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Aggregation of human blood platelets in the presence of the pyridoindole stobadine
Life Scimce.$Vol. 65, Nos. 18119,pp. 1983-1986,1999 Copyright0 1999Ekevier ScienceInc. Printedin theUSA. All rightsreserved 0024_3205/99/fsee hmt matter
PIISOO24-3205(99)00460-9
ELSEVIER
AGGREGATION
OF HUMAN BLOOD PLATELETS IN THE PRESENCE OF THE PYFUDOINDOLE STOBADINE
Viera JanGnova, Rado No&l and *Edita Danihelova Institute of Experimental Pharmacology, Slovak Academy of Sciences and *Institute for Hematology and Transfkiology, Bratislava, Slovak Republic
Summary The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadii for 30 s inhibited StimuIated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 umoyl (adrenaline), 200 umol/l (ADP), and 1,000 umol/l (PMA). Aggregation induced with thrombin or Ca*+-ionophore A23 187 was not changed in the presence of stobadine even in the concentration of 1,000 umol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 umol/l) or prolonged onset of its second phase (10 umol/l). The presented experknts showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity. Key Words:
stobadine, human blood platelets, inhibition of platelet aggregation
Stobadiie is an original cardioprotective drug with antihypoxic and antiarrhythmic effects, which has undergone phase II clinical trial [l]. Moreover, it strikingly prolonged survival of dogs exposed to severe experimental brain ischemia. The latter effect was supposed to result not only from the direct protection of nervous structures but also from eliminated thrombotization of capillaries observed in the presence of stobadine [2]. It was hypothesized that the antithrombotic activity may result from its protective effect on blood vessels, endothelium and erythrocytes, counteracting the destructive action of reactive oxygen species. As blood platelets and especially platelet aggregates can participate substantiahy in thrombus formation, the effect of stobadine on human platelet aggregation was investigated. To characterize the site of stobadine action more precisely, its effect on platelet aggregation induced by 5 stimuli and initiated via difkrent pathways was compared. Methods Suspensions of human blood platelets were isolated as described previously [3]. Platelet aggregation was measured by the turbidiitric method in a dual channel aggregometer (Chrono-log aggrometer, USA). After 2-min stabilization at 37°C the platelets were incubated with stobadine for 30 s. Aggregation, initiated by addition of adrenaline (4 umol/l), ADP
1984
Stobadine and Human Platelet Aggregation
Vol. 65, Nos. 18/19, 1999
(2 umol/l), PMA (50 nmoYl), thrombin (0.05 NIH U/ml) or Ca”-ionophore A23 187 (1.8 umoVl), was recorded for 2.5 min by means of aggregation curves. In separate experiments, stobadine was added 30 or 120 s after platelet stimulation. Aggregation was measured as the amplitude of the aggregation curve; the differences between aggregation of control and stobadine-treated platelets (run in parallel) were evaluated by Student’s t-test. Materials ADP (adenosine-S-diphosphate): Serva, Germany; human thrombin: Imuna, Slovak Republic; PMA (phorbol 12-my&ate 13-acetate) and adrenaline (epinephrine bitartrate): Sigma, USA and calcium ionophore A23187: Calbiochem, Switzerland. Fresh blood was obtained at the blood bank by venepuncture from healthy male donors (20 - 50 years) who had not received any medication for at least 7 days. Stobadine, (-)-cis-2,8-Dimethyl-2,3,4,4a,5,9b-hexahydro-lHpyrido[4,3-blindole dihydrochloride, was developed at the Institute of Experimental Pharmacology SAS, Bratislava. Results The 30-second pretreatment of human platelets with stobadine in the concentration range from 1 10 pmol/l significantly inhibited adrenaline-induced aggregation (left part of Table 1). Amplitudes of both the first and second phase of aggregation curves were decreased and the onset of the second phase was signikantly prolonged. Application of stobadine 30 seconds after adrenaline resulted in terminated aggregation (100 and 1,000 umol/l) or in prolonged onset of its second phase (10 umol/l). However, when stobadine was added in the 120th second of aggregation, only the 1,000 urnoF concentration was inhibitory and induced significant desaggregation.
Dose-dependent
inhibition
TABLE 1 of human platelet aggregation
by stobadine
ST0 = stobadine, ADR = adrenaline, ADP = adenosine-5’-diphosphate, PMA = phorbol 12myristate13-acetate, THR = thrombin.Mean values from lo-12 measurements (6 donors) f SEM are given.Significant changes in comparison to the control (0 umoVl), *p < 0.05, **p < 0.01.
