Platelets ( 2002 ) 13, 479– 485
Antiplatelet activity of carvedilol in comparison to propranolol M. Petr´õ kov´a, V. Jancinov´ a, R. Nos´al’, M. M´ajekov´a, E. Danihelov´a a
The non-selective vasodilating b-blocker carvedilol was found to inhibit platelet aggregation as well as thromboxane B2 formation more effectively than propranolol. The antiaggregatory activity of carvedilol decreased, depending on the stimulus used, in the following rank order of potency ( in parentheses the respective mean inhibitory concentrations of carvedilol and propranolol are given in m mol/ l): PMA ( 19 and 34) > thrombin (55 and 77) > Ca2+ -ionophore A23187 ( 58 and 81) > epinephrine ( 86 and 118 ). However, aggregation of platelets activated with ADP was not affected by carvedilol in concentrations up to 100 m mol/ l. In platelets stimulated with thrombin, carvedilol ( 10 m mol/ l) reduced thromboxane B2 formation by 64%, whereas propranolol was ineffective at this concentration. Moreover, A23187-induced formation of thromboxane B2 , not affected by propranolol, was completely blocked by 100 m mol/ l carvedilol. In comparison to propranolol, the molecule of carvedilol is more lipophilic and possesses lower dipole moment and higher molar refractivity, thus penetrating into platelet membranes readily and in large quantities. The antiplatelet effect was assumed to result from interactions of carvedilol with membrane macromolecules ( phospholipids, ion channels, enzymes, etc.) rather than from blockade of a- and b-adrenergic receptors.
Introduction Activated blood platelets were found to participate substantially in the genesis of many pathological states. Among them cardio- and cerebrovascular diseases and their complications are of utmost importance.1 Several drugs from different pharmacological groups can correct increased platelet aggregability,2– 4 e.g., b-blockers whose ability to inhibit aggregation was confirmed under in vitro conditions by many authors.5–11 Above all, propranolol was studied in detail for its antiplatelet effect. Extensive series of in vitro studies12–17 demonstrated reduced aggregation, thromboxane A2 formation, as well as decreased serotonin uptake and liberation in the presence of propranolol. These effects Margita Petr´õ kov´a, Viera Jancinov a´, Rado Nos´al’, Magdal´ena M´ajea kov a´, Institute of Experimental Pharmacology, Slovak Academy of Sciences, D´ubravsk a´ cesta 9, 842 16 Bratislava; Edita Danihelov´a, Institute of Haematology and Transfusiology, Partiz´anska 4, 811 03 Bratislava, Slovak Republic. Correspondence to: Margita Petr´õ kov a´ , Institute of Experimental Pharmacology, Slovak Academy of Sciences, D´ubravsk a´ cesta 9, 842 16 Bratislava. Fax: + 421-2-54-775-928; E-mail:
[email protected] k
were found to occur independently of b-adrenergic receptor blockade and result from the structure of propranolol and from the interference of this drug with microenvironment of platelet membranes.18 Carvedilol is a non-selective b-blocker of the new generation, possessing a vasodilating effect mediated by blockade of a1-adrenergic receptors. Cardioprotection, observed both experimentally19– 21 and in patients,22,23 was ascribed to the anti-oxidant activity of carvedilol and of its metabolites. Despite the fact that carvedilol is successfully used in the therapy of cardio- and cerebrovascular diseases, i.e., in pathological states accompanied with platelet hyperreactivity, information concerning its antiplatelet activity is rather scarce.24 The aim of this study was to analyse the in vitro effect of carvedilol on aggregation of human blood platelets stimulated with five different stimuli, to compare this effect with the effect of propranolol, and to find out whether inhibited aggregation was accompanied with reduced thromboxane B2 formation. Moreover, some physico-chemical parameters characterising affinity of carvedilol to platelet membranes – partition coefficient,
ISSN 0953-7104 print/ ISSN 1369-1635 online/ 02/ 080479-07 © 2002 Taylor & Francis Ltd DOI: 10.1080/ 0953710021000057848
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ANTIPLATELET ACTIVITY OF CARVEDILOL
Figure 1. moment.
