Clinical Hemorheology and Microcirculation 57 (2014) 385–394 DOI 10.3233/CH-131795 IOS Press
385
Cocaine in high concentrations inhibits platelet aggregation in vitro F. Cagienarda , T. Schulzkib and W.H. Reinharta,∗ a b
Department of Internal Medicine, Kantonsspital Graub¨unden, Chur, Switzerland Division of Transfusion Medicine, Kantonsspital Graub¨unden, Chur, Switzerland
Abstract. Complications of cocaine administration are acute vascular occlusions such as myocardial infarction and stroke. We have studied the influence of cocaine on platelet function in vitro. For that purpose, citrated blood from healthy volunteers was incubated with cocaine concentrations of 0 (control), 10, 100, 1000, 2500, and 10’000 mol/l plasma. Platelet aggregation was measured in whole blood under high shear flow conditions with a platelet function analyzer PFA-100 using either epinephrine (EPI) or ADP as a platelet activator, as well as in non-flowing blood measuring the change of impedance after the addition of either collagen or ADP (Chronolog-700 Aggregometer). In addition, platelet aggregation was measured by the change in light transmission in platelet rich plasma containing the same cocaine concentrations (Chronolog-700). Platelet aggregation in flowing whole blood (PFA-100) was not affected by cocaine up to 1000 mol/l, partially inhibited by 2500 mol/l and completely inhibited by 10’000 mol/l cocaine. In non-flowing blood, platelet aggregation was decreased already at cocaine concentrations of 1000 mol/l with ADP and 2500 mol/l with collagen as a platelet activator. In platelet-rich plasma, aggregation was partially inhibited by 1000 and 2500 mol/l and completely inhibited by 10’000 mol/l cocaine. We conclude that platelet aggregation is inhibited by cocaine in vitro. This occurs, however, at concentrations above those measurable in vivo. These observations make it very unlikely that a direct platelet activation plays a role in vascular events complicating cocaine consumption. Keywords: Cocaine, platelet aggregation
1. Introduction Cocaine is extracted from the leaves of the coca plant. The purified drug has been used as a local anaesthetic, especially for surgery of the eye, nose and throat. Today, it is primarily known as a drug of abuse with an increasing consumption worldwide. It is the most common cause of illicit drug-related emergencies [3] which are due to vascular occlusive events such as myocardial infarction [10] or stroke [6]. Renal [33] and mesenterial [19] artery thrombosis have also been described. Autopsies of such cases have shown platelet-rich arterial thrombi suggesting platelet activation as the underlying pathophysiological mechanism [18, 31]. We have recently observed that high concentrations of cocaine affected the erythrocyte shape by inducing a concentration – dependent, reversible stomatocytosis [2]. Therefore, an influence of cocaine on platelet shape and function is conceivable. Studies on the mechanism of action of cocaine on platelets have not given consistent results, but have shown a rather complex picture with either platelet activation, platelet inhibition, or no effect, which depended on the cocaine concentration, type of application, as well as on aggregation tests and type of agonist used. Generally speaking, low concentrations tended to activate platelets at least in some ∗ Corresponding author: W.H. Reinhart, MD, Department of Internal Medicine, Kantonsspital Graub¨unden, CH-7000 Chur, Switzerland. Tel.: +41 81 256 63 05; Fax: +41 81 256 63 81; E-mail: [email protected].
1386-0291/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
386
F. Cagienard et al. / Cocaine in high concentrations inhibits platelet aggregation in vitro
individuals, which has been shown for ADP-induced aggregation in vitro [24]. Cocaine applied either intranasally [9] or intravenously [25] increased platelet factor 4 and thromboglobulin [9] and P-selectin expression [25]. Some cocaine users had elevated P-selectin expression [25]. An infusion of cocaine or placebo in healthy volunteers, however, yielded a similar percentage of stimulated platelets for both [25], suggesting that this ␣-granule release is not caused by a direct action of cocaine. It was the aim the present study to investigate the influence of cocaine on platelet aggregation in vitro with the help of different platelet aggregation tests using either whole blood or platelet rich plasma. Furthermore, platelet aggregation in whole blood was tested either under flowing conditions through a glass capillary into a collagen-coated pore or in non-flowing blood.