The stimulatory effect of ADP and PMA was inhibited to a lesser extent and at 20 - 100 times higher concentrations of stobadine than the effect of adrenaline; aggregation induced with thrombin or A23 187 was not changed in the presence of stobadine.
Vol. 65, Nos. 18/19, 1999
Stobadine and Human Platelet Aggregation
1985
Discussion Comparison of the effect of stobadine on platelets activated with 5 diierent stimuli showed preferential inhibition of adrenaline-induced aggregation, where not only pretreatment of platelets with stobadine but also its addition during aggregation was effective. Thus the interference of stobadine with adrenaline-stimulated processes, as listed in [4], may account for the mechanism of its antiaggregatory effect. Interaction of stobadme with platelet a,-adrenergic receptors was indicated by relatively low effective concentrations of the drug, by the rapid onset of its effect, as well as by the abii of stobaclme to compete with adrenaline during the initial steps of aggregation Moreover, a,-adrenolytic properties of stobadme were observed in canine arteries [5]. Inhibition ofprotein kinase C by stobadme may be due to its ability to antagonize aggregation induced with a direct activator of this enzyme, i.e. with PMA. On the other hand, interference of stobadme with processes initiated by calcium mobilization seems to be less likely, as Ca*‘ionophore A23187-stimulated aggregation was not affected in the presence of this drug. The abii of stobadine to inhibit adrenaline- as well as ADP-stimulated aggregation indicates its interference with a mechanism actively involved in both types of activation, with inhibition of stimulus-induced inhibition of adenylate cyclase as an eligible candidate. Our preliminary experiments showed decreased thromboxane B, formation in the presence of 1 mmoV1 stobadine, indicating inhibition of arachidonic acid cascade, though only at high stobadine concentrations. The non-specific membrane-stabilizing effect and destruction of platelet integriw do not seem to be involved in the antiaggregatory effect of stobadme since tbrombin- and A23187-induced aggregation occurred normally in the presence of the highest stobadine concentration used. In view of the fact that reactive oxygen species stimulate blood platelets, assistance of the antioxidant properties of stobadme in its antiaggregatory effect cannot be excluded, although the formation of oxygen free radicals was found in platelets stimulated with thrombm rather than with adrenaline [6]. The present study, a pilot direct analysis of stobadine effects on human cells, showed the special abii of this drug to inhibit platelet aggregation induced with adrenaline. In comparison to other cationic amphiphilic drugs, stobadine was 2 - 50 times more effective than the H,-histamine antagonist dithiaden [3] and the antimalarial drug chloroquine [7]. Its inhibitory effect started at 1 pmol/l concentration, which corresponds well with the level of stobadme found in volunteer sera after a single dose of 2.5 rngkg [8]. Moreover, the experiments suggest that in vivo stobadine would be more effective in the initial steps of platelet activation (e.g. during stress-induced adrenaline release or after discharge of ADP from erythrocytes) rather than in platelet aggregation associated with activation of coagulation proteins and with thrombin formation. The direct inhibitory effect of stobadine on blood platelets, along with its protective effect on blood vessels, the endothelium and erythrocytes, may participate in the antithrombotic effect of stobadine observed during experimental ischemia. Acknowledgement This work was supported in part by the research grant S.G.A. 97/5305/l 52.
1. 2. 3. 4. 5.
References L.BENES and S.STOLC, Drugs ofthe Future 14 135-137 (1989). M.POMFY, A.NICdLK, J.MOJ&S, J.H&XA and L.BENES, Mol. Chem. Neuropathol. 115-122 (1995). R.NOS&.Z, V.JANCINOVA and E.DANIHELOVA, Platelets 8 175- 180 (1997). D.BLOCKMANS, H.DECKMYN and J.VERMYLEN, Blood Rev. 9 143-156 (1995). R.SOTNIKOV% P.GIBALA and J.DI&IAL, Bra&l. lek. Listy 84 536-541 (1985).
25
1986
6. 7. 8.
Stobadine and Human Platelet Aggregation
Vol. 65, Nos. 18/19, 1999
L.IULIANO, A.R.COLAVITA, R.LEO, D.PRATICO and F.VIOLI, Free Radical Biol. Med. 22 999- 1006 ( 1997). V.JAN&NOVA, R.NOS& and PMATEJKA, Scripta medica 70 Suppl. 4 65-68 (1997). L.sOLTlb, Z.Kh.LLAY, &BEZEK and V.FEDELESOVk Biopharm. Drug Disposition 12 29-35 (1991).
