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Chloroquine
Thrombosis Research 98 (2000) 411–421
ORIGINAL ARTICLE
Chloroquine: A Multipotent Inhibitor of Human Platelets in Vitro Rado Nosa´l’1, Viera Jancˇinova´1 and Edita Danihelova´2 Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava; 2Institute of Haematology and Transfusiology, Bratislava, Slovak Republic.
1
(Received 28 September 1999 by Editor J.E. Dyr; revised/accepted 6 January 2000)
Abstract Chloroquine inhibited human platelet aggregation in vitro both at receptor- and nonreceptor-operated stimuli. The inhibition was dose-dependent, recorded on isolated platelets as well as in plateletrich plasma, and followed the rank order of stimuli: adrenaline (second phase)⬎phorbol 12-myristate 13 acetate⬎adenosine diphosphate⬎adrenaline (first phase)⬎thrombin⬎calcium ionophore A23187. In thrombin-activated platelets, chloroquine decreased in a dose-dependent manner phospholipase A2-induced arachidonic acid liberation from membrane phospholipids, malondialdehyde formation (a marker of membrane phospholipid peroxidation), and thromboxane generation, considered the most potent autoaggregatory agent. Chloroquine only slightly altered the arachidonic acid cascade of platelets stimulated with A23187 and phorbol 12-myristate 13 acetate. Histamine formation and liberation induced with thrombin and A23187 were not affected by chloroquine. On the other hand, thrombin-stimulated serotonin seAbbreviations: CQ, chloroquine; 5-HT, 5-hydroxytryptamine; ADP, adenosine diphosphate; PRP, platelet-rich plasma; PPP, platelet-poor plasma; EDTA, ethylenediaminetetraacetic acid disodium salt; PMA, phorbol 12-myristate 13 acetate; [3H]AA, [3H] arachidonic acid; MDA, malondialdehyde; TXB2, thromboxane B2; RIA, radio immunoassay; SDS, sodium dodecylsulphate; GP, glycoprotein. Corresponding author: Dr. Rado Nosa´l’, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska 2, 842 16 Bratislava, Slovak Republic. Tel: ⫹421 (7) 59410 662; E-mail: ⬍[email protected]⬎.
cretion was significantly decreased with chloroquine in the concentration of 10 mol/L. This indicated that chloroquine might interfere with stimulated secretion from platelets. The results suggest that chloroquine inhibited activated platelets: first, intracellularly; second, in a close relationship to the intraplatelet Ca2⫹ mobile pool; and third, most probably at the site of platelet phospholipase A2 activation. 2000 Elsevier Science Ltd. All rights reserved. Key Words: Chloroquine; Platelets; Aggregation; Arachidonate cascade; Histamine; Serotonin
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hloroquine (CQ), one of the most potent antimalarial drugs with anti-inflammatory and immunomodulatory properties, has been intensively studied, and its effect on blood platelets has been investigated for more than 20 years [1–3]. As a pharmacological tool and model for drugs with a cationic amphiphilic structure similar to other antimalarials, CQ was used to elucidate nonreceptor mediated 5-hydroxytryptamine (5HT) liberation from platelets with concomitant accumulation of the drug in dense bodies [4]. CQ possesses a high affinity for human platelets both in vitro and in vivo with high dose-dependent accumulation [5,6]. An antiplatelet effect of CQ was described in normal subjects as well as in patients infected with malaria both in vitro and ex vivo [7,8]. The affinity of CQ to rat platelets in vitro resulted in a dose-dependent inhibition of aggregation stimulated with agonists of different signal
0049-3848/00 $–see front matter 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00200-0
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transduction pathway [9]. Of these, the CQinduced inhibition of arachidonic acid cascade was demonstrated to be the essential step of arachidonic acid liberation (inhibition of phospholipase A2), malondialdehyde formation (corresponding to membrane phospholipid peroxidation), and thromboxane generation (the end product of the arachidonic acid cascade) [10]. These findings supported the suggested high-affinity of CQ for negatively charged membrane phospholipids and positioned CQ as a selective inhibitor of PLA2 [11,12]. In addition, CQ significantly diminished membrane signal transduction of stimulated platelets below the adenosine diphosphate (ADP) receptor, close to the active sites utilising intraplatelet calcium for aggregation [9]. Yet the high capacity of platelets to accumulate CQ with concomitant displacement of 5-HT on one hand and the inhibition of aggregation on the other hand are stimulating since any receptor specificity for CQ pharmacology has not been defined as yet. So far CQ represents a unique multipotent inhibitor of activated platelets, while its binary effect requires further analysis.
1. Methods and Materials 1.1. Blood Sampling Blood was taken at the blood bank from healthy volunteers (men, aged 20 to 50 years) by antecubital venepuncture and was immediately mixed with 3.8% v/w trisodium citrate dihydrate, ratio 9 mL of blood to 1 mL of citrate in polypropylene centrifuge tubes.
1.2. Centrifugation Blood was centrifuged 30 to 40 minutes after venepuncture for 15 minutes at 200⫻g at 22⬚C. Plateletrich plasma (PRP) was removed and the blood was recentrifuged for 30 minutes at 2200⫻g at 22⬚C to obtain platelet-poor plasma (PPP). This was used to adjust the reference point in the aggregometer and to dilute the PRP to obtain a count of 200,000 platelets/L of sample. The number of platelets was adjusted after counting in a Thrombocounter Coulter (Coulter Electronics, Luton, Beds., UK).
1.3. Platelet Aggregation in PRP PRP (450 L) was stabilised in an aggregometer (Aggregometer Chrono-log, Harveston, MI, USA) at 37⬚C for l minute. Subsequently the drug tested was added in 20-L amount and incubated with the sample for 30 seconds. The aggregation was induced by adding the stimulus (ADP, 2 mol/L, or adrenaline, 4 mol/L; final concentration) in the amount of 20 L, and the aggregation was recorded for 2.5 minutes.
1.4. Aggregation of Isolated Platelets Whole blood with 3.8% w/v trisodium citrate dihydrate was centrifuged for 15 minutes at 200⫻g at 22⬚C. PRP was transferred and mixed with a mixture of 4.5% w/v citric acid and 6.6% w/v dextrose at 50 L per l mL of PRP. After centrifugation at 980⫻g for 10 minutes at 22⬚C, PPP was decanted and platelets were resuspended in an equal volume of PPP with Tyrode solution ⫹ ethylenediaminetetraacetic acid disodium salt (EDTA) (see above). After stabilising the suspension of platelets for 10 minutes at room temperature, the samples were centrifuged for 6 minutes at 980⫻g at 22⬚C. Sedimented platelets were resuspended in an equal volume of Tyrode solution without EDTA and the suspension was adjusted with Tyrode solution to obtain 200,000 platelets per 1 L. For aggregation studies 450 L of platelet suspension/sample was stabilised for 2 minutes at 37⬚C, and the drug tested was added (20 L) and incubated for 30 seconds. Aggregation was induced either with calcium ionophore A23187 (20 L, final concentration 1.8 mol/L), phorbolmyristateacetate (PMA; 20 L, final concentration 50 nmol/L), or thrombin (20 L, final concentration 0.05 NIH U/mL), and data was recorded for 2.5 minutes.
