THROMBOSIS RESEARCH 66; 101-l 10 1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
LOW CONCENTRATIONS THROMBOXANE
Eric Rubiiein,
OF SODIUM AZIDE SPECIFICALLY
INHIBIT A
A2 PATHWAY IN HUMAN PLATELETS.
Irene Ursa and Roger C. Carroll
Department of Medical Biology, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville TN 37920, USA (Received 10.7.1991; accepted in revised form 4.12.1991 by Editor J. Soria) (Received by Executive Editorial Office 20.2.1992)
ABSTRACT Sodium azide completely inhibits the serotonin release induced by ADP, arachidonic acid and the thromboxane A2 mimetic U46619, but does npt have any effect on the activation by PMA. Collagen and thrombin are inhibited when used at low concentrations, but not at high concentration. This pa tern of activation suggests that the inhibition by azide is not a metabolic in J ‘bition. The antagonism of U46619-induced secretion was further studied and was shown to be non-competitive. It is selective for certain components of the U46619 stimulus-response coupling: aggregation, serotonin secretion and the activation of protein kinase C are completely or almost completely inhibited by 300 PM sodium azide. Shape change, calcium elevation, cyto lasmic alkalinization and phosphorylation of myosin light chain are only k ‘ally modified. This suggests that azide may specifically inhibit one of the different forms of thromboxane A2 receptors present in platelets.
INTRODUCTION
Sodium azide (NaN3) is a classical inhibitor of cytochrome oxidase and thereby inhibits mitochondrial respiratory energy production. Another effect of this compound on energetic metabolism is inhibition of ATPase, a coupling factor of oxidative phosphorylation, in procarytic and eucaryotic cells (1). Sodium azide is also an activator of guanylate cyclase in brain, liver (2) and platelets (3). In this latter system, it was concluded that cGMP and agents Key words: Platelet activation; azide; thromboxanes. 101
102
THROMBOXANE
A2 AND AZIDE
Vol. 66, Nos. 213
increasing cGMP concentration, including azide, did not affect platelet activation by thrombin. The effect of sodium azide on platelet aggregation was further studied by Stibbe and Holmsen (4). Sodium azide was shown to partklly inhibit the first wave of aggregation induced by ADP, epinephrine (in the presence of indomethacin), the ionophore A 23 187, and thrombin (in presence of indomethacin and ADP scavenger). Serotonin secretion induced by ADP, epinephrine and collagen was also inhibited. This effect was not due to an inhibition of energy metabolism. We show here that sodium azide inhibits specific pathways and propose a mechanism of platelet inactivation by azide.
Platelet preparation: Blood was collected from volunteers giving informed consent who denied taking any medication for the previous two weeks. The blood was drawn from the antecubital vein into syringes containing 0.1 volume of CCD (100 mM sodium citrate and 130 mM glucose, pH 6.5). The blood was centrifuged 3 min at 1000 x g at room temperature to separate the platelet-rich plasma (PRP). For certain experiments, platelets were gel-filtered (GFP) on a Sepharose-2B column (Sigma) equilibrated with a modified Tangen-HEPES-BSA buffer @H 7.4) containing 145 mM sodium chloride, 5 mM potassium chloride, 0.1 mM magnesium chloride, 0.05 mM calcium chloride, 5.5 mM glucose, 15 mM HEPES and 1 mg/ml BSA (5). Aggregometry and serotouin secretion: PRP or GFP were incubated for 15 min with 0.05 PCilml 14Cserotonin (Amersham). Platelet aggregation was monitored by a Payton dual channel aggregometer and triplicate 0.1 ml aliquots were taken after maximum aggregation was reached. These were immediately mixed with 0.9 ml of ice-cold PBS plus 10 mM EGTA and 0.2% glutaraldehyde to stop release. The platelets were removed by centrifugation for 3 min at 10,008 x g before taking 0.5 ml aliquots of the supematant for scintillation counting of released t4C-serotonin secretion. Similarly diluted aliquots, which were not centrifuged, were counted for total label. Control cpm, which were less than 10% of the total cpm, were subtracted from both the sample cpm and total cpm. Percent release was calculated by dividing the corrected sample cpm by total cpm and multiplying by 100 to calculate % release. Cytoplasmic calcium and PI-Imeasurements: Platelets in plasma were loaded for 15 min with 2 PM of the the calcium specific dye Fura- (acetoxymethyl ester; Molecular Probes) or 10 PM of the proton specific dye BCECF (acetoxymethyl ester; Molecular Probes), and then gel-filtered at room temperature. Fura- fluorescence emission at 510 nm was monitored with a Shimadzu fluorometer in plastic cuvettes thermostated at 37OC, under constant stirring. Excitation fluorescences were 335 and 360 nm (isobestic point). BCECF fluorescence was similarly monitored for emission at 530 nm with 495 nm excitation. Platelet protein phosphorylation: PRP was chilled on ice for 15 min before adding one-half volume of 4OC CCD buffer and spining at 900 x g for 10 min at 4OC. The