PIISOO24-3205(99)00460-9
ELSEVIER
AGGREGATION
OF HUMAN BLOOD PLATELETS IN THE PRESENCE OF THE PYFUDOINDOLE STOBADINE
Viera JanGnova, Rado No&l and *Edita Danihelova Institute of Experimental Pharmacology, Slovak Academy of Sciences and *Institute for Hematology and Transfkiology, Bratislava, Slovak Republic
Summary The antiarrhythmic and cardioprotective drug stobadine, possessing antioxidant and neuroprotective properties, was studied as to its in vitro effect on aggregation of human blood platelets. Pretreatment of platelets with stobadii for 30 s inhibited StimuIated platelet aggregation in a dose-dependent way. Depending on the aggregation stimulus used, the minimal effective concentrations of the drug were 1 umoyl (adrenaline), 200 umol/l (ADP), and 1,000 umol/l (PMA). Aggregation induced with thrombin or Ca*+-ionophore A23 187 was not changed in the presence of stobadine even in the concentration of 1,000 umol/l. Addition of stobadine 30 s after adrenaline was also effective and terminated aggregation (100 and 1,000 umol/l) or prolonged onset of its second phase (10 umol/l). The presented experknts showed stobadine as a potent inhibitor of adrenaline-induced aggregation, indicating its involvement in the observed antithrombotic and cytoprotective activity. Key Words:
stobadine, human blood platelets, inhibition of platelet aggregation
Stobadiie is an original cardioprotective drug with antihypoxic and antiarrhythmic effects, which has undergone phase II clinical trial [l]. Moreover, it strikingly prolonged survival of dogs exposed to severe experimental brain ischemia. The latter effect was supposed to result not only from the direct protection of nervous structures but also from eliminated thrombotization of capillaries observed in the presence of stobadine [2]. It was hypothesized that the antithrombotic activity may result from its protective effect on blood vessels, endothelium and erythrocytes, counteracting the destructive action of reactive oxygen species. As blood platelets and especially platelet aggregates can participate substantiahy in thrombus formation, the effect of stobadine on human platelet aggregation was investigated. To characterize the site of stobadine action more precisely, its effect on platelet aggregation induced by 5 stimuli and initiated via difkrent pathways was compared. Methods Suspensions of human blood platelets were isolated as described previously [3]. Platelet aggregation was measured by the turbidiitric method in a dual channel aggregometer (Chrono-log aggrometer, USA). After 2-min stabilization at 37°C the platelets were incubated with stobadine for 30 s. Aggregation, initiated by addition of adrenaline (4 umol/l), ADP
1984
Stobadine and Human Platelet Aggregation
Vol. 65, Nos. 18/19, 1999
(2 umol/l), PMA (50 nmoYl), thrombin (0.05 NIH U/ml) or Ca”-ionophore A23 187 (1.8 umoVl), was recorded for 2.5 min by means of aggregation curves. In separate experiments, stobadine was added 30 or 120 s after platelet stimulation. Aggregation was measured as the amplitude of the aggregation curve; the differences between aggregation of control and stobadine-treated platelets (run in parallel) were evaluated by Student’s t-test. Materials ADP (adenosine-S-diphosphate): Serva, Germany; human thrombin: Imuna, Slovak Republic; PMA (phorbol 12-my&ate 13-acetate) and adrenaline (epinephrine bitartrate): Sigma, USA and calcium ionophore A23187: Calbiochem, Switzerland. Fresh blood was obtained at the blood bank by venepuncture from healthy male donors (20 - 50 years) who had not received any medication for at least 7 days. Stobadine, (-)-cis-2,8-Dimethyl-2,3,4,4a,5,9b-hexahydro-lHpyrido[4,3-blindole dihydrochloride, was developed at the Institute of Experimental Pharmacology SAS, Bratislava. Results The 30-second pretreatment of human platelets with stobadine in the concentration range from 1 10 pmol/l significantly inhibited adrenaline-induced aggregation (left part of Table 1). Amplitudes of both the first and second phase of aggregation curves were decreased and the onset of the second phase was signikantly prolonged. Application of stobadine 30 seconds after adrenaline resulted in terminated aggregation (100 and 1,000 umol/l) or in prolonged onset of its second phase (10 umol/l). However, when stobadine was added in the 120th second of aggregation, only the 1,000 urnoF concentration was inhibitory and induced significant desaggregation.
Dose-dependent
inhibition
TABLE 1 of human platelet aggregation
by stobadine
ST0 = stobadine, ADR = adrenaline, ADP = adenosine-5’-diphosphate, PMA = phorbol 12myristate13-acetate, THR = thrombin.Mean values from lo-12 measurements (6 donors) f SEM are given.Significant changes in comparison to the control (0 umoVl), *p < 0.05, **p < 0.01.