1.5. Phospholipase A2 Activation (Measured as 3H-Arachidonic Acid Liberation) The PRP was mixed with a mixture of 4.5 w/v citric acid and 6.6% w/v dextrose in the amount of 50 L per 1 mL of PRP and with [3H]-arachidonic acid (3H-AA) 1.85⫻104 Bq/mL of PRP. After 60 minutes of incubation at 37⬚C, the samples were centrifuged for 10 minutes at 980⫻g and 22⬚C and the platelets were washed two times by centrifuga-
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tion in an equal volume of Tyrode buffer with EDTA for 6 minutes at 980⫻g at 22⬚C. The supernatant was removed and the platelets were resuspended in Tyrode buffer without EDTA and after counting it was adjusted with Tyrode buffer to get 400,000 platelets per 1 L of sample. The 1-mL samples were stabilised for 2 minutes at 37⬚C, and the drug tested was added (20 L) and incubated for 30 seconds. The stimulation was induced with A23187 (20 L, final concentration 1.8 mol/L), PMA (20 L; 10, 50, 100, and 500 nmol/L), or thrombin (20 L, final concentration 0.05 NIH U/ mL) for 5 minutes at 37⬚C. The incubation was terminated by centrifugation of the samples at 12000⫻g for 3 minutes at 4⬚C. After removal of the supernatant the sedimented platelets were resuspended in 0.5 mL of deionised H2O and 2 mL of the extraction mixture, which consisted of chloroform, methanol, and deionised H2O in the ratio of 20:40:1. After vigorous mixing 0.5 mL of deonised H2O and 0.5 mL of chloroform were added to each sample and the tubes were centrifuged at 2200⫻g for 30 minutes at 22⬚C. The chloroform layer was removed and 400 L from both the supernatant and the sediment extract were transformed to 5 mL of Bray’s scintillation fluid to determine the radioactivity in a Packard Tricarb 2500 scintillation counter (Packard TriCARB 2500 TR; Packard Canberra Co., Downers Grove, IL, USA). The amount of 3H-AA liberation was evaluated as percentage from the total radioactivity in the sample, as described previously [13].
1.6. Malondialdehyde Formation A modification of the method described by Nosa´l’ et al. [14] was used. Briefly platelets were isolated as described above. In the final step isolated platelets were diluted with EDTA-free Tyrode solution to get 700,000 platelets per 1 L of sample. Samples of 450 L were stabilised for 2 minutes at 37⬚C followed by incubation with the drug tested (20 L). After 30 seconds the incubation was continued with a stimulus. This was either A23187 (20 L, 1.8 mol/L) or thrombin (20 L, final concentration 1 NIH U/mL) for 5 minutes at 37⬚C. To stop the incubation, 500 L of sodium dodecylsulphate (0.81 g/10 mL) was added and two 400 L samples were taken for malondialdehyde (MDA) determination. To each sample 1500 L of 20% w/v acetic
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acid, 0.8% w/v thiobarbituric acid, and 600 L of deionised H2O were added. Samples were heated for 60 minutes at 95⬚C, then cooled to room temperature, and after addition of 1 mL of deionised H2O and 5 mL of n-butanol, the content of the tubes was mixed vigorously for 30 seconds on vortex. This was followed by centrifugation at 2000⫻g for 10 minutes at 22⬚C. The fluorescence was measured in the butanol layer at 515-nm excitation and 553-nm emission spectra in a Perkin-Elmer (Hitachi, Tokyo, Japan) spectrofluorometer (model 203). The amount of MDA formation was evaluated from a standard calibration curve of thiobarbituric acid.
1.7. Thromboxane Generation Platelets were isolated and prepared by the same procedure as described for 3H-AA liberation. For the experiment the platelets were diluted with EDTA-free Tyrode buffer to get 10000 platelet/ L of the sample. Tubes with 450 L of platelet suspension were stabilised at 37⬚C for 2 minutes and treated subsequently with 20 L of the drug tested for 30 seconds. Stimulation with 20 L of A23187 (1.8 mol/L final concentration), PMA (50 nmol/L final concentration), or thrombin (0.05 NIH U/mL final concentration) followed for 5 minutes at 37⬚C. Incubation was stopped by addition of indomethacine (final concentration 0.1 mmol/ L). After centrifugation at 14000⫻g for 2 minutes at 4⬚C, the generation of thromboxane was determined in 15 L of the supernatant by means of a [125I]-thromboxane B2 (TXB2) radioimmunoasssay (RIA) kit in an RIA Multidetector Counter JNG 402 (Tesla, Brno, Czech Republic).
1.8. 5-HT Determination Platelet suspension (2 mL) in calcium-free Tyrode solution (2⫻105 platelets/L of the sample) were incubated for 2 minutes at 37⬚C. CQ in the amount of 53 L was added 30 seconds or 15 minutes before the stimulus. After thrombin administration (50 L, final concentration 0.05 NIH U/mL) the samples were incubated for 5 minutes at 37⬚C. Incubation was stopped by cooling the samples to 0⬚C and the tubes were immediately centrifuged for 2 minutes at 14000⫻g at 4⬚C. The supernatant was decanted and sedimented platelets resuspended in
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2 mL of 0.02 N HCl and subsequently sonicated for 20 minutes in a bath at 22⬚C. For 5-HT determination 800 L of supernatant and sediment were taken. To all samples 2200 L of 0.02 N HCl and 200 L of 10% w/v ZnSO4 were added. After vortexing, the samples were treated with 100 L of 1 N NaOH to precipitate any protein left and tubes were centrifuged for 10 minutes at 2500 rpm at 22⬚C. The native fluorescence of 5-HT was measured in Perkin Elmer Fluorescence Spectrometer 203 at 300-nm excitation and 332-nm emission wavelength. In each measurement internal and external standard curves were evaluated. The recovery of 5-HT for internal:external standard was between 92 to 98%. The lowest measurable amount of 5-HT was between 4 to 6 ng/sample. All experiments and measures were run in parallels.
1.9. Determination of Histamine The number of isolated platelets in calcium-free Tyrode solution was adjusted to 4⫻105/L. Samples of 2 mL were stabilised for 2 minutes at 37⬚C. The incubation was continued with CQ (53 L) for 30 seconds and after addition of thrombin (20 L, final concentration 0.05 NIH/mL) for 5 minutes. Then the samples were cooled to 0⬚C and centrifuged for 2 minutes at 14000⫻g at 4⬚C. The supernatant was decanted and sedimented platelets were disrupted in 0.02 N HCl and sonicated for 20 minutes at 22⬚C. For histamine determination a spectrofluorometric method described earlier was modified [15]. To 400 L of both supernatant and sediment 1900 L of 0.02 N HCl was added. Samples were treated with 400 L of 1 N NaOH and after vortexing 100 L of 1% w/v ortophtaldialdehyde in methanol was added. After further vortexing for 4 minutes, the condensating reaction was stopped with 200 L of 3 N HCl. Histamine was measured at 360-nm excitation and 450-nm emission spectra in Perkin Elmer Fluorescence Spectrometer 203. The percentage of histamine release in supernatant was calculated from the total histamine content in the sample.
1.10. Materials Materials used were: CQ phosphate (ACO, Molndal, Sweden), ADP, sodium dodecylsulphate (SDS), thiobarbituric acid (Serva, Heidelberg,
Germany), adrenaline (epinephrine bitartrate) and PMA (Sigma, St. Louis, MO, USA), calcium ionophore A23187 (Calbiochem, La Jolla, CA, USA), human thrombin (Imuna Sˇ. Michal’any Slovakia), histamine 2 HCl and 5-hydroxytryptamine kreatinine sulphate (Koch Light Ltd, Colbrook, Bucks, UK), o-phthaldialdehyde (Merck, Darmstadt, Germany), and Bray’s solution (Spolana Neratovice, Czech Republic). [5,6,8,9,11,14,15 (N)3H]-arachidonic acid (7 TBq/mmol/L) and 125-TXB2 RIA kit were kind gifts from Dr. I. Mucha, Institute of Isotopes, Budapest, Hungary. All other chemicals of analytical grade were from available commercial sources. Tyrode buffer consisted of: 137 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO3, 0.4 mmol/L NaH2PO4⫻2H2O, 1 mmol/L MgCl2⫻6H2O, 5.4 mmol/L EDTA, and 5.6 mmol/L dextrose, pH 6.9.
1.11. Statistical Evaluation The data from measurements were calculated in the MS Windows 98, operating PC programs (Excel, Statistica), and evaluated statistically by means of the Student’s t test.
