NEFROLOGÍA. Vol. XXII. Suplemento 2. 2002 Inhibition of platelet aggregability by losartan in essential hypertension J. Pavel Levy, C. Yunis, J. Owen, K. Bridget Brosnihan, R. Smith and C. M. Ferrario From The Hypertension & Vascular Disease Center, and the Department of Hematology/Oncology, Wake Forest University School of Medicine, Winston-Salem, North Carolina. This study was supported by an unrestricted Medical School Grant from Merck Human Health Division, Merck, Co., Inc., West Point, New Jersey; and National Institutes of Health Grant PO1-HL-51952, Bethesda, Maryland. Manuscript received March 8, 2000; revised manuscript received and accepted June 15, 2000. Address for reprints: Carlos M. Ferrario, MD, Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157. E-mail cferrari@wfubmc.edu. Epidemiologic evidence documents that there is a link between hypertension and coagulation disorders 1, 2. Among the latter are increased platelet aggregability 3 and vascular endothelial dysfunction 4. Evidence suggests a contribution of the renin-angiotensin system to the pathogenesis of atherosclerosis 5 and to the development of a prothrombotic stage in hypertensive subjects 6. Angiotensin II (Ang II) increases production and secretion of plasminogen activator inhibitor type 1 (PAI-1) from vascular endothelial cells 7, whereas losartan inhibits the vasoconstrictor and platelet aggregation effects induced by thromboxane A2 (TxA2) 8-10. The possibility that chronic therapy with an Ang II antagonist may decrease platelet ag-gregability, thus contributing to amelioration of the prothrombogenic stage associated with hypertension has not been studied in human subjects. Therefore, we assessed the effects of 4week losartan therapy on thrombin-mediated platelet aggregation and plasma markers of coagulation and fibrinolysis in subjects with stages I and II hypertension. METHODS Patients This study was performed in 10 subjects (4 women and 6 men, mean age [mean ± SD] 53 ± 9 years, range 43 to 69) with stages I to II essential hypertension. Nine other healthy normotensive ( 140/90 mmHg) volunteers (39 ± 11 years, range 24 to 55) participated as a time-control group. All subjects were recruited from the Hypertension and Vascular Disease Clinic in compliance with guidelines approved by the Wake Forest University Institutional Review Board/Human Subject Review Committee and after each subject had signed a consent form. Blood pressure measurements were obtained and venous blood withdrawn (10 ml) after subjects rested 24 in the supine position for a minimum of 15 minutes in a quiet examining room maintained at an ambient temperature between 25° C and 28° C. Four of the 9 subjects had newly diagnosed hypertension and had not received prior antihypertensive therapy. Exclusion criteria were: subjects with known secondary causes for hypertension; an acute illness of any type; previously documented thrombotic events (e.g., deep vein thrombosis, myocardial infarction, or stroke), or defined hypercoagulable state; major surgery in the past 3 months; current or past exposure to antiplatelet therapy, nonsteroidal anti-inflammatory drugs, lipid-lowering therapy, antidepressants, or oral contraceptives. Current smokers, subjects with diabetes mellitus, chronic renal insufficiency (serum creatinine 1.8 mg/dl), known allergy to losartan potassium, and subjects with worsening of kidney function while receiv-ing angiotensin-converting enzyme inhibitor therapy were also excluded from the study. Study design The effects of 4 weeks of losartan therapy on arterial pressure, thrombin receptor agonist-induced platelet aggregation, plasma levels of the von Willebrand factor (vWF) antigen, and PAI-1 antigen concentrations were determined in a prospective, double-blind (patients and hematology laboratory), placebo-controlled study. All subjects underwent a screening physical examination, urinalysis, and labo-ratory tests (blood counts and serum biochemistry, including renal function and electrolytes). During the study, participants were asked to abstain from taking aspirin or aspirin-containing medications and antide-pressants, and were instructed to report any acute illness. Weekly visits were scheduled to assess changes in blood pressure, compliance with medica-tions (pill counting), and adherence to avoidance of using over-the- INHIBITION OF PLATELET AGGREGABILITY BY LOSARTAN IN ESSENTIAL HYPERTENSION counter medications. After a 4-week washout period, all subjects entered a 2-week placebo run-in period, followed by a 30-day period of losartan therapy (50 mg once daily). Venous blood was obtained at the end of the washout period, both at weeks ­2 and 0 of the placebo run-in period, and at week 4 of losartan therapy. In healthy time-control volunteers, blood sampling was obtained at the beginning of the study and again 30 days after. Laboratory studies Platelet responsiveness to the thrombin receptoractivating peptide SFLRRN-NH2 (PMIS387, Bachem, California) was determined from platelet-rich plasma prepared by centrifugation of the blood at 900 rpm for 15 minutes at 20° C, as described elsewhere11, Platelet aggregations were performed from plateletrich plasma incubated at 37° C for 2 minutes and stirred for 60 seconds (1,100 rpm) in an aggregometer (PAP 4-C, Biodata, Horsham, Pennsylvania). Changes in optical density were recorded for 4 minutes after platelets were stimulated with the thrombin receptor agonist at concentrations ranging from 5 to 1,000 µmol/L. A 4-parameter logistic dosere-sponse curve, expressing the relation between percent aggregation, rate, and final concentration of the thrombin receptor agonist was calculated for each subject's sample. Both maximal extent of aggregation and rate were determined for each concentration. The fitted curve allowed determination of the agonist concentration required to produce half-maximal response (EC50)11. Plasma concentrations of the PAI-1 and von Willebrand protein were determined using the Asserachrome enzyme-linked immunosorbent assay kits from Diagnostica Stage (Parsippany, New Jersey). Statistical analysis The primary end point of the study was the measurement of changes in thrombin receptor agonistinduced platelet aggregation between losartan and placebo periods. Descriptive analyses were performed by the calculation of means ± SD for continuous variables and proportions for categorical variables using the Student's t test and chi-square or Fisher 's exact test. Multicolinearity between primary end points and potential confounders was excluded by correlation analysis. The primary end point was evaluated by analysis of variance with effects for period and treatment groups. Analysis was performed using the SAS statistical package (Cary, North Carolina). RESULTS Nine of the 10 patients initially enrolled in the study completed the trial without either adverse reactions or side effects. One subject was excluded from the study because of failure to discontinue antidepres-sant therapy. Treatment with losartan produced an 11.2% decrease in systolic blood pressure that was statistically lower (p = 0.01) than the values recorded during both the preceding placebo and washout periods (table 1). Changes in diastolic blood pressure were not significantly different between the end of the placebo and treatment periods (p > 0.05). The EC50 for extent and rate of platelet aggregation induced by the thrombin receptor agonist averaged 0.95 ± 0.23 µmol/L and 1.04 ± 0.24% (mean ± SD), respectively, in 9 healthy volunteers at the initiation of the study. These values were not different from those recorded in study patients at weeks ­2 and 0 of the placebo period (table 1). Table I. Effects of losartan treatment on hemodynamic variables and platelet aggregability Placebo Period Washout Period (wk -6) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Heart rate (beats/min) TRA EC50 extent (µmol/L) TRA EC50 rate (%) 147 ± 15 87 ± 7 72 ± 10 ND ND Wk -2 149 89 75 0.95 1.05 ± ± ± ± ± 15 7 11 0.33 0.28 Wk 0 152 90 73 0.95 1.03 ± ± ± ± ± 18 6 8 0.34 0.31 Losartan therapy (wk 4) 135 87 75 1.14 1.14 ± ± ± ± ± 9 10 8 0.29 0.28 p Value* 0.01 0.23 0.48 0.05 0.05 *The values denote statistical differenes at week 4 of losartan treatment compared with weeks -2 and 0 of placebo therapy. Values are expressed as mean ± SD of data obtained in 9 essential hypertensive subjects of corresponding time periods. ND: not determined; TRA: thrombin receptor agonist. 