ORIGINAL ARTICLE
Effect of sildenafil on platelet function and platelet cGMP of patients with erectile dysfunction 3 € Yaman3, G. Erisßgen4, D. Tekin4 & E. Ozdiler € M. Akand1, E. Gencer2, O.
1 2 3 4
Department Department Department Department
of of of of
Urology, School of Medicine, Selcuk University, Konya, Turkey; €lent Ecevit University, Zonguldak, Turkey; Physiology, School of Medicine, Bu Urology, School of Medicine, Ankara University, Ankara, Turkey; Physiology, School of Medicine, Ankara University, Ankara, Turkey
Keywords cGMP—erectile dysfunction— phosphodiesterase—platelet—sildenafil Correspondence € Murat Akand, MD, FEBU, Selcßuk Universitesi, € € €ltesi Alaaddin Keykubat Kampusu, Tıp Faku € i, Hastanesi, E-Blok, Kat:1, Uroloji Poliklinig €rkiye. Konya 42075, Tu Tel.: +90 532 7438333; Fax: +90 332 2412184; E-mail: [email protected] Accepted: October 12, 2014 doi: 10.1111/and.12387
Summary To investigate the effect of sildenafil on platelet function and cyclic guanosine monophosphate (cGMP) levels in patients with erectile dysfunction, we evaluated the association between erectile function and platelet responses after administration of 100 mg sildenafil. Erectile responses were monitored after 8 daily doses of the drug. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation and simultaneous adenosine triphosphate (ATP) release and cGMP levels were determined before and after sildenafil therapy. Basal levels for platelet aggregation, ATP release and cGMP were compared with age-matched controls. There was no difference among basal levels of platelet responses between patients and controls, except for ADP-induced platelet aggregation (P = 0.04). It was significantly higher in the patient group. Analysis of the responses to sildenafil revealed that for the patients who showed a positive erectile response, there was a significant increase in platelet cGMP (P = 0.028) and a decrease in ADP-induced platelet aggregation (P = 0.04). However, for those who showed a negative or poor erectile response, there was no change in platelet cGMP levels and platelet functions. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. It is concluded that sildenafil increases platelet cGMP in the patients with positive erectile response. Therefore, it has been speculated that platelet cGMP may be used as an index for erectile response.
Introduction Phosphodiesterases (PDEs) are a large group of enzymes, whose catalytic function is to hydrolyse 30 ,50 -cyclic nucleotides. Cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) has a particular importance due to wide use of sildenafil citrate, an orally active selective PDE5 inhibitor (PDE5I), for the treatment of erectile dysfunction (ED) (Boolell et al., 1996; Steers et al., 1998). As well as corporeal smooth muscle, PDE5 is found in the smooth muscle cells of vasculature, gastrointestinal tract and platelets. Human platelet and corpus cavernosum PDE5s have similar sensitivity to several pharmacological inhibitors (Wang et al., 2001). Saenz de Tejada et al. (2001) evaluated the selectivity of PDE5Is using PDE5 isolated from platelets. © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
Associations of ED with endothelial dysfunction and cardiovascular diseases have prompted considerable interest on the effect of sildenafil on the cardiovascular system. Inhibition of PDE5 produces a modest reduction in blood pressure, a decrease in pulmonary resistance and an increase in coronary blood flow (Boswell-Smith et al., 2006; Reffelmann & Kloner, 2006). Sildenafil was originally investigated as an anti-anginal agent, and it was noted that it is beneficial for patients with cardiovascular diseases (Gudmundsd ottir et al., 2005). Platelets play a key role in the pathogenesis of cardiovascular diseases. It was reported that sildenafil does not have a direct effect on platelets in vitro but potentiates the inhibitory effect of nitric oxide (NO) on platelet aggregation (Wallis et al., 1999; Gudmundsd ottir et al., 2005). There are some conflicting results which suggested that sildenafil increases the aggregatory response of 1
Sildenafil and platelet functions
platelets or has independent antithrombotic effect (Halcox et al., 2002; Li et al., 2003). Li et al. (2003) reported that cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulation and subsequent inhibition. They indicated the stimulatory effect of sildenafil on thrombin-induced platelet aggregation. Halcox et al. (2002) observed that oral sildenafil inhibits platelet activation in patients with coronary artery disease (CAD). It is known that 20–30% of patients are expected to report a failure in sildenafil (McMahon, 2002). The cumulative probability of achieving successful intercourse in men with ED treated with sildenafil increases with the number of attempts and reaches a plateau at the eighth attempt (McCullough et al., 2002). The aim of the present study was to evaluate the effect of sildenafil on platelet aggregation and adenosine triphosphate (ATP) release in comparison with cGMP level in the patients with ED. We evaluated the association between erectile and platelet responses after administration of eight doses of 100 mg sildenafil.