2. Results Figure 1 summarises the effect of CQ on aggregation in PRP (panel A) or isolated platelets (panel B). In the concentration of 10 mol/L, CQ significantly decreased the amplitude of the second wave of adrenaline-induced aggregation by 33.7%. This effect was more pronounced with 20 mol/L CQ, yielding a 56.2% inhibition. Moreover, this concentration significantly decreased ADP-induced aggregation by 22.3%. With 50 mol/L, CQ potentiated the inhibitory effect on ADP-stimulated platelets by 34.6%, and a significant decrease was recorded in the first phase of adrenaline aggregation to 59.0% of the control (⫽100%). By increasing the concentration to 100 and 1000 mol/L, CQ evidently shortened the amplitude of ADP- induced aggregation curves and the first wave of adrenaline-stimulated aggregation by 84 and 79.7%, respectively. In isolated platelets, CQ in 10 mol/L concentra-
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Fig. 1. Dose-dependent effect of chloroquine on platelet aggregation. (Panel A) Effect on platelets in plasma (PRP) stimulated with ADP and adrenaline (ADRE). ADRE 1⫽amplitude of the first wave of aggregation and ADRE 2⫽amplitude of the second wave of aggregation. (Panel B) Effect on isolated platelets stimulated with calcium ionophore A23187, PMA, and thrombin (THROMB). n⫽6 to 8, mean⫾SEM; *p⬍0.05, **p⬍0.01.
tion significantly decreased the aggregation of PMAstimulated platelets by 10%. By increasing the concentration to 50 and 100 mol/L, the inhibition increased by 20.4 and 34.3%, respectively. CQ in concentrations of 100 and 1000 mol/L decreased thrombin-stimulated aggregation by 50.7 and 93.2%, respectively. In A23187-stimulated platelets CQ was effective only in the 1000 mol/L concentration, resulting in an inhibition to 50.8% of the control. The parameters characterising changes in the dynamics of aggregation events are summarised in Table 1. As evident from the data, CQ did not alter the desaggregation onset (“recovery”) of platelets stimulated with ADP. On the other hand, CQ in concentrations of 10 and 20 mol/L prolonged the onset of the second wave of adrenaline-induced aggregation from 95 to 118 and 141 seconds, respectively. The onset of PMA-induced aggregation was prolonged with 50 and 100 mol/L of CQ from 36
to 53.6 and 70.4 seconds, respectively, representing a 48 and 95% increase in the delay of aggregation onset. In our experimental setup resting platelets liberated 2.83⫾0.21% (mean value) of the total incorporated arachidonic acid. Figure 2 shows that stimulation with A23187, PMA, and thrombin resulted in 31.4, 2.88, and 16.6% 3H-AA liberation from platelet membrane phospholipids, respectively. The effect of CQ on 3H-AA liberation and MDA formation in platelets stimulated with A23187 and thrombin is demonstrated in Figure 3. In A23187stimulated platelets, CQ in all concentrations tested failed to alter either 3H-AA liberation or MDA formation, though with 1000 mol/L CQ a slight decrease of both parameters tested was recorded. On the other hand, CQ in concentrations of 10, 100, and 1000 mol/L significantly decreased 3HAA liberation in thrombin-stimulated platelets from
Table 1. Effect of chloroquine on evaluated parameters of aggregation curves of platelets in plasma stimulated with ADP (2 mol/L, onset of desaggregation) or adrenaline (4 mol/L, onset of the second phase of aggregation), or in isolated platelets stimulated with PMA (20 mol/ L, onset of the aggregation after stimulus)a Chloroquine (mol/L) 0 1 10 20 50 100 a
Disaggregation ADP
Second phase, adrenaline
Aggregation, PMA
57.24⫾2.40 54.41⫾2.83 55.20⫾4.04 51.38⫾3.02 53.89⫾2.40 61.32⫾3.52
94.86⫾2.27 95.23⫾3.15 118.81⫾6.06* 141.10⫾5.60*
36.11⫾1.37 36.96⫾1.16 40.91⫾1.73 42.14⫾1.67 53.59⫾2.67* 70.36⫾2.70*
b b
Each value is the mean from six to eight measurements⫾SEM. b Aggregation occurred without the second phase. * p⬍0.01.
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Fig. 2. Effect of different stimuli on 3H-AA from membrane phospholipids of isolated platelets. A23187⫽calcium ionophore, THROM⫽thrombin. n⫽5 to 6, mean⫾SEM; *p⬍0.05, **p⬍0.01.
16.6% to 12.4, 4.8, and 3.0%, respectively. Similarly, CQ in concentrations of 50, 100, and 1000 mol/L significantly decreased thrombin-stimulated formation of MDA from 106.3 nmol/L (control value) to 50, 33, and 8 nmol/L, respectively. Figure 4 shows the effect of CQ on thromboxane generation in A23187-, PMA-, and thrombin-stimulated platelets. On the average, the spontaneous generation of TXB2 was 45.6⫾6.6 g/106 platelets. It is evident from the figure that CQ had no effect on TXB2 generation in PMA-stimulated platelets. In A23187-stimulated platelets CQ in concentrations of 100 mol/L significantly decreased TXB2 generation to 69% of the control value. In platelets stimulated with thrombin, CQ in the concentrations of 3, 10, and 100 mol/L significantly decreased TXB2 generation to 70, 41, and 10% of the total, respectively.
Fig. 4. TXB2 generation in isolated platelets pretreated with CQ and subsequently stimulated with calcium ionophore A23187, PMA, or thrombin. n⫽4 to 6, mean⫾ SEM, **p⬍0.01.
Human platelets stimulated with A23187 and thrombin liberated 61 and 73% of the total histamine, respectively. It is evident from Figure 5 that CQ in the concentration range from 1 to 1000 mol/ L did not alter stimulated histamine release from platelets. A dose-dependent liberation of 5-HT from isolated platelets due to CQ is demonstrated in Figure 6. CQ in the concentrations of 5, 10, and 50 mol/ L liberated 11, 19, and 29% of the total 5-HT, respectively. An increase of CQ concentration to 100 mol/L did not result in any further 5-HT liberation. The mean amount of 5-HT liberated from isolated platelets stimulated with thrombin was 48% of the total amount. Figure 7 shows the effect of CQ preincubated for 30 seconds or 15 minutes on isolated platelets stimulated with thrombin. CQ in the concentrations of 10 and 50 mol/L decreased thrombin-stimulated 5-HT liberation from platelets to values of 41 and
Fig. 3. Dose-dependent effect of chloroquine on 3H-AA liberation (panel A) and malondialdehyde formation (panel B) in isolated blood platelets stimulated with calcium ionophore A23187 or thrombin (THRO). n⫽4 to 6, mean⫾SEM; **p⬍0.05, **p⬍0.01.
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Fig. 5. Dose-dependent effect of chloroquine on histamine liberation from isolated platelets stimulated with calcium ionophore A23187 or thrombin (THR). n⫽6 to 8, mean⫾SEM; *p⬎0.05.
31.5% of the total amount, respectively. As evident from the figure, no difference was found between the 5-HT amounts liberated from platelets exposed to CQ for 30 seconds or 15 minutes. This indicates that the inhibitory effect of CQ was operative within a very short time after exposure.
3. Discussion CQ inhibited human platelet aggregation in vitro induced with different stimuli. The inhibition was dose-dependent and concerned both PRP and isolated platelets. No substantial interspecies difference was found between human and rat platelets in the inhibitory effect of CQ on PRP or isolated platelets
Fig. 6. Serotonin (5-HT) liberation from isolated platelets treated with increasing concentrations of chloroquine. n⫽6 to 8, mean⫾SEM; **p⬍0.01.