Chemical formulas and optimal structures of carvedilol and propranolol. Marks -/+ denotes the orientation of dipole
dipole moment and molar refractivity – were calculated and compared with propranolol. Materials and methods Materials Adenosine-5 9-diphosphate ( ADP) was from Serva ( Germany), Ca2 + -ionophore A23187 (A23187 ) from Calbiochem (Switzerland ), epinephrine bitartrate salt (EPI) and 4b-phorbol-12 b-myristate- a13-acetate ( PMA) from Sigma– Aldrich Chemie ( Germany ) and human thrombin ( THROM) from Imuna ( Slovakia). Carvedilol ( 1-[carbazolyl-( 4)-oxy]-3-[ ( 2-methoxy-phenoxyethyl ) amino]propanol, CAR) was a kind gift from Dr. Sven Hauptmann, Roche Mannheim (Germany ), propranolol ( 1-isopropylamino-3- ( 1-naphthyloxy )-2-propanol, PRO) was obtained from ICI (England ) and thromboxane B2 kits from the Institute of Radioizotopes ( Hungary ). All other chemicals of analytical grade were from available commercial sources. Tyrode’s solution ( pH 7.4) contained 136.9 mmol/ l NaCl, 11.9 mmol NaHCO3 , 0.4 mmol/ l NaH2 PO4·2H2 O, 1 mmol/ l MgCl2·6H2 O and 5.6 mmol/ l glucose. Platelet preparation Platelets were isolated as described earlier.10 In brief, fresh blood of healthy volunteers was anticoagulated
with 3.8% trisodium citrate ( ratio blood:citrate, 9:1) and centrifuged at 200´ g and 22°C for 15 min. Platelet-rich plasma ( PRP) was removed and an aliquot was recentrifuged for 30 min at 980´ g to obtain platelet-poor plasma ( PPP). After adjustment of platelet count to 2´ 105/ ml by autologous PPP ( Thrombocounter C, Coulter Electronics, England), PRP was used for the study of ADP- or epinephrine-stimulated aggregation. For all other studies, isolated platelets were prepared as follows. PRP was mixed with a solution containing 4.5% citric acid and 6.6% dextrose ( 50 ml/ ml) and centrifuged at 980´ g for 10 min. Platelets were resusupended in an equal volume of Tyrode’s solution containing 5.4 mmol/ l ethylenediamine tetraacetic acid ( EDTA), pH 6.5. After 10 min stabilisation at room temperature, the suspension was centrifuged for 6 min at 980´ g and platelets were resuspended in the same buffer free of EDTA ( pH 7.4) to obtain 2´ 105 ( aggregation ) or 1´ 104 ( thromboxane ) platelets per 1 ml. Platelet aggregation Platelet aggregation was measured turbidimetrically,10 in a dual channel aggregometer (Chrono-log aggrometer, USA). After 2 min stabilisation at 37°C, platelets ( 450 ml) were incubated with b-blocker tested ( 20 ml) for 30 s. Aggregation was initiated by addition of 20 ml epinephrine ( final concentration 4 mmol/ l), ADP
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Figure 2. Inhibition of phorbolmyristate acetate- (PMA), thrombin- (THROM) or Ca2+-ionophore A23187-stimulated platelet aggregation by carvedilol and propranolol. Mean ± SEM, n = 6, *P thrombin > Ca2 + -ionophore A23187 > epinephrine. In accordance with the findings of Gasser et al.,24 carvedilol was least effective in platelets stimulated with ADP – in concentrations up to 100 mmol/ l no significant inhibition of aggregation was found. This is indicative of three facts: ( i) the mechanism of platelet activation by ADP differs from that of other stimuli used,27 ( ii) carvedilol, even at high concentra-
Figure 4. Thromboxane B2 (TXB2 ) formation in platelets stimulated with thrombin or Ca2+-ionophore A23187. Effect of carvedilol (striped columns) and propranolol (open columns). Mean±SEM, n = 6, **P