M. Akand et al.
(poor erectile response or nonresponder group) and from E4 to E5 indicate the erections firm enough for intercourse (positive erectile response or responder group). Interviewers were blinded with regard to the results of platelet analysis. Blood sampling The blood samples of the patients and the control subjects were obtained from antecubital vein, through a 19-gauge needle. Samples were collected in siliconised tubes containing 3.8% sodium citrate. The second samples of the patients were taken within 12 h after the 8th dosage. Platelet functions
A total of 25 men aged between 40 and 71 with ED were included in this study. Patients were evaluated based on general medical history, sexual history, physical examination, endocrine blood tests (prolactin and testosterone) and fasting glucose. Patients with unstable CAD, low output congestive cardiac failure, hypertension treated with two or more drugs, currently prescribed organic nitrate medication and patients with diabetes as well as those who had previous pelvic or prostate surgery were excluded from the study. Control group was formed of 16 age-matched healthy volunteers who had not taken any medication for at least 2 weeks. All the patients and controls gave written informed consent for participation in the study.
Platelet aggregation was evaluated within 1 h of blood collection by impedance technique using a Chrono-Log Model 560 WB aggregometer in whole blood. A pair of platinum electrodes was placed in 450 ll of blood, diluted with 450 ll of 0.9% saline solution. The aggregation pen was moved to the 90 line on the top of the recorder chart. The impedance gain was set so that 20 ohms referred to was shown as 40 small divisions on the chart. The chart was allowed to run 2 cm min1. Collagen (2 lg ml1) and adenosine diphosphate (ADP) (10 lM) were added in the samples. The increase in the impedance of electrodes due to the adhesion and aggregation of platelets was recorded. The maximal aggregation intensity (MAI) was determined by measuring maximal increase in the impedance. Platelet ATP release was measured by bioluminescence technique. Hundred microlitre of luciferin–luciferase was added to the samples just after the aggregation was completed. The change in the light emission, detected for ATP standard (2 nM of final concentration), was used to correlate to the light emission detected for ADP or collagen-induced ATP release (Ficicilar et al., 2003). ADP, collagen, ATP standard and luciferin–luciferase reagents were obtained from Chronolog Corp. (Havertown, PA, USA).
Sildenafil administration
Platelet preparation and measurement of platelet cGMP
Sildenafil was administered to each patient at a dose of 100 mg, and the instructions on the optimal use of the drug were repeated. Within 12 h after the 8th dose of sildenafil, drug efficacy was assessed by interviewing the patients according to erection scale (E1–E5) (Gudmundsd ottir et al., 2005): E1: no tumescence, no rigidity; E2: little tumescence, no rigidity; E3: some tumescence, no rigidity; E4: full tumescence, rigidity firm enough for intercourse; and E5: full erection. Values from E1 to E3 refer to erections not sufficiently rigid for intercourse
Platelet-rich plasma (PRP) was obtained by centrifugation of citrated blood at 300 g for 15 min. Platelets were separated by centrifugation at 1200 g for 15 min and were washed twice before being resuspended in a Hepes-buffered Tyrode’s solution, containing 138 mM NaCl, 0.36 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose and 20 mM Hepes (pH 7.4). The platelet count was adjusted to 5–8 9 108 per ml. The platelet pellet was resuspended and lysed using 10% TCA. The lysate was centrifuged at 10 000 g for 10 min. The supernatant was
Patients and methods Patients
2
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
M. Akand et al.
Sildenafil and platelet functions
removed for the measurement of cGMP by the EIA kit obtained from Cayman Chemical (Ann Arbor, MI, USA) (Anfossi et al., 2001). Statistical analysis Platelet cGMP, the maximal intensity of platelet aggregation and ATP release of the patients with erection insufficiently rigid (E1–E3), and firm enough for intercourse (E4–5) before and after sildenafil administration were analysed using the Wilcoxon signed rank test. The Pearson correlation coefficient was used to measure the relationship between platelet aggregation, platelet ATP release and cGMP levels. Differences were considered significant at a level of P < 0.05. Results The median age of the patients was 63 years. There was no difference among basal levels of platelet responses between the patients and controls, except for ADPinduced platelet aggregation (P = 0.04). It was significantly higher in the patient group (Table 1).
Table 1 Platelet cGMP, maximal intensity of ADP (ADP-MI) and collagen-induced (Coll-MI) platelet aggregation, and ADP (ADP-ATP release) and collagen-induced (Coll-ATP release) platelet ATP release of the controls and the patients Controls cGMP (pol/1010 plat) ADP-MI (ohm) ADP-ATP release (nM) Coll-MI (ohm) Coll-ATP release (nM)
1318.7 4.94 1.75 6.51 1.68
Patients 664.86 1.7 0.7 1.54 0.92
905.88 9.16 1.93 6.84 1.97
7.44 5.22* 1.41 1.14 1.8
*P = 0.04 control versus patients.