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Fig. 7. Dose-dependent effect of CQ pretreated with isolated platelets for 30 seconds or 15 minutes and subsequently stimulated with thrombin (THR) on serotonin (5-HT) liberation. n⫽6 to 8, mean⫾SEM; **p⬍0.01.
in vitro [16]. In adrenaline-stimulated platelets a decrease in the second “secretory” phase appeared prior to changes in the first phase of aggregation initiated via ␣2-receptors coupled to G-proteins and activation of protein kinase C [17]. Similarly, the ADP-stimulated aggregation decreased proportionally to the increasing CQ concentrations. Since CQ did not show any affinity to specific purinergic receptors in platelets, its inhibitory effect should be localised distal to the thienopyridine sites [18]. This suggestion is supported by the finding that CQ diminished platelet aggregation even when added 120 seconds after ADP application [9]. In isolated platelets, CQ was most effective against PMA-induced aggregation. As PMA activates platelets by stimulating protein kinase C intracellularly, the interaction with CQ may result from its intervention in intracellular protein phosphorylation, from mobilisation of intracellular Ca2⫹ stores, expression of the fibrinogen receptor, outside-in transmembrane signaling, and/or agonist-receptor desensitisation [19]. Of these possible mechanisms, intracellular Ca2⫹ mobilisation seems to be most relevant. The aggregation induced by calcium ionophore A23187, which bypasses any membrane receptors, was decreased with CQ only in the highest concentration used. This indicates that the transport of extracellular calcium for aggregation is affected much less by CQ than the suggested mobilisation of intraplatelet calcium stores with PMA [20,21]. Since thrombin-stimulated aggregation is a rather complex event, its inhibition by CQ requires detailed analysis. CQ is suggested to rapidly pass the mem-
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brane region intracellularly and since no transmembrane affinity domain G-protein-coupled receptors were demonstrated, the interaction of CQ with intraplatelet second messenger pathway could be speculated. As consecutive steps of phospholipase C activation, thrombin mobilises intraplatelet Ca2⫹ and induces arachidonate release [22]. CQ may not inhibit platelets via surface specific glycoproteins. CQ significantly reduced the expression of HLA class I antigens, while the density of the molecules glycoprotein GPIa/IIa, GPIIb, and GPIIb/IIIa carrying thrombocyte-specific antigens was not altered [23]. As a consequence of intraplatelet calcium mobilisation, A23187 and thrombin most probably liberate arachidonic acid from membrane phospholipids due to the activation of phospholipase A2, which was shown to be calcium-dependent, have a pH optimum between 8 and 10, and show a striking preference for the substrate [24]. Moreover, phospholipase A2 (PLA2) does not necessarily require previous activation of phosphoinositide phospholipase C and subsequent increase in diacylglycerol formation. Mepacrine, a CQ-related antimalarial, was demonstrated to inhibit the effect of PLA2 [25–27]. In addition, CQ stabilised the interior phospholipid bilayer of human erythrocyte membrane against diacylglycerol-induced perturbation and PLA2 activation [28]. The higher the concentration of CQ, the lower the liberation of 3H-AA, MDA formation, and TXB2 generation. CQ-induced inhibition of these three crucial steps of arachidonic acid cascade corroborated the assumed inhibition of thrombin- induced aggregation, while the effect of CQ on A23187-stimulated arachidonate cascade was much less pronounced. A similar effect of CQ on platelet aggregation and AA pathway was demonstrated in rat platelets [9,10]. Since CQ is considered a selective inhibitor of phospholipase A2 in different cells and tissues [29,30], its antiplatelet effect may result from inactivation of this enzyme. Yet its inhibition of PMAinduced aggregation, which seems to bypass arachidonate pathway, does not support this hypothesis. Cinchonine, a structurally different antimalarial, also inhibited platelet aggregation induced with PMA but had no effect on AA-induced platelet aggregation and thromboxane synthesis. Since pro-
tein kinase C (PKC) was demonstrated to act in synergy with Ca2⫹ to amplify the aggregation response, drugs such as CQ and cinchonine may act also by affecting Ca2⫹ signals inside platelets [31,32]. Since the mechanism of calcium mobilisation in platelets is rather complex, more detailed investigations are needed to elucidate the mode of CQ interaction with platelet calcium. Histamine is formed and liberated from platelets after stimulation [33] and was considered to be an intracellular messenger for platelet aggregation [34]. Moreover, histamine appeared in A23187-stimulated platelets, indicating a calcium-dependent formation [35]. The failure to inhibit stimulated histamine liberation indicated that CQ most probably did not affect the intraplatelet nongranular calcium pool necessary for histamine formation, as suggested for thrombin-, PMA-, collagen-, thapsigargin-, or A23187-stimulated platelets [35–37]. CQ dose-dependently liberated 5-HT from isolated human platelets. Since CQ accumulated in dense granules, it may displace 5-HT by a nonspecific mechanism demonstrated for amine displacement by cationic amphiphilic drugs from secretory cells [4,38]. The inhibition of thrombin-induced 5-HT liberation indicated that CQ may interact with platelet secretion. This “binary” effect of CQ on histamine secretion was also described in isolated mast cells [39,40]. The mechanism of storage and secretion of 5-HT from platelets was recognized a long time ago [41]. In thrombin-stimulated platelets, 5-HT is liberated during the second secretory phase of aggregation and 5-HT serves as an amplifier to potentiate activation [42,43]. The onset of this second phase was abolished by CQ given 10 to 30 seconds after thrombin-induced aggregation. Moreover, this effect of CQ was exaggerated in platelets stimulated with ADP [9]. CQ uptake in cells increased with increasing extracellular pH and resulted in increased intracellular pH. Increase in intraplatelet pH due to CQ would expect the induction of Na⫹/H⫹-antiport, which facilitates Ca2⫹-regulated platelet activation [1,44,45]. As demonstrated, CQ possessed an opposite, inhibitory effect, suggesting suppression of platelet PLA2. Nevertheless, the interaction of CQ with calcium mobilisation in platelets seems to be crucial in explaining its antiplatelet mechanism of action and should be the focus of more detailed investigations.
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We are grateful to Dr. Istvan Mucha from the Institute of Isotopes, Budapest, Hungary, for supplying us with 3H-AA and TXB2 RIA kits. This work was supported in part by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences (grant no. 2/5019/98). We wish to thank Professor Magda Kourˇilova´-Urbanczik for correcting the English in the manuscript.
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9. Jancˇinova´ V, Nosa´l; R, Petrı´kova´ M. On the inhibitory effect of chloroquine on blood platelet aggregation. Thromb Res 1994;74:495–504. 10. Nosa´l’ R, Jancˇinova´ V, Petrı´kova´ M. Chloroquine inhibits stimulated platelets at the arachidonic acid pathway. Thromb Res 1995;77: 531–42. 11. Joshi UM, Mehendale HM. Drug-induced pulmonary phospholipidosis. Comm Toxicol 1989;3:91–115. 12. Nosa´l’ R. Blood platelets and chloroquine. Platelets 1995;6:310–6. 13. Jancˇinova´ V, Petrı´kova´ M, Nosa´l’ R. Difference among beta-adrenoceptor blocking drugs in modifying platelet aggregation and arachidonic acid liberation under thrombin stimulation. Thromb Res 1989;54:687–98. 14. Nosa´l’ R, Petrı´kova´ M, Jancˇinova´ V. Alteration in lipid peroxidation of thrombin-stimulated rat platelets treated with -adrenoceptor blocking drugs. J Lipid Med 1993;8:121–32. 15. Nosa´l’ R, Dra´bikova´ K, Rekalov V. Currentvoltage relationship in isolated mast cells during histamine liberation and membrane fluidization. Inflamm Res 1998;47:S13–5. 16. Nosa´l’ R, Jancˇinova´ V, Danihelova´ E. Species differences in the pharmacology of blood platelets. Platelets 1998;9:419–20. 17. Nieuwland R, Wijburg OZC, Van Willigen G, Akkerman JWN. ␣2-adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin-sensitive G-protein. FEBS Lett 1994;339:79–83. 18. Gachet C, Cattaneao M, Ohlmann P, Lecchi A, Hechler B, Chevalier J, Cassel D, Manucci P, Cezenave JP. Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors. Br J Haematol 1995;91:434–44. 19. Ware JA, Chang JD. Protein kinase C and its interactions with other serine-threonine kinases. In: von Bruchhausen F, Walter U, editors. Platelets and Their Functions. Handbook of Experimental Pharmacology, Vol. 126. New York: Springer Verlag; 1998. p. 247–62. 20. Nosa´l’ R, Jancˇinova´ V, Petrı´kova´ M. The role of intracellular calcium in A23187-stimulated and beta-adrenoceptor blocking drug treated blood platelets. Biochem Pharmacol 1994; 47:2207–11.