2. Materials and methods A stock solution of cocaine was prepared by dissolving cocaine hydrochloride (Sigma Chemicals, Fluka, Buchs, Switzerland) in phosphate buffered saline (PBS: 122 mmol/l NaCl, 25 mmol/l Na2 P04 x 2H2 0, 5 mmol/l K2 HPO4 , 2 g/l glucose) to obtain a cocaine concentration of 280 mmol/l. Serial 10-fold dilutions in PBS were then prepared to obtain cocaine concentrations of 28, 2.8 and 0.28 mmol/l. Blood was drawn from healthy volunteers into tubes containing 0.105 mol/l buffered sodium citrate (BD Vacutainers, Franklin Lakes, NJ, USA). The tubes were incubated at room temperature for 30 min before experiments were started. Hematological parameters were checked with an electronic particle counter (Sysmex XT-1800i, Sysmex Digitana Co, Horgen, Switzerland). For the assessment of platelet aggregation in flowing whole blood under high shear rates, mimicking in vivo flow conditions, a Platelet Function Analyser (PFA-100® Dade Behring, D¨udingen, Switzerland) was used. Whole blood was incubated with appropriate volumes of stock solutions to obtain cocaine concentrations of 0, 10, 100, 1000, 2500, and 10’000 mol/l plasma. The assessment of platelet function in the PFA-100® -instrument occurs as follows. The blood is aspirated at a high shear rate (5000–6000 s−1 ) through a glass capillary (diameter 200 m) into a membrane pore (diameter 150 m), which is coated with collagen and either epinephrine (EPI) or ADP as an additional platelet activator. Platelets adhere to the collagen-coated pore wall, become activated and aggregate among each other, gradually forming a platelet plug, which finally occludes the pore and stops blood flow, which is measured as the closure time (CT). Measurements were performed in duplicate and mean values were calculated. Platelet aggregation in whole blood under non-flowing conditions was assessed with a Chrono-log 700 optical Lumi-Aggregometer with Aggro/Link 8 software (Chrono-log Corp. Havertown, USA). This instrument assesses platelet aggregation by measuring the change in impedance between a pair of metal electrodes immersed in whole blood after the addition of either collagen or ADP as a platelet agonist. Prewarmed electrodes (37◦ C) were immersed in 1000 l prewarmed, diluted whole blood (500 l whole blood and 500 l PBS) to which the same cocaine concentrations as described above had been added. Sedimentation was prevented by constant stirring. After a brief electrical calibration, aggregation was started by the addition of 2 l/ml collagen or 10 l/ml ADP and impedance was recorded for 5 min. Platelet aggregation in plasma was assessed with the same instrument Chrono-log 700 optical LumiAggregometer. For that purpose, platelet rich plasma (PRP) was prepared by gentle centrifugation of whole blood at 140× g for 10 min. The PRP (lower part of the plasma column) was aspirated. The remaining blood was centrifuged at 3920× g for 5 min in order to yield platelet poor plasma (PPP) for calibration purposes. The basic principle of the Lumi-Aggregometer is to measure platelet aggregation by the increase of light transmittance through platelet rich plasma (PRP), which is increased when platelets
F. Cagienard et al. / Cocaine in high concentrations inhibits platelet aggregation in vitro
387
aggregate. The experiments were performed with a volume of 300 l PRP and a stir bar speed of 1000 rpm. Platelets were stimulated with 1 g/ml collagen, or 5 mol/l ADP. Light transmission was then recorded for 5 min. The amplitude of the light transmission curve was compared with the light transmission through PPP without any platelet aggregation and expressed as percentage. Platelet morphology was analyzed by scanning electron microscopy. For that purpose PRP was fixed with an equal volume of 2% glutaraldehyde in PBS. The specimens were then vortexed briefly. 50 l of each sample was placed centrally on a glass coverslip in a 12 well plate and were left to attach for a minimum of 2 h at room temperature. Samples were then washed with 0.1 M PIPES (pH 7.4). Fixation involved the addition of 1 ml of 2.5% glutaraldehyde in 0.1 M PIPES (pH 7.4) to the samples for 5 min. Samples were washed twice for 2 min with 0.1 M PIPES (pH 7.4). For post-fixation, samples were rinsed with 1 ml/well of 1% osmium tetroxide in 0.1 M PIPES (pH 6.8) for 1 h at room temperature and then washed three times for 2 min with double distilled water. Samples were dehydrated with ethanol series: 50, 60, 70, 80, 90, 96 and 100% for 5 min each, respectively. Samples were dried at 45◦ C for at least 1 day and subsequently mounted. Finally, samples were sputter-coated with 10 nm Gold/Palladium (MED 020 unit, Baltec, Balzers, Liechtenstein). Scanning electron microscopical examination was done with a Hitachi S-4700 Field Emission SEM (Hitachi, Tokyo, Japan) operating at 3 kV and 30 A. Statistical analysis was performed with STATISTICA for Windows, Version 9.1 (Statsoft Inc., Tulsa, OK, USA, www.statsoft.com). The statistics were compiled by non-parametric methods with a Friedmanns-ANOVA (analysis of variance) and a Wilcoxon’s signed rank test for paired data, as appropriate. The results are presented as mean values ± standard deviation. A p-value of