Thirteen of the 25 patients completed the experiment. Six of them had a positive erectile response (E4–E5 scale) and seven of them had poor erectile response (E1–E3 scale) to sildenafil. Twelve of the patients refused blood sampling after the sildenafil therapy. Of these twelve patients, seven were in nonresponder group (E1–E3), and five were in responder group (E4–E5). In the patients with positive erectile response, sildenafil increased platelet cGMP (P = 0.028) and inhibited ADPinduced platelet aggregation (P = 0.046). ADP-induced ATP release and collagen-induced platelet responses did not alter significantly after sildenafil administration in both E1–3 and E4–5 groups (P = 0.091 for E1–3 and P = 0.342 for E4–5 in ADP-ATP release; P = 0.177 for E1–3 and P = 0.073 for E4–5 in Coll-MI) (Table 2). No significant changes were observed in cGMP levels in the seven patients with a negative or poor erectile response to sildenafil (P = 0.247) (Table 2). Discussion Sildenafil citrate, the first PDE5I, has launched ED therapy into a new era. PDE5Is are considered to be the firstline treatment with a high response rate around 70%. It is known that 20–30% of patients are expected to report a failure in sildenafil (McMahon, 2002). The cumulative probability of achieving successful intercourse in men with ED treated with sildenafil increases with the number of attempts and reaches a plateau at the eighth attempt (McCullough et al., 2002). Therefore, in the present study, drug efficacy was assessed after the eighth dose. Seven of the thirteen patients failed to respond sildenafil. However, as we did not have results for all the patients, it is not possible to draw any conclusions about responsiveness of the therapy in our study. Sexual arousal results in the release of NO from cavernous nonadrenergic noncholinergic nerves, endothelial
Table 2 Platelet cGMP; maximal intensity of ADP (ADP-MI) and collagen-induced (Coll-MI) platelet aggregation; and ADP (ADP-ATP release) and collagen-induced (Coll-ATP release) platelet ATP release of controls, group E1–3 and E4–5 before and after sildenafil E1–3 (n = 7) Control cGMP (pmol/1010 plat) ADP-MI (ohm) ADP-ATP release (nM) Coll-MI (ohm) Coll-ATP release (nM)
1318.7 4.94 1.75 6.51 1.68
Before
664.86 1.7 0.7 1.54 0.92
993 5.7 2.24 5.67 2.39
E4–5 (n = 6) After
795 3.2 1.75 2.29 2.09
1314 2.56 0.37 4.9 1.57
Before
1250 2.09 0.17 2.03 1.31
333.3 12.12 1.35 8.82 1.9
After
206.6* 2.5♯ 1.2 3.6 0.88
1680 3.55 1.7 12.25 0.92
1080** 2.75§ 1.6 6.39 0.88
*P = 0.028 control versus patients. P = 0.046 control versus patients. **P = 0.007 before versus after sildenafil. § P = 0.026 before versus after sildenafil. #
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
3
Sildenafil and platelet functions
and smooth muscle cells. NO causes the relaxation of corporeal smooth muscle by cGMP-mediated decline in intracellular calcium (Uckert et al., 2004). Sildenafil enhances the relaxant effect of NO by inhibiting degradation of cGMP in corporeal smooth muscle. There are conflicting results about the effect of sildenafil on platelets. The studies are generally focused on CAD, and platelet aggregation has been evaluated after a single dose of sildenafil. Besides the inhibition of PDE and platelet activation, several reports have indicated that sildenafil alone has no direct effect on platelet aggregation and is potentiated the effect of NO donors (Wallis et al., 1999; Halcox et al., 2002). Berkels et al. (2001) reported that sildenafil did not inhibit ADP-induced aggregation; whereas collagen-induced aggregation was markedly reduced 1 h after and significantly inhibited 4 h after the application. In the present study, platelet functions were analysed 12 h after the 8th dose. We did not observe any effect of sildenafil on collagen-induced aggregation and secretion. Secretory responses of platelets were similar to aggregatory responses, but differences were not statistically significant. The discrepancies between ADP- and collagen-induced platelet aggregatory and secretory responses suggest that the signalling pathways do not have similar sensitivities to cGMP. It has been reported that there is a synergistic inhibition of platelet aggregation by cAMP and cGMP, and inhibition of collagen-induced aggregation correlated with an increase in platelet cAMP rather than cGMP (Jang et al., 2002). As various mediators can activate and inhibit platelets, the balance between platelet activators and inhibitors in ED and sildenafil treatment needs to be examined. It is interesting that platelet cGMP levels of the responders were lower than those of controls and nonresponders, while ADP-induced platelet aggregation was higher before sildenafil treatment. We thought that high platelet aggregatory response and low cGMP levels of the patients were indicators of the endothelial dysfunction. Platelet cGMP levels increased and MAI of ADP-induced platelets decreased after sildenafil therapy. Halcox et al. (2002) reported that sildenafil improved endothelial dysfunction and inhibited platelet activation in patients with CAD. Our results confirmed the improvement of endothelial dysfunction by sildenafil. Sildenafil did not alter cGMP levels and ADP-induced platelet aggregation in nonresponders. ED of nonresponders may result from endothelial independent impairment of smooth muscle relaxation. Our study has certainly some limitations, which should be listed here. The main limitation is the relatively small patient numbers in the study and control groups. Besides the initial small numbers, exclusion of some patients both in responder and nonresponder groups has caused the final numbers being fewer that can make the significance/ 4