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21. Haynes DH. Effects of cyclic nucleotides and protein kinases on platelet calcium homeostasis and mobilization. Platelets 1993;4:231–42. 22. Watson SP, Ruggiero M, Abrahams SL, Lapetina EG. Inositol 1,4,5-triphosphate induced aggregation and release of 5-hydroxytryptamine from saponine-permeabilized human platelets. J Biol Chem 1986;261:5368–72. 23. Neumuller J, Tohidast-Akrad M, Fischer M, Mayr WR. Influence of chloroquine or acid treatment of human platelets on the antigenicity of HLA and the thrombocyte-specific glycoproteins Ia/IIa, IIb and IIb/IIIa. Vox Sang 1993;65:223–31. 24. Kramer RM, Hession C, Johansen B, Hayes G, McGray P, Chow Pingchang E, Tizard R, Pepinsky RB. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem 1989;264:5768–75. 25. Goracci G. Activation of phospholipase A2 and -thromboglobulin release in human platelets: comparative effects of thrombin and fluoroaluminate stimulation. Biochim Biophys Acta 1992;1124:279–87. 26. Mehadevapa VG, Sicilia F. Mobilization of arachidonic acid in thrombin-stimulated human platelets. Biochim Cell Biol 1990;68:520–7. 27. Hashizume T, Yamaguchi H, Kawamoto A, Tamura A, Sato T, Tatsuzo F. Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation. Arch Biochem Biophys 1991;289:47–52. 28. Zidovetzki R, Sherman IW, Cardenas M, Borchardt DB. Chloroquine stabilization of phospholipid membranes against diacylglycerolinduced perturbation. Biochem Pharmacol 1993;45:183–9. 29. Grabner R. Influence of cationic amphiphilic drugs on the phosphatidylcholine hydrolysis by phospholipase A2. Biochem Pharmacol 1987; 36:1063–7. 30. Hostetler KY, Matsuzawa Y. Studies on the mechanism of drug-induced lipidosis. Cationic amphiphilic drug inhibition of lysosomal phospholipases A and C. Biochem Pharmacol 1981;30:1121–6. 31. Shah BH, Safdar B, Virani SS, Nazaw Z, Saeed SA, Gilani AH. The antiplatelet aggregatory
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ORIGINAL ARTICLE
Chloroquine: A Multipotent Inhibitor of Human Platelets in Vitro Rado Nosa´l’1, Viera Jancˇinova´1 and Edita Danihelova´2 Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava; 2Institute of Haematology and Transfusiology, Bratislava, Slovak Republic.
1
(Received 28 September 1999 by Editor J.E. Dyr; revised/accepted 6 January 2000)
Abstract Chloroquine inhibited human platelet aggregation in vitro both at receptor- and nonreceptor-operated stimuli. The inhibition was dose-dependent, recorded on isolated platelets as well as in plateletrich plasma, and followed the rank order of stimuli: adrenaline (second phase)⬎phorbol 12-myristate 13 acetate⬎adenosine diphosphate⬎adrenaline (first phase)⬎thrombin⬎calcium ionophore A23187. In thrombin-activated platelets, chloroquine decreased in a dose-dependent manner phospholipase A2-induced arachidonic acid liberation from membrane phospholipids, malondialdehyde formation (a marker of membrane phospholipid peroxidation), and thromboxane generation, considered the most potent autoaggregatory agent. Chloroquine only slightly altered the arachidonic acid cascade of platelets stimulated with A23187 and phorbol 12-myristate 13 acetate. Histamine formation and liberation induced with thrombin and A23187 were not affected by chloroquine. On the other hand, thrombin-stimulated serotonin seAbbreviations: CQ, chloroquine; 5-HT, 5-hydroxytryptamine; ADP, adenosine diphosphate; PRP, platelet-rich plasma; PPP, platelet-poor plasma; EDTA, ethylenediaminetetraacetic acid disodium salt; PMA, phorbol 12-myristate 13 acetate; [3H]AA, [3H] arachidonic acid; MDA, malondialdehyde; TXB2, thromboxane B2; RIA, radio immunoassay; SDS, sodium dodecylsulphate; GP, glycoprotein. Corresponding author: Dr. Rado Nosa´l’, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska 2, 842 16 Bratislava, Slovak Republic. Tel: ⫹421 (7) 59410 662; E-mail: ⬍[email protected]⬎.
cretion was significantly decreased with chloroquine in the concentration of 10 mol/L. This indicated that chloroquine might interfere with stimulated secretion from platelets. The results suggest that chloroquine inhibited activated platelets: first, intracellularly; second, in a close relationship to the intraplatelet Ca2⫹ mobile pool; and third, most probably at the site of platelet phospholipase A2 activation. 2000 Elsevier Science Ltd. All rights reserved. Key Words: Chloroquine; Platelets; Aggregation; Arachidonate cascade; Histamine; Serotonin
C
hloroquine (CQ), one of the most potent antimalarial drugs with anti-inflammatory and immunomodulatory properties, has been intensively studied, and its effect on blood platelets has been investigated for more than 20 years [1–3]. As a pharmacological tool and model for drugs with a cationic amphiphilic structure similar to other antimalarials, CQ was used to elucidate nonreceptor mediated 5-hydroxytryptamine (5HT) liberation from platelets with concomitant accumulation of the drug in dense bodies [4]. CQ possesses a high affinity for human platelets both in vitro and in vivo with high dose-dependent accumulation [5,6]. An antiplatelet effect of CQ was described in normal subjects as well as in patients infected with malaria both in vitro and ex vivo [7,8]. The affinity of CQ to rat platelets in vitro resulted in a dose-dependent inhibition of aggregation stimulated with agonists of different signal
0049-3848/00 $–see front matter 2000 Elsevier Science Ltd. All rights reserved. PII S0049-3848(00)00200-0
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transduction pathway [9]. Of these, the CQinduced inhibition of arachidonic acid cascade was demonstrated to be the essential step of arachidonic acid liberation (inhibition of phospholipase A2), malondialdehyde formation (corresponding to membrane phospholipid peroxidation), and thromboxane generation (the end product of the arachidonic acid cascade) [10]. These findings supported the suggested high-affinity of CQ for negatively charged membrane phospholipids and positioned CQ as a selective inhibitor of PLA2 [11,12]. In addition, CQ significantly diminished membrane signal transduction of stimulated platelets below the adenosine diphosphate (ADP) receptor, close to the active sites utilising intraplatelet calcium for aggregation [9]. Yet the high capacity of platelets to accumulate CQ with concomitant displacement of 5-HT on one hand and the inhibition of aggregation on the other hand are stimulating since any receptor specificity for CQ pharmacology has not been defined as yet. So far CQ represents a unique multipotent inhibitor of activated platelets, while its binary effect requires further analysis.
1. Methods and Materials 1.1. Blood Sampling Blood was taken at the blood bank from healthy volunteers (men, aged 20 to 50 years) by antecubital venepuncture and was immediately mixed with 3.8% v/w trisodium citrate dihydrate, ratio 9 mL of blood to 1 mL of citrate in polypropylene centrifuge tubes.
1.2. Centrifugation Blood was centrifuged 30 to 40 minutes after venepuncture for 15 minutes at 200⫻g at 22⬚C. Plateletrich plasma (PRP) was removed and the blood was recentrifuged for 30 minutes at 2200⫻g at 22⬚C to obtain platelet-poor plasma (PPP). This was used to adjust the reference point in the aggregometer and to dilute the PRP to obtain a count of 200,000 platelets/L of sample. The number of platelets was adjusted after counting in a Thrombocounter Coulter (Coulter Electronics, Luton, Beds., UK).
1.3. Platelet Aggregation in PRP PRP (450 L) was stabilised in an aggregometer (Aggregometer Chrono-log, Harveston, MI, USA) at 37⬚C for l minute. Subsequently the drug tested was added in 20-L amount and incubated with the sample for 30 seconds. The aggregation was induced by adding the stimulus (ADP, 2 mol/L, or adrenaline, 4 mol/L; final concentration) in the amount of 20 L, and the aggregation was recorded for 2.5 minutes.