385
Cocaine in high concentrations inhibits platelet aggregation in vitro F. Cagienarda , T. Schulzkib and W.H. Reinharta,∗ a b
Department of Internal Medicine, Kantonsspital Graub¨unden, Chur, Switzerland Division of Transfusion Medicine, Kantonsspital Graub¨unden, Chur, Switzerland
Abstract. Complications of cocaine administration are acute vascular occlusions such as myocardial infarction and stroke. We have studied the influence of cocaine on platelet function in vitro. For that purpose, citrated blood from healthy volunteers was incubated with cocaine concentrations of 0 (control), 10, 100, 1000, 2500, and 10’000 mol/l plasma. Platelet aggregation was measured in whole blood under high shear flow conditions with a platelet function analyzer PFA-100 using either epinephrine (EPI) or ADP as a platelet activator, as well as in non-flowing blood measuring the change of impedance after the addition of either collagen or ADP (Chronolog-700 Aggregometer). In addition, platelet aggregation was measured by the change in light transmission in platelet rich plasma containing the same cocaine concentrations (Chronolog-700). Platelet aggregation in flowing whole blood (PFA-100) was not affected by cocaine up to 1000 mol/l, partially inhibited by 2500 mol/l and completely inhibited by 10’000 mol/l cocaine. In non-flowing blood, platelet aggregation was decreased already at cocaine concentrations of 1000 mol/l with ADP and 2500 mol/l with collagen as a platelet activator. In platelet-rich plasma, aggregation was partially inhibited by 1000 and 2500 mol/l and completely inhibited by 10’000 mol/l cocaine. We conclude that platelet aggregation is inhibited by cocaine in vitro. This occurs, however, at concentrations above those measurable in vivo. These observations make it very unlikely that a direct platelet activation plays a role in vascular events complicating cocaine consumption. Keywords: Cocaine, platelet aggregation
1. Introduction Cocaine is extracted from the leaves of the coca plant. The purified drug has been used as a local anaesthetic, especially for surgery of the eye, nose and throat. Today, it is primarily known as a drug of abuse with an increasing consumption worldwide. It is the most common cause of illicit drug-related emergencies [3] which are due to vascular occlusive events such as myocardial infarction [10] or stroke [6]. Renal [33] and mesenterial [19] artery thrombosis have also been described. Autopsies of such cases have shown platelet-rich arterial thrombi suggesting platelet activation as the underlying pathophysiological mechanism [18, 31]. We have recently observed that high concentrations of cocaine affected the erythrocyte shape by inducing a concentration – dependent, reversible stomatocytosis [2]. Therefore, an influence of cocaine on platelet shape and function is conceivable. Studies on the mechanism of action of cocaine on platelets have not given consistent results, but have shown a rather complex picture with either platelet activation, platelet inhibition, or no effect, which depended on the cocaine concentration, type of application, as well as on aggregation tests and type of agonist used. Generally speaking, low concentrations tended to activate platelets at least in some ∗ Corresponding author: W.H. Reinhart, MD, Department of Internal Medicine, Kantonsspital Graub¨unden, CH-7000 Chur, Switzerland. Tel.: +41 81 256 63 05; Fax: +41 81 256 63 81; E-mail: [email protected].
1386-0291/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
386
F. Cagienard et al. / Cocaine in high concentrations inhibits platelet aggregation in vitro
individuals, which has been shown for ADP-induced aggregation in vitro [24]. Cocaine applied either intranasally [9] or intravenously [25] increased platelet factor 4 and thromboglobulin [9] and P-selectin expression [25]. Some cocaine users had elevated P-selectin expression [25]. An infusion of cocaine or placebo in healthy volunteers, however, yielded a similar percentage of stimulated platelets for both [25], suggesting that this ␣-granule release is not caused by a direct action of cocaine. It was the aim the present study to investigate the influence of cocaine on platelet aggregation in vitro with the help of different platelet aggregation tests using either whole blood or platelet rich plasma. Furthermore, platelet aggregation in whole blood was tested either under flowing conditions through a glass capillary into a collagen-coated pore or in non-flowing blood.