Vol. 66, Nos. 213
THROMBOXANE
A2 AND AZIDE
103
supematant was decanted and the platelets resuspended in the modified Tangen-HEPES-BSA buffer. The platelet suspension was warmed to 37OC, incubated with 0.5 mCi/ml 32Porthophosphate (New England Nuclear) for 45 min, chilled again and gel-filtered at 4OC. Platelets were rewarmed at 37OC for at least 30 min prior to addition of azide and U46619. Aliquots (50 ~1) were taken at indicated times, immediately denatured with 25 ~1 of 6% sodium dodecyl sulfate, 6% &mercaptoeJhanol, 15 mM EGTA, 30% glycerol, 0.03% bromophenol blue, 150 mM Tris-HCl, pH 6.8, electrophoresed and autoradiographed to visualize 32P-labeled peptides, as previously described (6). Label associated with the 20 kDa myosin light chain (p20) and a 47 kDa cytosolic phosphoprotein of unknown function (p47) was quantified by a Zeineh laser densitometer with adjustable slit width set to cover the entire width of the gel lane. The peak area associated with the p20 or p47 bands was quantified on an interfaced Shimadzu CR3-A recording integrator.
RESULTS
Effect of sodium azide on a set of platelet activators. In a first set of experiments, platelets were incubated with relatively high concentrations (300 PM) of NaN3 before addition of different platelet activators. Thrombin was tested in GFP to avoid the formation of a thrombus. PMA was studied in parallel to thrombin whereas all other agonists were tested in PRP.
TABLE I Effect of NaNs on Serotonin Release Induced by Different Agonists
Agonist:
Serotonin - NaN3
ADP (10 l.rM) Arachidonic acid (60 PM) U46619 (1 //M) PMA (1 NM) Collagen (10 pg/ml) Collagen (0.5 pg/ml) Thrombin (1 U/ml) Thrombin (0.1 U/ml)
44*7 46k5 24klO 14zt2 82k3 46k9 89*5 39*26
14*5 74k8 14k12 71*9
LOW CONCENTRATIONS THROMBOXANE
Eric Rubiiein,
OF SODIUM AZIDE SPECIFICALLY
INHIBIT A
A2 PATHWAY IN HUMAN PLATELETS.
Irene Ursa and Roger C. Carroll
Department of Medical Biology, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville TN 37920, USA (Received 10.7.1991; accepted in revised form 4.12.1991 by Editor J. Soria) (Received by Executive Editorial Office 20.2.1992)
ABSTRACT Sodium azide completely inhibits the serotonin release induced by ADP, arachidonic acid and the thromboxane A2 mimetic U46619, but does npt have any effect on the activation by PMA. Collagen and thrombin are inhibited when used at low concentrations, but not at high concentration. This pa tern of activation suggests that the inhibition by azide is not a metabolic in J ‘bition. The antagonism of U46619-induced secretion was further studied and was shown to be non-competitive. It is selective for certain components of the U46619 stimulus-response coupling: aggregation, serotonin secretion and the activation of protein kinase C are completely or almost completely inhibited by 300 PM sodium azide. Shape change, calcium elevation, cyto lasmic alkalinization and phosphorylation of myosin light chain are only k ‘ally modified. This suggests that azide may specifically inhibit one of the different forms of thromboxane A2 receptors present in platelets.
INTRODUCTION
Sodium azide (NaN3) is a classical inhibitor of cytochrome oxidase and thereby inhibits mitochondrial respiratory energy production. Another effect of this compound on energetic metabolism is inhibition of ATPase, a coupling factor of oxidative phosphorylation, in procarytic and eucaryotic cells (1). Sodium azide is also an activator of guanylate cyclase in brain, liver (2) and platelets (3). In this latter system, it was concluded that cGMP and agents Key words: Platelet activation; azide; thromboxanes. 101
102
THROMBOXANE
A2 AND AZIDE
Vol. 66, Nos. 213
increasing cGMP concentration, including azide, did not affect platelet activation by thrombin. The effect of sodium azide on platelet aggregation was further studied by Stibbe and Holmsen (4). Sodium azide was shown to partklly inhibit the first wave of aggregation induced by ADP, epinephrine (in the presence of indomethacin), the ionophore A 23 187, and thrombin (in presence of indomethacin and ADP scavenger). Serotonin secretion induced by ADP, epinephrine and collagen was also inhibited. This effect was not due to an inhibition of energy metabolism. We show here that sodium azide inhibits specific pathways and propose a mechanism of platelet inactivation by azide.