The stimulatory effect of ADP and PMA was inhibited to a lesser extent and at 20 - 100 times higher concentrations of stobadine than the effect of adrenaline; aggregation induced with thrombin or A23 187 was not changed in the presence of stobadine.
Vol. 65, Nos. 18/19, 1999
Stobadine and Human Platelet Aggregation
1985
Discussion Comparison of the effect of stobadine on platelets activated with 5 diierent stimuli showed preferential inhibition of adrenaline-induced aggregation, where not only pretreatment of platelets with stobadine but also its addition during aggregation was effective. Thus the interference of stobadine with adrenaline-stimulated processes, as listed in [4], may account for the mechanism of its antiaggregatory effect. Interaction of stobadme with platelet a,-adrenergic receptors was indicated by relatively low effective concentrations of the drug, by the rapid onset of its effect, as well as by the abii of stobaclme to compete with adrenaline during the initial steps of aggregation Moreover, a,-adrenolytic properties of stobadme were observed in canine arteries [5]. Inhibition ofprotein kinase C by stobadme may be due to its ability to antagonize aggregation induced with a direct activator of this enzyme, i.e. with PMA. On the other hand, interference of stobadme with processes initiated by calcium mobilization seems to be less likely, as Ca*‘ionophore A23187-stimulated aggregation was not affected in the presence of this drug. The abii of stobadine to inhibit adrenaline- as well as ADP-stimulated aggregation indicates its interference with a mechanism actively involved in both types of activation, with inhibition of stimulus-induced inhibition of adenylate cyclase as an eligible candidate. Our preliminary experiments showed decreased thromboxane B, formation in the presence of 1 mmoV1 stobadine, indicating inhibition of arachidonic acid cascade, though only at high stobadine concentrations. The non-specific membrane-stabilizing effect and destruction of platelet integriw do not seem to be involved in the antiaggregatory effect of stobadme since tbrombin- and A23187-induced aggregation occurred normally in the presence of the highest stobadine concentration used. In view of the fact that reactive oxygen species stimulate blood platelets, assistance of the antioxidant properties of stobadme in its antiaggregatory effect cannot be excluded, although the formation of oxygen free radicals was found in platelets stimulated with thrombm rather than with adrenaline [6]. The present study, a pilot direct analysis of stobadine effects on human cells, showed the special abii of this drug to inhibit platelet aggregation induced with adrenaline. In comparison to other cationic amphiphilic drugs, stobadine was 2 - 50 times more effective than the H,-histamine antagonist dithiaden [3] and the antimalarial drug chloroquine [7]. Its inhibitory effect started at 1 pmol/l concentration, which corresponds well with the level of stobadme found in volunteer sera after a single dose of 2.5 rngkg [8]. Moreover, the experiments suggest that in vivo stobadine would be more effective in the initial steps of platelet activation (e.g. during stress-induced adrenaline release or after discharge of ADP from erythrocytes) rather than in platelet aggregation associated with activation of coagulation proteins and with thrombin formation. The direct inhibitory effect of stobadine on blood platelets, along with its protective effect on blood vessels, the endothelium and erythrocytes, may participate in the antithrombotic effect of stobadine observed during experimental ischemia. Acknowledgement This work was supported in part by the research grant S.G.A. 97/5305/l 52.
1. 2. 3. 4. 5.
References L.BENES and S.STOLC, Drugs ofthe Future 14 135-137 (1989). M.POMFY, A.NICdLK, J.MOJ&S, J.H&XA and L.BENES, Mol. Chem. Neuropathol. 115-122 (1995). R.NOS&.Z, V.JANCINOVA and E.DANIHELOVA, Platelets 8 175- 180 (1997). D.BLOCKMANS, H.DECKMYN and J.VERMYLEN, Blood Rev. 9 143-156 (1995). R.SOTNIKOV% P.GIBALA and J.DI&IAL, Bra&l. lek. Listy 84 536-541 (1985).
25
1986
6. 7. 8.
Stobadine and Human Platelet Aggregation
Vol. 65, Nos. 18/19, 1999
L.IULIANO, A.R.COLAVITA, R.LEO, D.PRATICO and F.VIOLI, Free Radical Biol. Med. 22 999- 1006 ( 1997). V.JAN&NOVA, R.NOS& and PMATEJKA, Scripta medica 70 Suppl. 4 65-68 (1997). L.sOLTlb, Z.Kh.LLAY, &BEZEK and V.FEDELESOVk Biopharm. Drug Disposition 12 29-35 (1991).