M. Akand et al.
insignificance and accuracy of the statistics questionable. Therefore, similar studies should be carried out with high caseloads. In conclusion, this study shows that there is a relationship between platelet cGMP and erectile response of patients with ED to sildenafil. As platelet and corpus cavernosum type PDE have similar characteristics, platelet cGMP might give an opinion about cavernosal GMP status, which plays an essential role leading to enhanced relaxation of the corpus cavernosum smooth muscle. Accordingly, platelet cGMP level may be used as an index for sildenafil effect. Mirone et al. (2009) suggested in a relatively simple assay that platelet cGMP could provide information on the activity of PDE5 inhibition. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. Further studies on the effect of sildenafil on platelet signalling pathways may explain the poor response of some patients to PDE5Is. References Anfossi G, Russo I, Massucco P, Mattiello L, Balbo A, Cavalot F, Trovati M (2001) Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content. Thromb Res 102:319–330. Berkels R, Klotz T, Sticht G, Englemann U, Klaus W (2001) Modulation of human platelet aggregation by the phosphodiesterase type 5 inhibitor sildenafil. J Cardiovasc Pharmacol 37:413–421. Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, Osterloh IH, Gingell C (1996) Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 8:47–52. Boswell-Smith V, Spina D, Page CP (2006) Phosphodiesterase inhibitors. Br J Pharmacol 147(Suppl 1):S252–S257. Ficicilar H, Zergeroglu AM, Tekin D, Ersoz G (2003) The effects of acute exercise on plasma antioxidant status and platelet response. Thromb Res 111:267–271. Gudmundsd ottir IJ, McRobbie SJ, Robinson SD, Newby DE, Megson IL (2005) Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation. Biochem Biophys Res Commun 337:382–385. Halcox JP, Nour KR, Zalos G, Mincemoyer RA, Waclawiw M, Rivera CE, Willie G, Ellahham S, Quyyumi AA (2002) The effect of sildenafil on human vascular function, platelet activation, and myocardial ischemia. J Am Coll Cardiol 40:1232–1240. Jang EK, Azzam JE, Dickinson NT, Davidson MM, Haslam RJ (2002) Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside. Br J Haematol 117:664–675.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
M. Akand et al.
Li Z, Xi X, Gu M, Feil R, Ye RD, Eigenthaler M, Hofmann F, Du X (2003) A stimulatory role for cGMP-dependent protein kinase in platelet activation. Cell 112:77–86. McCullough AR, Barada JH, Fawzy A, Guay AT, Hatzichristou D (2002) Achieving treatment optimisation with sildenafil citrate (Viagra) in patients with erectile dysfunction. Urology 60(2 Suppl 2):28–38. McMahon CG (2002) High dose sildenafil citrate as a salvage therapy for severe erectile dysfunction. Int J Impot Res 14:533–538. Mirone V, d’Emmanuele di Villa Bianca R, Mitidieri E, Imbimbo C, Fusco F, Verze P, Vitale DF, Sorrentino R, Cirino G (2009) Platelet cyclic guanosine monophosphate as a biomarker of phosphodiesterase type 5 inhibitor efficacy in the treatment of erectile dysfunction: a randomized placebocontrolled study. Eur Urol 56:1067–1073. Reffelmann T, Kloner RA (2006) Cardiovascular effects of phosphodiesterase 5 inhibitors. Curr Pharm Des 12:3485– 3494. Saenz de Tejada I, Angulo J, Cuevas P, Fernandez A, Moncada I, Allona A, Lled o E, K€ orschen HG, Niew€ ohner U, Haning
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
Sildenafil and platelet functions
H, Pages E, Bischoff E (2001) The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil. Int J Impot Res 13:282– 290. Steers WD et al. (1998) Sildenafil (Viagra) is effective in the treatment of severe erectile dysfunction. In: Proceedings of the Second Meeting of the European Society for Impotence Research, Madrid. Int J Impot Res 10:S49. Uckert S, Hedlund P, Waldkirch E, Sohn M, Jonas U, Andersson KE, Stief CG (2004) Interactions between cGMPand cAMP-pathways are involved in the regulation of penile smooth muscle tone. World J Urol 22:261–266. Wallis RM, Corbin JD, Francis SH, Ellis P (1999) Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro. Am J Cardiol 83:3C–12C. Wang P, Wu P, Myers JG, Stamford A, Egan RW, Billah MM (2001) Characterization of human, dog and rabbit corpus cavernosum type 5 phosphodiesterases. Life Sci 68:1977– 1987.