1.4. Aggregation of Isolated Platelets Whole blood with 3.8% w/v trisodium citrate dihydrate was centrifuged for 15 minutes at 200⫻g at 22⬚C. PRP was transferred and mixed with a mixture of 4.5% w/v citric acid and 6.6% w/v dextrose at 50 L per l mL of PRP. After centrifugation at 980⫻g for 10 minutes at 22⬚C, PPP was decanted and platelets were resuspended in an equal volume of PPP with Tyrode solution ⫹ ethylenediaminetetraacetic acid disodium salt (EDTA) (see above). After stabilising the suspension of platelets for 10 minutes at room temperature, the samples were centrifuged for 6 minutes at 980⫻g at 22⬚C. Sedimented platelets were resuspended in an equal volume of Tyrode solution without EDTA and the suspension was adjusted with Tyrode solution to obtain 200,000 platelets per 1 L. For aggregation studies 450 L of platelet suspension/sample was stabilised for 2 minutes at 37⬚C, and the drug tested was added (20 L) and incubated for 30 seconds. Aggregation was induced either with calcium ionophore A23187 (20 L, final concentration 1.8 mol/L), phorbolmyristateacetate (PMA; 20 L, final concentration 50 nmol/L), or thrombin (20 L, final concentration 0.05 NIH U/mL), and data was recorded for 2.5 minutes.
1.5. Phospholipase A2 Activation (Measured as 3H-Arachidonic Acid Liberation) The PRP was mixed with a mixture of 4.5 w/v citric acid and 6.6% w/v dextrose in the amount of 50 L per 1 mL of PRP and with [3H]-arachidonic acid (3H-AA) 1.85⫻104 Bq/mL of PRP. After 60 minutes of incubation at 37⬚C, the samples were centrifuged for 10 minutes at 980⫻g and 22⬚C and the platelets were washed two times by centrifuga-
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tion in an equal volume of Tyrode buffer with EDTA for 6 minutes at 980⫻g at 22⬚C. The supernatant was removed and the platelets were resuspended in Tyrode buffer without EDTA and after counting it was adjusted with Tyrode buffer to get 400,000 platelets per 1 L of sample. The 1-mL samples were stabilised for 2 minutes at 37⬚C, and the drug tested was added (20 L) and incubated for 30 seconds. The stimulation was induced with A23187 (20 L, final concentration 1.8 mol/L), PMA (20 L; 10, 50, 100, and 500 nmol/L), or thrombin (20 L, final concentration 0.05 NIH U/ mL) for 5 minutes at 37⬚C. The incubation was terminated by centrifugation of the samples at 12000⫻g for 3 minutes at 4⬚C. After removal of the supernatant the sedimented platelets were resuspended in 0.5 mL of deionised H2O and 2 mL of the extraction mixture, which consisted of chloroform, methanol, and deionised H2O in the ratio of 20:40:1. After vigorous mixing 0.5 mL of deonised H2O and 0.5 mL of chloroform were added to each sample and the tubes were centrifuged at 2200⫻g for 30 minutes at 22⬚C. The chloroform layer was removed and 400 L from both the supernatant and the sediment extract were transformed to 5 mL of Bray’s scintillation fluid to determine the radioactivity in a Packard Tricarb 2500 scintillation counter (Packard TriCARB 2500 TR; Packard Canberra Co., Downers Grove, IL, USA). The amount of 3H-AA liberation was evaluated as percentage from the total radioactivity in the sample, as described previously [13].
1.6. Malondialdehyde Formation A modification of the method described by Nosa´l’ et al. [14] was used. Briefly platelets were isolated as described above. In the final step isolated platelets were diluted with EDTA-free Tyrode solution to get 700,000 platelets per 1 L of sample. Samples of 450 L were stabilised for 2 minutes at 37⬚C followed by incubation with the drug tested (20 L). After 30 seconds the incubation was continued with a stimulus. This was either A23187 (20 L, 1.8 mol/L) or thrombin (20 L, final concentration 1 NIH U/mL) for 5 minutes at 37⬚C. To stop the incubation, 500 L of sodium dodecylsulphate (0.81 g/10 mL) was added and two 400 L samples were taken for malondialdehyde (MDA) determination. To each sample 1500 L of 20% w/v acetic
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acid, 0.8% w/v thiobarbituric acid, and 600 L of deionised H2O were added. Samples were heated for 60 minutes at 95⬚C, then cooled to room temperature, and after addition of 1 mL of deionised H2O and 5 mL of n-butanol, the content of the tubes was mixed vigorously for 30 seconds on vortex. This was followed by centrifugation at 2000⫻g for 10 minutes at 22⬚C. The fluorescence was measured in the butanol layer at 515-nm excitation and 553-nm emission spectra in a Perkin-Elmer (Hitachi, Tokyo, Japan) spectrofluorometer (model 203). The amount of MDA formation was evaluated from a standard calibration curve of thiobarbituric acid.
1.7. Thromboxane Generation Platelets were isolated and prepared by the same procedure as described for 3H-AA liberation. For the experiment the platelets were diluted with EDTA-free Tyrode buffer to get 10000 platelet/ L of the sample. Tubes with 450 L of platelet suspension were stabilised at 37⬚C for 2 minutes and treated subsequently with 20 L of the drug tested for 30 seconds. Stimulation with 20 L of A23187 (1.8 mol/L final concentration), PMA (50 nmol/L final concentration), or thrombin (0.05 NIH U/mL final concentration) followed for 5 minutes at 37⬚C. Incubation was stopped by addition of indomethacine (final concentration 0.1 mmol/ L). After centrifugation at 14000⫻g for 2 minutes at 4⬚C, the generation of thromboxane was determined in 15 L of the supernatant by means of a [125I]-thromboxane B2 (TXB2) radioimmunoasssay (RIA) kit in an RIA Multidetector Counter JNG 402 (Tesla, Brno, Czech Republic).
1.8. 5-HT Determination Platelet suspension (2 mL) in calcium-free Tyrode solution (2⫻105 platelets/L of the sample) were incubated for 2 minutes at 37⬚C. CQ in the amount of 53 L was added 30 seconds or 15 minutes before the stimulus. After thrombin administration (50 L, final concentration 0.05 NIH U/mL) the samples were incubated for 5 minutes at 37⬚C. Incubation was stopped by cooling the samples to 0⬚C and the tubes were immediately centrifuged for 2 minutes at 14000⫻g at 4⬚C. The supernatant was decanted and sedimented platelets resuspended in
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2 mL of 0.02 N HCl and subsequently sonicated for 20 minutes in a bath at 22⬚C. For 5-HT determination 800 L of supernatant and sediment were taken. To all samples 2200 L of 0.02 N HCl and 200 L of 10% w/v ZnSO4 were added. After vortexing, the samples were treated with 100 L of 1 N NaOH to precipitate any protein left and tubes were centrifuged for 10 minutes at 2500 rpm at 22⬚C. The native fluorescence of 5-HT was measured in Perkin Elmer Fluorescence Spectrometer 203 at 300-nm excitation and 332-nm emission wavelength. In each measurement internal and external standard curves were evaluated. The recovery of 5-HT for internal:external standard was between 92 to 98%. The lowest measurable amount of 5-HT was between 4 to 6 ng/sample. All experiments and measures were run in parallels.
1.9. Determination of Histamine The number of isolated platelets in calcium-free Tyrode solution was adjusted to 4⫻105/L. Samples of 2 mL were stabilised for 2 minutes at 37⬚C. The incubation was continued with CQ (53 L) for 30 seconds and after addition of thrombin (20 L, final concentration 0.05 NIH/mL) for 5 minutes. Then the samples were cooled to 0⬚C and centrifuged for 2 minutes at 14000⫻g at 4⬚C. The supernatant was decanted and sedimented platelets were disrupted in 0.02 N HCl and sonicated for 20 minutes at 22⬚C. For histamine determination a spectrofluorometric method described earlier was modified [15]. To 400 L of both supernatant and sediment 1900 L of 0.02 N HCl was added. Samples were treated with 400 L of 1 N NaOH and after vortexing 100 L of 1% w/v ortophtaldialdehyde in methanol was added. After further vortexing for 4 minutes, the condensating reaction was stopped with 200 L of 3 N HCl. Histamine was measured at 360-nm excitation and 450-nm emission spectra in Perkin Elmer Fluorescence Spectrometer 203. The percentage of histamine release in supernatant was calculated from the total histamine content in the sample.