2. Materials and methods A stock solution of cocaine was prepared by dissolving cocaine hydrochloride (Sigma Chemicals, Fluka, Buchs, Switzerland) in phosphate buffered saline (PBS: 122 mmol/l NaCl, 25 mmol/l Na2 P04 x 2H2 0, 5 mmol/l K2 HPO4 , 2 g/l glucose) to obtain a cocaine concentration of 280 mmol/l. Serial 10-fold dilutions in PBS were then prepared to obtain cocaine concentrations of 28, 2.8 and 0.28 mmol/l. Blood was drawn from healthy volunteers into tubes containing 0.105 mol/l buffered sodium citrate (BD Vacutainers, Franklin Lakes, NJ, USA). The tubes were incubated at room temperature for 30 min before experiments were started. Hematological parameters were checked with an electronic particle counter (Sysmex XT-1800i, Sysmex Digitana Co, Horgen, Switzerland). For the assessment of platelet aggregation in flowing whole blood under high shear rates, mimicking in vivo flow conditions, a Platelet Function Analyser (PFA-100® Dade Behring, D¨udingen, Switzerland) was used. Whole blood was incubated with appropriate volumes of stock solutions to obtain cocaine concentrations of 0, 10, 100, 1000, 2500, and 10’000 mol/l plasma. The assessment of platelet function in the PFA-100® -instrument occurs as follows. The blood is aspirated at a high shear rate (5000–6000 s−1 ) through a glass capillary (diameter 200 m) into a membrane pore (diameter 150 m), which is coated with collagen and either epinephrine (EPI) or ADP as an additional platelet activator. Platelets adhere to the collagen-coated pore wall, become activated and aggregate among each other, gradually forming a platelet plug, which finally occludes the pore and stops blood flow, which is measured as the closure time (CT). Measurements were performed in duplicate and mean values were calculated. Platelet aggregation in whole blood under non-flowing conditions was assessed with a Chrono-log 700 optical Lumi-Aggregometer with Aggro/Link 8 software (Chrono-log Corp. Havertown, USA). This instrument assesses platelet aggregation by measuring the change in impedance between a pair of metal electrodes immersed in whole blood after the addition of either collagen or ADP as a platelet agonist. Prewarmed electrodes (37◦ C) were immersed in 1000 l prewarmed, diluted whole blood (500 l whole blood and 500 l PBS) to which the same cocaine concentrations as described above had been added. Sedimentation was prevented by constant stirring. After a brief electrical calibration, aggregation was started by the addition of 2 l/ml collagen or 10 l/ml ADP and impedance was recorded for 5 min. Platelet aggregation in plasma was assessed with the same instrument Chrono-log 700 optical LumiAggregometer. For that purpose, platelet rich plasma (PRP) was prepared by gentle centrifugation of whole blood at 140× g for 10 min. The PRP (lower part of the plasma column) was aspirated. The remaining blood was centrifuged at 3920× g for 5 min in order to yield platelet poor plasma (PPP) for calibration purposes. The basic principle of the Lumi-Aggregometer is to measure platelet aggregation by the increase of light transmittance through platelet rich plasma (PRP), which is increased when platelets
F. Cagienard et al. / Cocaine in high concentrations inhibits platelet aggregation in vitro
387
aggregate. The experiments were performed with a volume of 300 l PRP and a stir bar speed of 1000 rpm. Platelets were stimulated with 1 g/ml collagen, or 5 mol/l ADP. Light transmission was then recorded for 5 min. The amplitude of the light transmission curve was compared with the light transmission through PPP without any platelet aggregation and expressed as percentage. Platelet morphology was analyzed by scanning electron microscopy. For that purpose PRP was fixed with an equal volume of 2% glutaraldehyde in PBS. The specimens were then vortexed briefly. 50 l of each sample was placed centrally on a glass coverslip in a 12 well plate and were left to attach for a minimum of 2 h at room temperature. Samples were then washed with 0.1 M PIPES (pH 7.4). Fixation involved the addition of 1 ml of 2.5% glutaraldehyde in 0.1 M PIPES (pH 7.4) to the samples for 5 min. Samples were washed twice for 2 min with 0.1 M PIPES (pH 7.4). For post-fixation, samples were rinsed with 1 ml/well of 1% osmium tetroxide in 0.1 M PIPES (pH 6.8) for 1 h at room temperature and then washed three times for 2 min with double distilled water. Samples were dehydrated with ethanol series: 50, 60, 70, 80, 90, 96 and 100% for 5 min each, respectively. Samples were dried at 45◦ C for at least 1 day and subsequently mounted. Finally, samples were sputter-coated with 10 nm Gold/Palladium (MED 020 unit, Baltec, Balzers, Liechtenstein). Scanning electron microscopical examination was done with a Hitachi S-4700 Field Emission SEM (Hitachi, Tokyo, Japan) operating at 3 kV and 30 A. Statistical analysis was performed with STATISTICA for Windows, Version 9.1 (Statsoft Inc., Tulsa, OK, USA, www.statsoft.com). The statistics were compiled by non-parametric methods with a Friedmanns-ANOVA (analysis of variance) and a Wilcoxon’s signed rank test for paired data, as appropriate. The results are presented as mean values ± standard deviation. A p-value of