5
Effect of sildenafil on platelet function and platelet cGMP of patients with erectile dysfunction 3 € Yaman3, G. Erisßgen4, D. Tekin4 & E. Ozdiler € M. Akand1, E. Gencer2, O.
1 2 3 4
Department Department Department Department
of of of of
Urology, School of Medicine, Selcuk University, Konya, Turkey; €lent Ecevit University, Zonguldak, Turkey; Physiology, School of Medicine, Bu Urology, School of Medicine, Ankara University, Ankara, Turkey; Physiology, School of Medicine, Ankara University, Ankara, Turkey
Keywords cGMP—erectile dysfunction— phosphodiesterase—platelet—sildenafil Correspondence € Murat Akand, MD, FEBU, Selcßuk Universitesi, € € €ltesi Alaaddin Keykubat Kampusu, Tıp Faku € i, Hastanesi, E-Blok, Kat:1, Uroloji Poliklinig €rkiye. Konya 42075, Tu Tel.: +90 532 7438333; Fax: +90 332 2412184; E-mail: [email protected] Accepted: October 12, 2014 doi: 10.1111/and.12387
Summary To investigate the effect of sildenafil on platelet function and cyclic guanosine monophosphate (cGMP) levels in patients with erectile dysfunction, we evaluated the association between erectile function and platelet responses after administration of 100 mg sildenafil. Erectile responses were monitored after 8 daily doses of the drug. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation and simultaneous adenosine triphosphate (ATP) release and cGMP levels were determined before and after sildenafil therapy. Basal levels for platelet aggregation, ATP release and cGMP were compared with age-matched controls. There was no difference among basal levels of platelet responses between patients and controls, except for ADP-induced platelet aggregation (P = 0.04). It was significantly higher in the patient group. Analysis of the responses to sildenafil revealed that for the patients who showed a positive erectile response, there was a significant increase in platelet cGMP (P = 0.028) and a decrease in ADP-induced platelet aggregation (P = 0.04). However, for those who showed a negative or poor erectile response, there was no change in platelet cGMP levels and platelet functions. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. It is concluded that sildenafil increases platelet cGMP in the patients with positive erectile response. Therefore, it has been speculated that platelet cGMP may be used as an index for erectile response.
Introduction Phosphodiesterases (PDEs) are a large group of enzymes, whose catalytic function is to hydrolyse 30 ,50 -cyclic nucleotides. Cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) has a particular importance due to wide use of sildenafil citrate, an orally active selective PDE5 inhibitor (PDE5I), for the treatment of erectile dysfunction (ED) (Boolell et al., 1996; Steers et al., 1998). As well as corporeal smooth muscle, PDE5 is found in the smooth muscle cells of vasculature, gastrointestinal tract and platelets. Human platelet and corpus cavernosum PDE5s have similar sensitivity to several pharmacological inhibitors (Wang et al., 2001). Saenz de Tejada et al. (2001) evaluated the selectivity of PDE5Is using PDE5 isolated from platelets. © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
Associations of ED with endothelial dysfunction and cardiovascular diseases have prompted considerable interest on the effect of sildenafil on the cardiovascular system. Inhibition of PDE5 produces a modest reduction in blood pressure, a decrease in pulmonary resistance and an increase in coronary blood flow (Boswell-Smith et al., 2006; Reffelmann & Kloner, 2006). Sildenafil was originally investigated as an anti-anginal agent, and it was noted that it is beneficial for patients with cardiovascular diseases (Gudmundsd ottir et al., 2005). Platelets play a key role in the pathogenesis of cardiovascular diseases. It was reported that sildenafil does not have a direct effect on platelets in vitro but potentiates the inhibitory effect of nitric oxide (NO) on platelet aggregation (Wallis et al., 1999; Gudmundsd ottir et al., 2005). There are some conflicting results which suggested that sildenafil increases the aggregatory response of 1
Sildenafil and platelet functions
platelets or has independent antithrombotic effect (Halcox et al., 2002; Li et al., 2003). Li et al. (2003) reported that cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulation and subsequent inhibition. They indicated the stimulatory effect of sildenafil on thrombin-induced platelet aggregation. Halcox et al. (2002) observed that oral sildenafil inhibits platelet activation in patients with coronary artery disease (CAD). It is known that 20–30% of patients are expected to report a failure in sildenafil (McMahon, 2002). The cumulative probability of achieving successful intercourse in men with ED treated with sildenafil increases with the number of attempts and reaches a plateau at the eighth attempt (McCullough et al., 2002). The aim of the present study was to evaluate the effect of sildenafil on platelet aggregation and adenosine triphosphate (ATP) release in comparison with cGMP level in the patients with ED. We evaluated the association between erectile and platelet responses after administration of eight doses of 100 mg sildenafil.