1.10. Materials Materials used were: CQ phosphate (ACO, Molndal, Sweden), ADP, sodium dodecylsulphate (SDS), thiobarbituric acid (Serva, Heidelberg,
Germany), adrenaline (epinephrine bitartrate) and PMA (Sigma, St. Louis, MO, USA), calcium ionophore A23187 (Calbiochem, La Jolla, CA, USA), human thrombin (Imuna Sˇ. Michal’any Slovakia), histamine 2 HCl and 5-hydroxytryptamine kreatinine sulphate (Koch Light Ltd, Colbrook, Bucks, UK), o-phthaldialdehyde (Merck, Darmstadt, Germany), and Bray’s solution (Spolana Neratovice, Czech Republic). [5,6,8,9,11,14,15 (N)3H]-arachidonic acid (7 TBq/mmol/L) and 125-TXB2 RIA kit were kind gifts from Dr. I. Mucha, Institute of Isotopes, Budapest, Hungary. All other chemicals of analytical grade were from available commercial sources. Tyrode buffer consisted of: 137 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO3, 0.4 mmol/L NaH2PO4⫻2H2O, 1 mmol/L MgCl2⫻6H2O, 5.4 mmol/L EDTA, and 5.6 mmol/L dextrose, pH 6.9.
1.11. Statistical Evaluation The data from measurements were calculated in the MS Windows 98, operating PC programs (Excel, Statistica), and evaluated statistically by means of the Student’s t test.
2. Results Figure 1 summarises the effect of CQ on aggregation in PRP (panel A) or isolated platelets (panel B). In the concentration of 10 mol/L, CQ significantly decreased the amplitude of the second wave of adrenaline-induced aggregation by 33.7%. This effect was more pronounced with 20 mol/L CQ, yielding a 56.2% inhibition. Moreover, this concentration significantly decreased ADP-induced aggregation by 22.3%. With 50 mol/L, CQ potentiated the inhibitory effect on ADP-stimulated platelets by 34.6%, and a significant decrease was recorded in the first phase of adrenaline aggregation to 59.0% of the control (⫽100%). By increasing the concentration to 100 and 1000 mol/L, CQ evidently shortened the amplitude of ADP- induced aggregation curves and the first wave of adrenaline-stimulated aggregation by 84 and 79.7%, respectively. In isolated platelets, CQ in 10 mol/L concentra-
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Fig. 1. Dose-dependent effect of chloroquine on platelet aggregation. (Panel A) Effect on platelets in plasma (PRP) stimulated with ADP and adrenaline (ADRE). ADRE 1⫽amplitude of the first wave of aggregation and ADRE 2⫽amplitude of the second wave of aggregation. (Panel B) Effect on isolated platelets stimulated with calcium ionophore A23187, PMA, and thrombin (THROMB). n⫽6 to 8, mean⫾SEM; *p⬍0.05, **p⬍0.01.
tion significantly decreased the aggregation of PMAstimulated platelets by 10%. By increasing the concentration to 50 and 100 mol/L, the inhibition increased by 20.4 and 34.3%, respectively. CQ in concentrations of 100 and 1000 mol/L decreased thrombin-stimulated aggregation by 50.7 and 93.2%, respectively. In A23187-stimulated platelets CQ was effective only in the 1000 mol/L concentration, resulting in an inhibition to 50.8% of the control. The parameters characterising changes in the dynamics of aggregation events are summarised in Table 1. As evident from the data, CQ did not alter the desaggregation onset (“recovery”) of platelets stimulated with ADP. On the other hand, CQ in concentrations of 10 and 20 mol/L prolonged the onset of the second wave of adrenaline-induced aggregation from 95 to 118 and 141 seconds, respectively. The onset of PMA-induced aggregation was prolonged with 50 and 100 mol/L of CQ from 36
to 53.6 and 70.4 seconds, respectively, representing a 48 and 95% increase in the delay of aggregation onset. In our experimental setup resting platelets liberated 2.83⫾0.21% (mean value) of the total incorporated arachidonic acid. Figure 2 shows that stimulation with A23187, PMA, and thrombin resulted in 31.4, 2.88, and 16.6% 3H-AA liberation from platelet membrane phospholipids, respectively. The effect of CQ on 3H-AA liberation and MDA formation in platelets stimulated with A23187 and thrombin is demonstrated in Figure 3. In A23187stimulated platelets, CQ in all concentrations tested failed to alter either 3H-AA liberation or MDA formation, though with 1000 mol/L CQ a slight decrease of both parameters tested was recorded. On the other hand, CQ in concentrations of 10, 100, and 1000 mol/L significantly decreased 3HAA liberation in thrombin-stimulated platelets from
Table 1. Effect of chloroquine on evaluated parameters of aggregation curves of platelets in plasma stimulated with ADP (2 mol/L, onset of desaggregation) or adrenaline (4 mol/L, onset of the second phase of aggregation), or in isolated platelets stimulated with PMA (20 mol/ L, onset of the aggregation after stimulus)a Chloroquine (mol/L) 0 1 10 20 50 100 a
Disaggregation ADP
Second phase, adrenaline
Aggregation, PMA
57.24⫾2.40 54.41⫾2.83 55.20⫾4.04 51.38⫾3.02 53.89⫾2.40 61.32⫾3.52
94.86⫾2.27 95.23⫾3.15 118.81⫾6.06* 141.10⫾5.60*
36.11⫾1.37 36.96⫾1.16 40.91⫾1.73 42.14⫾1.67 53.59⫾2.67* 70.36⫾2.70*
b b
Each value is the mean from six to eight measurements⫾SEM. b Aggregation occurred without the second phase. * p⬍0.01.
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Fig. 2. Effect of different stimuli on 3H-AA from membrane phospholipids of isolated platelets. A23187⫽calcium ionophore, THROM⫽thrombin. n⫽5 to 6, mean⫾SEM; *p⬍0.05, **p⬍0.01.
16.6% to 12.4, 4.8, and 3.0%, respectively. Similarly, CQ in concentrations of 50, 100, and 1000 mol/L significantly decreased thrombin-stimulated formation of MDA from 106.3 nmol/L (control value) to 50, 33, and 8 nmol/L, respectively. Figure 4 shows the effect of CQ on thromboxane generation in A23187-, PMA-, and thrombin-stimulated platelets. On the average, the spontaneous generation of TXB2 was 45.6⫾6.6 g/106 platelets. It is evident from the figure that CQ had no effect on TXB2 generation in PMA-stimulated platelets. In A23187-stimulated platelets CQ in concentrations of 100 mol/L significantly decreased TXB2 generation to 69% of the control value. In platelets stimulated with thrombin, CQ in the concentrations of 3, 10, and 100 mol/L significantly decreased TXB2 generation to 70, 41, and 10% of the total, respectively.
Fig. 4. TXB2 generation in isolated platelets pretreated with CQ and subsequently stimulated with calcium ionophore A23187, PMA, or thrombin. n⫽4 to 6, mean⫾ SEM, **p⬍0.01.
