐like factor from endogenous L‐arginine and glyceryl trinitrate: effect of E. coli lipopolysaccharide

1 The inhibitory activity of astrocytoma cells (0.25–3 × 105) treated with indomethacin (10 μm) on platelet aggregation was enhanced by incubating the cells with E. coli lipopolysaccharide (LPS, 0.5 μg ml−1) for 18 h. This effect was attenuated when cycloheximide (10 μg ml−1) was incubated together with LPS. The inhibition of platelet aggregation by cells treated with LPS was potentiated by superoxide dismutase (60 u ml−1) and ablated by oxyhaemoglobin (oxyHb, 10 μm) or NG‐monomethyl‐l‐arginine (l‐NMMA, 30–300 μm). The effects of l‐NMMA were reversed by co‐incubation with l‐arginine (l‐Arg, 100 μm) but not d‐arginine (d‐Arg, 100 μm). LPS also increased the levels of nitrite in the culture media and this increase was ablated by co‐incubation with l‐NMMA (300 μm) or cycloheximide (10 μg ml−1). 2 Astrocytoma cells (0.5 × 105) treated with indomethacin (10 μm) enhanced the platelet inhibitory activity of glyceryl trinitrate (GTN, 11–352 μm) but not that of sodium nitroprusside (4 μm). Furthermore, when incubated with GTN (200 μm) a 4 fold increase in the levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) was observed. These effects were abrogated by co‐incubation with oxyHb (10 μm) but not with l‐NMMA (300 μm). Treatment of the cells with LPS (0.5 μg ml−1) for 18 h did not enhance their capacity to form NO from GTN. 3 Thus, in cultured astrocytoma cells, LPS enhances the formation of nitric oxide from endogenous l‐arginine. In addition, these cells can metabolize GTN to nitric oxide but this process is not enhanced by LPS stimulation.

Impairment of the L-arginine-nitric oxide pathway in mast cells from spontaneously hypertensive rats

Serosal mast cells (MC) from 6 month old spontaneously hypertensive rats (SHR) were compared to MC from 6 month old Wistar Kyoto rats (WKYR) for their ability to release nitric oxide (NO). The relationship between histamine release and NO-like activity from these cells was also investigated. MC from SHR released less NO-like factor than MC from WKYR as assessed by the use of platelet aggregation and soluble guanylate cyclase activation as bioassays for NO. Sodium nitroprusside elevated the concentrations of cGMP to a similar extent in MC from SHR or WKYR. No changes in the levels of cAMP were observed. The release of histamine from MC induced by compound 48/80 or the calcium ionophore A23187 was greater in MC from SHR than in MC from WKYR. Thus, MC from SHR show a decreased production of NO-like activity which is reflected by a decreased ability to inhibit platelet aggregation. The decreased production of cGMP in the MC leads to an increased stimulated release of histamine.