M. Akand et al.
(poor erectile response or nonresponder group) and from E4 to E5 indicate the erections firm enough for intercourse (positive erectile response or responder group). Interviewers were blinded with regard to the results of platelet analysis. Blood sampling The blood samples of the patients and the control subjects were obtained from antecubital vein, through a 19-gauge needle. Samples were collected in siliconised tubes containing 3.8% sodium citrate. The second samples of the patients were taken within 12 h after the 8th dosage. Platelet functions
A total of 25 men aged between 40 and 71 with ED were included in this study. Patients were evaluated based on general medical history, sexual history, physical examination, endocrine blood tests (prolactin and testosterone) and fasting glucose. Patients with unstable CAD, low output congestive cardiac failure, hypertension treated with two or more drugs, currently prescribed organic nitrate medication and patients with diabetes as well as those who had previous pelvic or prostate surgery were excluded from the study. Control group was formed of 16 age-matched healthy volunteers who had not taken any medication for at least 2 weeks. All the patients and controls gave written informed consent for participation in the study.
Platelet aggregation was evaluated within 1 h of blood collection by impedance technique using a Chrono-Log Model 560 WB aggregometer in whole blood. A pair of platinum electrodes was placed in 450 ll of blood, diluted with 450 ll of 0.9% saline solution. The aggregation pen was moved to the 90 line on the top of the recorder chart. The impedance gain was set so that 20 ohms referred to was shown as 40 small divisions on the chart. The chart was allowed to run 2 cm min1. Collagen (2 lg ml1) and adenosine diphosphate (ADP) (10 lM) were added in the samples. The increase in the impedance of electrodes due to the adhesion and aggregation of platelets was recorded. The maximal aggregation intensity (MAI) was determined by measuring maximal increase in the impedance. Platelet ATP release was measured by bioluminescence technique. Hundred microlitre of luciferin–luciferase was added to the samples just after the aggregation was completed. The change in the light emission, detected for ATP standard (2 nM of final concentration), was used to correlate to the light emission detected for ADP or collagen-induced ATP release (Ficicilar et al., 2003). ADP, collagen, ATP standard and luciferin–luciferase reagents were obtained from Chronolog Corp. (Havertown, PA, USA).
Sildenafil administration
Platelet preparation and measurement of platelet cGMP
Sildenafil was administered to each patient at a dose of 100 mg, and the instructions on the optimal use of the drug were repeated. Within 12 h after the 8th dose of sildenafil, drug efficacy was assessed by interviewing the patients according to erection scale (E1–E5) (Gudmundsd ottir et al., 2005): E1: no tumescence, no rigidity; E2: little tumescence, no rigidity; E3: some tumescence, no rigidity; E4: full tumescence, rigidity firm enough for intercourse; and E5: full erection. Values from E1 to E3 refer to erections not sufficiently rigid for intercourse
Platelet-rich plasma (PRP) was obtained by centrifugation of citrated blood at 300 g for 15 min. Platelets were separated by centrifugation at 1200 g for 15 min and were washed twice before being resuspended in a Hepes-buffered Tyrode’s solution, containing 138 mM NaCl, 0.36 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose and 20 mM Hepes (pH 7.4). The platelet count was adjusted to 5–8 9 108 per ml. The platelet pellet was resuspended and lysed using 10% TCA. The lysate was centrifuged at 10 000 g for 10 min. The supernatant was
Patients and methods Patients
2
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
M. Akand et al.
Sildenafil and platelet functions
removed for the measurement of cGMP by the EIA kit obtained from Cayman Chemical (Ann Arbor, MI, USA) (Anfossi et al., 2001). Statistical analysis Platelet cGMP, the maximal intensity of platelet aggregation and ATP release of the patients with erection insufficiently rigid (E1–E3), and firm enough for intercourse (E4–5) before and after sildenafil administration were analysed using the Wilcoxon signed rank test. The Pearson correlation coefficient was used to measure the relationship between platelet aggregation, platelet ATP release and cGMP levels. Differences were considered significant at a level of P < 0.05. Results The median age of the patients was 63 years. There was no difference among basal levels of platelet responses between the patients and controls, except for ADPinduced platelet aggregation (P = 0.04). It was significantly higher in the patient group (Table 1).
Table 1 Platelet cGMP, maximal intensity of ADP (ADP-MI) and collagen-induced (Coll-MI) platelet aggregation, and ADP (ADP-ATP release) and collagen-induced (Coll-ATP release) platelet ATP release of the controls and the patients Controls cGMP (pol/1010 plat) ADP-MI (ohm) ADP-ATP release (nM) Coll-MI (ohm) Coll-ATP release (nM)
1318.7 4.94 1.75 6.51 1.68
Patients 664.86 1.7 0.7 1.54 0.92
905.88 9.16 1.93 6.84 1.97
7.44 5.22* 1.41 1.14 1.8
*P = 0.04 control versus patients.
