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Doxazosin, an 1-Adrenoceptor Antagonist, Inhibits Serotonin-Induced Shape Change in Human Platelets

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Doxazosin, an 1-Adrenoceptor Antagonist, Inhibits Serotonin-Induced Shape Change in Human Platelets Journal of Human Hypertension (2001) 15, 203–207 2001 Nature Publishing Group All rights reserved 0950-9240/01 $15.00 www.nature.com/jhh ORIGINAL ARTICLE ␣ Doxazosin, an 1-adrenoceptor antagonist, inhibits serotonin-induced shape change in human platelets IA Jagroop and DP Mikhailidis Department of Molecular Pathology and Clinical Biochemistry, Royal Free and University College Medical School (University of London), Royal Free campus, London, UK Patients with peripheral vascular disease or diabetes and aliquots were removed at specific time points for mellitus tend to have elevated circulating levels of nat- median platelet volume (MPV) measurement (using a urally occurring platelet agonists like serotonin (5 hyd- high-resolution channelyser). The MPV was used as an roxytryptamine; 5-HT). This bioamine can induce plate- indicator of PSC. PRP was also incubated with doxazo- let shape change (PSC) an early phase of platelet sin (final concentration: 0.33 ␮M, a concentration similar activation, which is essentially aspirin resistant. In to therapeutic plasma levels) prior to the addition of addition, 5-HT exerts other harmful effects (eg stimulat- each of the above-mentioned agonists. Doxazosin sig- at 30 sec ,0.008 ؍ and P 0.007 ؍ ing vascular smooth muscle proliferation and inducing nificantly inhibited (P vasoconstriction in atheromatous coronary vessels). and 60 sec, respectively) the 5-HT-induced increase in The aim of this study was to determine whether doxazo- MPV. Doxazosin did not significantly inhibit ADP- or sin inhibits 5-HT-induced PSC. Doxazosin is a long act- U46619-induced PSC. The inhibitory effect of doxazosin ␣ ing 1-adrenoceptor antagonist, used in the treatment of seems to be specific to platelet 5-HT2 receptors, since essential hypertension and/or benign prostatic hyper- there was no effect on ADP- or U46619-induced PSC. plasia (BPH). Platelet rich plasma (PRP) was prepared This inhibition of platelet activation may be an five males and three additional, clinically relevant, advantage. Future in vivo ;8 ؍ from healthy volunteers (n females with a median age of 32 years, range: 26–57). studies should consider assessing the effect of doxazo- Agonists (5-HT, 0.06–0.5; ADP, 0.1–0.2 ␮mol/l or U46619, sin on 5-HT-induced platelet activation. ␮ a TXA2 analogue, 0.025–0.05 mol/l) were added to PRP Journal of Human Hypertension (2001) 15, 203–207 Keywords: doxazosin; platelet shape change; human platelets Introduction humans.5–7 This is a relevant topic for further research ␣ because patients with peripheral vascular disease Doxazosin is a long acting 1-adrenoceptor antagonist, (PVD) or diabetes mellitus have elevated circulating used in the treatment of essential hypertension and/or levels of 5-HT.8–11 These conditions are associated 1–3 benign prostatic hyperplasia (BPH). These effects with a high prevalence of hypertension and vascular are complemented, by favourable changes in serum 12 1–3 events. PVD is associated with platelet hyperactiv- lipids, haemostatic factors and insulin sensitivity. ity.13 Elevated plasma levels of 5-HT were also asso- Hypertensive patients are at a risk of thrombotic 4 ciated with an increased incidence of graft reoc- events. It follows that it would be an advantage for clusion after infrainguinal by-pass surgery.9 These antihypertensive drugs to possess anti-thrombotic associations may be attributed to the harmful proper- properties.4 In this context, there is evidence that 5–7 ties of 5-HT that include stimulating vascular muscle doxazosin inhibits platelet activation. However, proliferation and platelet aggregation as well as doxazosin did not inhibit serotonin (5-hydroxy- inducing vasoconstriction in atheromatous coronary tryptamine; 5-HT)-induced platelet aggregation in vessels.9–11 Platelets are the main (95%) store of circulating 5-HT.9–11 This bioamine is released from activated Correspondence: Dr Dimitri P Mikhailidis MD FRCPath, Reader platelets8 and can induce platelet shape change and Honorary Consultant, Department of Molecular Pathology (PSC), an early phase of platelet activation, which is and Clinical Biochemistry, Royal Free and University College essentially aspirin resistant.14–27 PSC can be induced Medical School (University of London), Royal Free campus, Pond Street, London NW3 2QG, UK. Fax: +44 (0) 171 830 2235 by considerably lower concentrations of 5-HT than 15 Received 26 May 2000; revised 25 July 2000; accepted 18 aggregation (0.06–0.5 vs 10.0 ␮mol/l, respectively). August 2000 Similarly, PSC can be inhibited by lower concen- Doxazosin and shape change in human platelets IA