Platelet preparation: Blood was collected from volunteers giving informed consent who denied taking any medication for the previous two weeks. The blood was drawn from the antecubital vein into syringes containing 0.1 volume of CCD (100 mM sodium citrate and 130 mM glucose, pH 6.5). The blood was centrifuged 3 min at 1000 x g at room temperature to separate the platelet-rich plasma (PRP). For certain experiments, platelets were gel-filtered (GFP) on a Sepharose-2B column (Sigma) equilibrated with a modified Tangen-HEPES-BSA buffer @H 7.4) containing 145 mM sodium chloride, 5 mM potassium chloride, 0.1 mM magnesium chloride, 0.05 mM calcium chloride, 5.5 mM glucose, 15 mM HEPES and 1 mg/ml BSA (5). Aggregometry and serotouin secretion: PRP or GFP were incubated for 15 min with 0.05 PCilml 14Cserotonin (Amersham). Platelet aggregation was monitored by a Payton dual channel aggregometer and triplicate 0.1 ml aliquots were taken after maximum aggregation was reached. These were immediately mixed with 0.9 ml of ice-cold PBS plus 10 mM EGTA and 0.2% glutaraldehyde to stop release. The platelets were removed by centrifugation for 3 min at 10,008 x g before taking 0.5 ml aliquots of the supematant for scintillation counting of released t4C-serotonin secretion. Similarly diluted aliquots, which were not centrifuged, were counted for total label. Control cpm, which were less than 10% of the total cpm, were subtracted from both the sample cpm and total cpm. Percent release was calculated by dividing the corrected sample cpm by total cpm and multiplying by 100 to calculate % release. Cytoplasmic calcium and PI-Imeasurements: Platelets in plasma were loaded for 15 min with 2 PM of the the calcium specific dye Fura- (acetoxymethyl ester; Molecular Probes) or 10 PM of the proton specific dye BCECF (acetoxymethyl ester; Molecular Probes), and then gel-filtered at room temperature. Fura- fluorescence emission at 510 nm was monitored with a Shimadzu fluorometer in plastic cuvettes thermostated at 37OC, under constant stirring. Excitation fluorescences were 335 and 360 nm (isobestic point). BCECF fluorescence was similarly monitored for emission at 530 nm with 495 nm excitation. Platelet protein phosphorylation: PRP was chilled on ice for 15 min before adding one-half volume of 4OC CCD buffer and spining at 900 x g for 10 min at 4OC. The
Vol. 66, Nos. 213
THROMBOXANE
A2 AND AZIDE
103
supematant was decanted and the platelets resuspended in the modified Tangen-HEPES-BSA buffer. The platelet suspension was warmed to 37OC, incubated with 0.5 mCi/ml 32Porthophosphate (New England Nuclear) for 45 min, chilled again and gel-filtered at 4OC. Platelets were rewarmed at 37OC for at least 30 min prior to addition of azide and U46619. Aliquots (50 ~1) were taken at indicated times, immediately denatured with 25 ~1 of 6% sodium dodecyl sulfate, 6% &mercaptoeJhanol, 15 mM EGTA, 30% glycerol, 0.03% bromophenol blue, 150 mM Tris-HCl, pH 6.8, electrophoresed and autoradiographed to visualize 32P-labeled peptides, as previously described (6). Label associated with the 20 kDa myosin light chain (p20) and a 47 kDa cytosolic phosphoprotein of unknown function (p47) was quantified by a Zeineh laser densitometer with adjustable slit width set to cover the entire width of the gel lane. The peak area associated with the p20 or p47 bands was quantified on an interfaced Shimadzu CR3-A recording integrator.
RESULTS
Effect of sodium azide on a set of platelet activators. In a first set of experiments, platelets were incubated with relatively high concentrations (300 PM) of NaN3 before addition of different platelet activators. Thrombin was tested in GFP to avoid the formation of a thrombus. PMA was studied in parallel to thrombin whereas all other agonists were tested in PRP.
TABLE I Effect of NaNs on Serotonin Release Induced by Different Agonists
Agonist:
Serotonin - NaN3
ADP (10 l.rM) Arachidonic acid (60 PM) U46619 (1 //M) PMA (1 NM) Collagen (10 pg/ml) Collagen (0.5 pg/ml) Thrombin (1 U/ml) Thrombin (0.1 U/ml)
44*7 46k5 24klO 14zt2 82k3 46k9 89*5 39*26
14*5 74k8 14k12 71*9