Thirteen of the 25 patients completed the experiment. Six of them had a positive erectile response (E4–E5 scale) and seven of them had poor erectile response (E1–E3 scale) to sildenafil. Twelve of the patients refused blood sampling after the sildenafil therapy. Of these twelve patients, seven were in nonresponder group (E1–E3), and five were in responder group (E4–E5). In the patients with positive erectile response, sildenafil increased platelet cGMP (P = 0.028) and inhibited ADPinduced platelet aggregation (P = 0.046). ADP-induced ATP release and collagen-induced platelet responses did not alter significantly after sildenafil administration in both E1–3 and E4–5 groups (P = 0.091 for E1–3 and P = 0.342 for E4–5 in ADP-ATP release; P = 0.177 for E1–3 and P = 0.073 for E4–5 in Coll-MI) (Table 2). No significant changes were observed in cGMP levels in the seven patients with a negative or poor erectile response to sildenafil (P = 0.247) (Table 2). Discussion Sildenafil citrate, the first PDE5I, has launched ED therapy into a new era. PDE5Is are considered to be the firstline treatment with a high response rate around 70%. It is known that 20–30% of patients are expected to report a failure in sildenafil (McMahon, 2002). The cumulative probability of achieving successful intercourse in men with ED treated with sildenafil increases with the number of attempts and reaches a plateau at the eighth attempt (McCullough et al., 2002). Therefore, in the present study, drug efficacy was assessed after the eighth dose. Seven of the thirteen patients failed to respond sildenafil. However, as we did not have results for all the patients, it is not possible to draw any conclusions about responsiveness of the therapy in our study. Sexual arousal results in the release of NO from cavernous nonadrenergic noncholinergic nerves, endothelial
Table 2 Platelet cGMP; maximal intensity of ADP (ADP-MI) and collagen-induced (Coll-MI) platelet aggregation; and ADP (ADP-ATP release) and collagen-induced (Coll-ATP release) platelet ATP release of controls, group E1–3 and E4–5 before and after sildenafil E1–3 (n = 7) Control cGMP (pmol/1010 plat) ADP-MI (ohm) ADP-ATP release (nM) Coll-MI (ohm) Coll-ATP release (nM)
1318.7 4.94 1.75 6.51 1.68
Before
664.86 1.7 0.7 1.54 0.92
993 5.7 2.24 5.67 2.39
E4–5 (n = 6) After
795 3.2 1.75 2.29 2.09
1314 2.56 0.37 4.9 1.57
Before
1250 2.09 0.17 2.03 1.31
333.3 12.12 1.35 8.82 1.9
After
206.6* 2.5♯ 1.2 3.6 0.88
1680 3.55 1.7 12.25 0.92
1080** 2.75§ 1.6 6.39 0.88
*P = 0.028 control versus patients. P = 0.046 control versus patients. **P = 0.007 before versus after sildenafil. § P = 0.026 before versus after sildenafil. #
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
3
Sildenafil and platelet functions
and smooth muscle cells. NO causes the relaxation of corporeal smooth muscle by cGMP-mediated decline in intracellular calcium (Uckert et al., 2004). Sildenafil enhances the relaxant effect of NO by inhibiting degradation of cGMP in corporeal smooth muscle. There are conflicting results about the effect of sildenafil on platelets. The studies are generally focused on CAD, and platelet aggregation has been evaluated after a single dose of sildenafil. Besides the inhibition of PDE and platelet activation, several reports have indicated that sildenafil alone has no direct effect on platelet aggregation and is potentiated the effect of NO donors (Wallis et al., 1999; Halcox et al., 2002). Berkels et al. (2001) reported that sildenafil did not inhibit ADP-induced aggregation; whereas collagen-induced aggregation was markedly reduced 1 h after and significantly inhibited 4 h after the application. In the present study, platelet functions were analysed 12 h after the 8th dose. We did not observe any effect of sildenafil on collagen-induced aggregation and secretion. Secretory responses of platelets were similar to aggregatory responses, but differences were not statistically significant. The discrepancies between ADP- and collagen-induced platelet aggregatory and secretory responses suggest that the signalling pathways do not have similar sensitivities to cGMP. It has been reported that there is a synergistic inhibition of platelet aggregation by cAMP and cGMP, and inhibition of collagen-induced aggregation correlated with an increase in platelet cAMP rather than cGMP (Jang et al., 2002). As various mediators can activate and inhibit platelets, the balance between platelet activators and inhibitors in ED and sildenafil treatment needs to be examined. It is interesting that platelet cGMP levels of the responders were lower than those of controls and nonresponders, while ADP-induced platelet aggregation was higher before sildenafil treatment. We thought that high platelet aggregatory response and low cGMP levels of the patients were indicators of the endothelial dysfunction. Platelet cGMP levels increased and MAI of ADP-induced platelets decreased after sildenafil therapy. Halcox et al. (2002) reported that sildenafil improved endothelial dysfunction and inhibited platelet activation in patients with CAD. Our results confirmed the improvement of endothelial dysfunction by sildenafil. Sildenafil did not alter cGMP levels and ADP-induced platelet aggregation in nonresponders. ED of nonresponders may result from endothelial independent impairment of smooth muscle relaxation. Our study has certainly some limitations, which should be listed here. The main limitation is the relatively small patient numbers in the study and control groups. Besides the initial small numbers, exclusion of some patients both in responder and nonresponder groups has caused the final numbers being fewer that can make the significance/ 4