Jagroop and DP Mikhailidis 204 trations of antagonists when compared to aggre- Table 1 Effect of doxazosin (0.33 ␮M) on a combination of ϩ = gation.15 We therefore used this technique to reas- 5HT ADP-induced PSC (n 1). PSC is expressed in terms of median platelet volume (MPV) in fl sess the effect of doxazosin on 5-HT-induced platelet activation in human platelets. This investi- ␣ Incubation Incubation gation is based on the evidence that -adrenoceptor Time (30 sec) Time (60 sec) antagonists also block 5-HT receptors.28,29 H2O (control) 5.42 5.36 ADP (only)a 5.61 5.61 Patients and methods 5HT (only)a 5.61 5.58 ADP+5HT 5.84 5.81 Venous blood was collected in 3.8% citrate (1 part ADP+5HT 5.62 5.58 citrate to 9 parts blood) from healthy volunteers + doxazosin (n = 8; five males, three females with a median age of 32 years, range: 26–57). Volunteers denied taking aThe dose of ADP alone (0.04 ␮M) and 5-HT alone (0.06 ␮M) was any drugs for at least 2 weeks prior to sampling. The selected to produce a middle range increase in MPV. This volunteers were fasting overnight and were all non- response could then be inhibited or enhanced. smokers. The citrated blood was centrifuged at 160 g for 15 min at room temperature. The platelet rich plasma (PRP) was then carefully collected with a Pasteur pipette. Aliquots of PRP (450 ␮l), in sili- inhibition was specific for any of the agonists. conised glass cuvettes, were placed in a Chronology Because a harmful synergistic effect of various agon- dual channel optical aggregometer (Coulter Elec- ists may occur in vivo, we examined the effect of tronics, Luton, Beds, UK) and kept at 37°C for doxazosin on a combination of two such agonists at 15 min prior to the start of the experiment.15–20,22–26 sub-maximal concentrations (ADP, 0.04 ␮mol/l + 5- Agonists (5-HT, 0.06–0.5; ADP, 0.1–0.2 ␮mol/l or HT, 0.06 ␮mol/l—see Table 1). ␮ U46619, a TXA2 analogue, 0.025–0.05 mol/l) were Doxazosin mesylate (mol. wt. 547.6) was prepared added (as 10–50 ␮l volumes) to PRP and stirred at by dissolving the compound in distilled water. 1000 rpm. These agonists concentrations and sam- Saline solutions, or other media-containing chloride pling times were determined in previous studies; ions, were avoided as this result in the precipitation they generate the most reproducible results.15–20,22–26 of the less soluble chloride salt of doxazosin.5 We adjusted the concentration of agonists to obtain a middle range response that could be either inhibited or enhanced.15–20,22–26 The volumes of distilled Statistical analysis and presentation of the results water (control) added to samples were matched to The results are presented as median and interquar- those of the agonist (as 10–50 ␮l volumes). ␮ tile range. Comparisons are by non-parametric PRP (100 l aliquots) was removed and mixed analysis (paired Wilcoxon tests, two-tailed). with 400 ␮l of fixative, (4% aqueous glutaraldehyde) at 30 sec, 60 sec (see ‘Results’ for details) after adding the agonists. This sample was further diluted Results 400-fold in Isoton II (Coulter Electronics). These samples were then processed by a Coulter counter Effect of doxazosin on 5-HT-induced PSC ␮ ZM (electrical impedance method using a 70- m The 5-HT-induced increase in MPV was signifi- diameter sampling tube orifice) coupled to a C-256 cantly inhibited (P = 0.007 and P = 0.008, at 30 sec channelyser (Coulter Electronics). The analyser sam- and 60 sec, respectively) by doxazosin (Table 2). 15– ple ‘window’ was set between 2.67 and 19.12 fl. Doxazosin alone did not affect the MPV (incubation 20,22–26 The counter was calibrated using latex-par- time: up to 4 min). ticles of 9 fl (Coulter Electronics). For particle analy- sis, data were accumulated to a maximum of 500 platelets in each of the 256 channels. The median Effect of doxazosin on ADP-and U46619-induced platelet volume (MPV) was the volume of the chan- PSC nel on each side of which 50% of the platelet popu- lation is distributed.15–20,22–26 The increase in MPV The increase in MPV induced by ADP and U46619 was taken as representing the PSC.15–20,22–26 was not inhibited by doxazosin (Table 3). The platelet count was monitored throughout the experiment to exclude PRP samples where signifi- + Ͼ Effect of doxazosin on a combination of ADP 5- cant ( 5%) aggregation occurred. HT-induced PSC PRP was also incubated for 4 min with doxazosin (final concentration 0.33 ␮mol/l, a concentration Doxazosin inhibited PSC induced by a combination similar to therapeutic plasma levels)1 prior to the of ADP + 5-HT (Table 1). In a representative experi- addition of each of the above-mentioned agonists. ment, the combined effect of ADP + 5-HT became This was carried out to determine whether doxazo- equivalent to the MPV response seen with ADP or sin could inhibit PSC. We also established if this 5-HT alone (at 30 and 60 sec).
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