Human platelets stimulated with A23187 and thrombin liberated 61 and 73% of the total histamine, respectively. It is evident from Figure 5 that CQ in the concentration range from 1 to 1000 mol/ L did not alter stimulated histamine release from platelets. A dose-dependent liberation of 5-HT from isolated platelets due to CQ is demonstrated in Figure 6. CQ in the concentrations of 5, 10, and 50 mol/ L liberated 11, 19, and 29% of the total 5-HT, respectively. An increase of CQ concentration to 100 mol/L did not result in any further 5-HT liberation. The mean amount of 5-HT liberated from isolated platelets stimulated with thrombin was 48% of the total amount. Figure 7 shows the effect of CQ preincubated for 30 seconds or 15 minutes on isolated platelets stimulated with thrombin. CQ in the concentrations of 10 and 50 mol/L decreased thrombin-stimulated 5-HT liberation from platelets to values of 41 and
Fig. 3. Dose-dependent effect of chloroquine on 3H-AA liberation (panel A) and malondialdehyde formation (panel B) in isolated blood platelets stimulated with calcium ionophore A23187 or thrombin (THRO). n⫽4 to 6, mean⫾SEM; **p⬍0.05, **p⬍0.01.
R. Nosa´l’ et al./Thrombosis Research 98 (2000) 411–421
Fig. 5. Dose-dependent effect of chloroquine on histamine liberation from isolated platelets stimulated with calcium ionophore A23187 or thrombin (THR). n⫽6 to 8, mean⫾SEM; *p⬎0.05.
31.5% of the total amount, respectively. As evident from the figure, no difference was found between the 5-HT amounts liberated from platelets exposed to CQ for 30 seconds or 15 minutes. This indicates that the inhibitory effect of CQ was operative within a very short time after exposure.
3. Discussion CQ inhibited human platelet aggregation in vitro induced with different stimuli. The inhibition was dose-dependent and concerned both PRP and isolated platelets. No substantial interspecies difference was found between human and rat platelets in the inhibitory effect of CQ on PRP or isolated platelets
Fig. 6. Serotonin (5-HT) liberation from isolated platelets treated with increasing concentrations of chloroquine. n⫽6 to 8, mean⫾SEM; **p⬍0.01.
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Fig. 7. Dose-dependent effect of CQ pretreated with isolated platelets for 30 seconds or 15 minutes and subsequently stimulated with thrombin (THR) on serotonin (5-HT) liberation. n⫽6 to 8, mean⫾SEM; **p⬍0.01.
in vitro [16]. In adrenaline-stimulated platelets a decrease in the second “secretory” phase appeared prior to changes in the first phase of aggregation initiated via ␣2-receptors coupled to G-proteins and activation of protein kinase C [17]. Similarly, the ADP-stimulated aggregation decreased proportionally to the increasing CQ concentrations. Since CQ did not show any affinity to specific purinergic receptors in platelets, its inhibitory effect should be localised distal to the thienopyridine sites [18]. This suggestion is supported by the finding that CQ diminished platelet aggregation even when added 120 seconds after ADP application [9]. In isolated platelets, CQ was most effective against PMA-induced aggregation. As PMA activates platelets by stimulating protein kinase C intracellularly, the interaction with CQ may result from its intervention in intracellular protein phosphorylation, from mobilisation of intracellular Ca2⫹ stores, expression of the fibrinogen receptor, outside-in transmembrane signaling, and/or agonist-receptor desensitisation [19]. Of these possible mechanisms, intracellular Ca2⫹ mobilisation seems to be most relevant. The aggregation induced by calcium ionophore A23187, which bypasses any membrane receptors, was decreased with CQ only in the highest concentration used. This indicates that the transport of extracellular calcium for aggregation is affected much less by CQ than the suggested mobilisation of intraplatelet calcium stores with PMA [20,21]. Since thrombin-stimulated aggregation is a rather complex event, its inhibition by CQ requires detailed analysis. CQ is suggested to rapidly pass the mem-
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brane region intracellularly and since no transmembrane affinity domain G-protein-coupled receptors were demonstrated, the interaction of CQ with intraplatelet second messenger pathway could be speculated. As consecutive steps of phospholipase C activation, thrombin mobilises intraplatelet Ca2⫹ and induces arachidonate release [22]. CQ may not inhibit platelets via surface specific glycoproteins. CQ significantly reduced the expression of HLA class I antigens, while the density of the molecules glycoprotein GPIa/IIa, GPIIb, and GPIIb/IIIa carrying thrombocyte-specific antigens was not altered [23]. As a consequence of intraplatelet calcium mobilisation, A23187 and thrombin most probably liberate arachidonic acid from membrane phospholipids due to the activation of phospholipase A2, which was shown to be calcium-dependent, have a pH optimum between 8 and 10, and show a striking preference for the substrate [24]. Moreover, phospholipase A2 (PLA2) does not necessarily require previous activation of phosphoinositide phospholipase C and subsequent increase in diacylglycerol formation. Mepacrine, a CQ-related antimalarial, was demonstrated to inhibit the effect of PLA2 [25–27]. In addition, CQ stabilised the interior phospholipid bilayer of human erythrocyte membrane against diacylglycerol-induced perturbation and PLA2 activation [28]. The higher the concentration of CQ, the lower the liberation of 3H-AA, MDA formation, and TXB2 generation. CQ-induced inhibition of these three crucial steps of arachidonic acid cascade corroborated the assumed inhibition of thrombin- induced aggregation, while the effect of CQ on A23187-stimulated arachidonate cascade was much less pronounced. A similar effect of CQ on platelet aggregation and AA pathway was demonstrated in rat platelets [9,10]. Since CQ is considered a selective inhibitor of phospholipase A2 in different cells and tissues [29,30], its antiplatelet effect may result from inactivation of this enzyme. Yet its inhibition of PMAinduced aggregation, which seems to bypass arachidonate pathway, does not support this hypothesis. Cinchonine, a structurally different antimalarial, also inhibited platelet aggregation induced with PMA but had no effect on AA-induced platelet aggregation and thromboxane synthesis. Since pro-
tein kinase C (PKC) was demonstrated to act in synergy with Ca2⫹ to amplify the aggregation response, drugs such as CQ and cinchonine may act also by affecting Ca2⫹ signals inside platelets [31,32]. Since the mechanism of calcium mobilisation in platelets is rather complex, more detailed investigations are needed to elucidate the mode of CQ interaction with platelet calcium. Histamine is formed and liberated from platelets after stimulation [33] and was considered to be an intracellular messenger for platelet aggregation [34]. Moreover, histamine appeared in A23187-stimulated platelets, indicating a calcium-dependent formation [35]. The failure to inhibit stimulated histamine liberation indicated that CQ most probably did not affect the intraplatelet nongranular calcium pool necessary for histamine formation, as suggested for thrombin-, PMA-, collagen-, thapsigargin-, or A23187-stimulated platelets [35–37]. CQ dose-dependently liberated 5-HT from isolated human platelets. Since CQ accumulated in dense granules, it may displace 5-HT by a nonspecific mechanism demonstrated for amine displacement by cationic amphiphilic drugs from secretory cells [4,38]. The inhibition of thrombin-induced 5-HT liberation indicated that CQ may interact with platelet secretion. This “binary” effect of CQ on histamine secretion was also described in isolated mast cells [39,40]. The mechanism of storage and secretion of 5-HT from platelets was recognized a long time ago [41]. In thrombin-stimulated platelets, 5-HT is liberated during the second secretory phase of aggregation and 5-HT serves as an amplifier to potentiate activation [42,43]. The onset of this second phase was abolished by CQ given 10 to 30 seconds after thrombin-induced aggregation. Moreover, this effect of CQ was exaggerated in platelets stimulated with ADP [9]. CQ uptake in cells increased with increasing extracellular pH and resulted in increased intracellular pH. Increase in intraplatelet pH due to CQ would expect the induction of Na⫹/H⫹-antiport, which facilitates Ca2⫹-regulated platelet activation [1,44,45]. As demonstrated, CQ possessed an opposite, inhibitory effect, suggesting suppression of platelet PLA2. Nevertheless, the interaction of CQ with calcium mobilisation in platelets seems to be crucial in explaining its antiplatelet mechanism of action and should be the focus of more detailed investigations.
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We are grateful to Dr. Istvan Mucha from the Institute of Isotopes, Budapest, Hungary, for supplying us with 3H-AA and TXB2 RIA kits. This work was supported in part by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences (grant no. 2/5019/98). We wish to thank Professor Magda Kourˇilova´-Urbanczik for correcting the English in the manuscript.
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