M. Akand et al.
insignificance and accuracy of the statistics questionable. Therefore, similar studies should be carried out with high caseloads. In conclusion, this study shows that there is a relationship between platelet cGMP and erectile response of patients with ED to sildenafil. As platelet and corpus cavernosum type PDE have similar characteristics, platelet cGMP might give an opinion about cavernosal GMP status, which plays an essential role leading to enhanced relaxation of the corpus cavernosum smooth muscle. Accordingly, platelet cGMP level may be used as an index for sildenafil effect. Mirone et al. (2009) suggested in a relatively simple assay that platelet cGMP could provide information on the activity of PDE5 inhibition. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. Further studies on the effect of sildenafil on platelet signalling pathways may explain the poor response of some patients to PDE5Is. References Anfossi G, Russo I, Massucco P, Mattiello L, Balbo A, Cavalot F, Trovati M (2001) Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content. Thromb Res 102:319–330. Berkels R, Klotz T, Sticht G, Englemann U, Klaus W (2001) Modulation of human platelet aggregation by the phosphodiesterase type 5 inhibitor sildenafil. J Cardiovasc Pharmacol 37:413–421. Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, Osterloh IH, Gingell C (1996) Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 8:47–52. Boswell-Smith V, Spina D, Page CP (2006) Phosphodiesterase inhibitors. Br J Pharmacol 147(Suppl 1):S252–S257. Ficicilar H, Zergeroglu AM, Tekin D, Ersoz G (2003) The effects of acute exercise on plasma antioxidant status and platelet response. Thromb Res 111:267–271. Gudmundsd ottir IJ, McRobbie SJ, Robinson SD, Newby DE, Megson IL (2005) Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation. Biochem Biophys Res Commun 337:382–385. Halcox JP, Nour KR, Zalos G, Mincemoyer RA, Waclawiw M, Rivera CE, Willie G, Ellahham S, Quyyumi AA (2002) The effect of sildenafil on human vascular function, platelet activation, and myocardial ischemia. J Am Coll Cardiol 40:1232–1240. Jang EK, Azzam JE, Dickinson NT, Davidson MM, Haslam RJ (2002) Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen-induced platelet aggregation by nitroprusside. Br J Haematol 117:664–675.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
M. Akand et al.
Li Z, Xi X, Gu M, Feil R, Ye RD, Eigenthaler M, Hofmann F, Du X (2003) A stimulatory role for cGMP-dependent protein kinase in platelet activation. Cell 112:77–86. McCullough AR, Barada JH, Fawzy A, Guay AT, Hatzichristou D (2002) Achieving treatment optimisation with sildenafil citrate (Viagra) in patients with erectile dysfunction. Urology 60(2 Suppl 2):28–38. McMahon CG (2002) High dose sildenafil citrate as a salvage therapy for severe erectile dysfunction. Int J Impot Res 14:533–538. Mirone V, d’Emmanuele di Villa Bianca R, Mitidieri E, Imbimbo C, Fusco F, Verze P, Vitale DF, Sorrentino R, Cirino G (2009) Platelet cyclic guanosine monophosphate as a biomarker of phosphodiesterase type 5 inhibitor efficacy in the treatment of erectile dysfunction: a randomized placebocontrolled study. Eur Urol 56:1067–1073. Reffelmann T, Kloner RA (2006) Cardiovascular effects of phosphodiesterase 5 inhibitors. Curr Pharm Des 12:3485– 3494. Saenz de Tejada I, Angulo J, Cuevas P, Fernandez A, Moncada I, Allona A, Lled o E, K€ orschen HG, Niew€ ohner U, Haning
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5
Sildenafil and platelet functions
H, Pages E, Bischoff E (2001) The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil. Int J Impot Res 13:282– 290. Steers WD et al. (1998) Sildenafil (Viagra) is effective in the treatment of severe erectile dysfunction. In: Proceedings of the Second Meeting of the European Society for Impotence Research, Madrid. Int J Impot Res 10:S49. Uckert S, Hedlund P, Waldkirch E, Sohn M, Jonas U, Andersson KE, Stief CG (2004) Interactions between cGMPand cAMP-pathways are involved in the regulation of penile smooth muscle tone. World J Urol 22:261–266. Wallis RM, Corbin JD, Francis SH, Ellis P (1999) Tissue distribution of phosphodiesterase families and the effects of sildenafil on tissue cyclic nucleotides, platelet function, and the contractile responses of trabeculae carneae and aortic rings in vitro. Am J Cardiol 83:3C–12C. Wang P, Wu P, Myers JG, Stamford A, Egan RW, Billah MM (2001) Characterization of human, dog and rabbit corpus cavernosum type 5 phosphodiesterases. Life Sci 68:1977– 1987.
5