25 J. PAVEL LEVY y cols. In contrast, table 1 also shows that losartan produced a significant increase in the EC50 of thrombin receptor agonist-induced platelet aggregation extent and rate in hypertensive subjects. The reduction in platelet aggregability and the prolongation of the time required for platelet aggregation were significantly greater than values determined for either the same subjects at the end of the placebo run-in period or normotensive time-control subjects (fig. 1). Three of 9 patients had an increase in the EC50 of thrombin receptor agonist-induced aggregation extent and rate during the placebo pretreatment period. Seven of 9 patients (78%) had a significant increase in thrombin receptor agonist-induced aggregation extent and rate 4 weeks after initiation of losartan therapy (fig. 1). In healthy volunteers, plasma concentrations of PAI1 (47.26 ± 26.30 ng/ml) and vWF (92.61 ± 71.83%) were within the range reported in other studies 12 and not different when measurements were repeated 30 days after (p < 0.05). Baseline plasma levels of PAI-1 in hypertensive subjects averaged 78.68 ± 50.40 ng/ml during the placebo period (fig. 2). Although these values were 66% higher than those found in healthy volunteers, the difference did not attain statistical significance (p = 0.12). Losartan had no effect on plasma levels of PAI-1 (fig. 2) and vWF (fig. 3) in hypertensive subjects. Correlation analysis of observed changes in thrombin receptor agonist EC50 extent, age, gender, and changes in arterial pressure, PAI-1, and vWF were not statistically significant (table 2). Plasminogen Activator Inhibitor-1 (ng/mL) 120 100 80 60 40 20 0 Normal Week 0 Week 4 Hypertensive subjects Fig. 2.--Differences in the plasma concentrations of plasminogen activator inhibitor-1 in 9 untreated healthy volunteers (stippled bar) and 9 hypertensive subjects at week 0 (solid bar, end of placebo period) and at week 4 of losartan treatment (solid bar, week 4). Values are expressed as mean ± 1 SEM. 140 von Willebrand factor (%) 120 100 80 60 40 20 0 Normal Week 0 Week 4 Hypertensive subjects 40 *# 30 Percent Change 20 10 0 -10 -20 Placebo Losartan Normal volunteers Fig. 3.--Differences in the plasma concentrations of von Willebrand factor in normal untreated healthy volunteers (stippled bar) and hypertensive subjects at week 0 (solid bar, end of placebo period) and at week 4 of losartan treatment (solid bar, week 4). Values are expressed as mean ± 1 SEM. DISCUSSION Treatment of hypertensive subjects over 4 weeks with losartan increased the EC50 for thrombin-induced platelet aggregation by 25%. This novel effect of an Ang II antag-onist after short-term standard-dose therapy in patients with mild to moderate essential hypertension was inferred from previous experimental studies 13. Its significance was not fully appreciated because high doses of losartan were used to demonstrate this event 8-10. This study now shows an antithrombotic effect of losartan in hypertensive subjects, which could be detected at initial treatment Fig. 1.--Increase in the EC50 extent of platelet aggregation mediated by a selective thrombin receptor agonist in 9 hypertensive subjects given 50 mg/day of losartan over 4 weeks. The EC50 for thrombin-induced platelet aggregation did not change in the same subjects over the 2-week placebo period. Thrombin-induced platelet aggregation remained unchanged in 9 normal volunteers during a 30-day time control period. Values are expressed as mean ± 1 SE. * ,#p < 0.05 compared with placebo and normal volunteers. 26 INHIBITION OF PLATELET AGGREGABILITY BY LOSARTAN IN ESSENTIAL HYPERTENSION Table II. Correlation analysis between changes in thrombin receptor agonist and laboratory variables Variable Thrombin receptor agonist % EC50 extent R coefficient p Values Thrombin receptor agonist EC50 rate R coefficient p Values SBP DBP Age Gender PAI-1 vWF -0.44 0.24 -0.33 0.39 -0,21 0.59 0.03 0.94 0.39 0.11 0.28 0.26 0.17 0.49 0.29 0.24 0.09 0.82 0.13 0.74 0.20 0.61 0.39 0.29 Regression analysis (R value) of data obtained in 9 essential hypertensive subjects. DBP: diastolic blood pressure; SBP: systolic blood pressure. doses and at the time at which the maximal antihypertensive effect of the drug was not fully achieved. The reduction in platelet aggregability was independent of changes in blood pressure and gender and not accompanied by modifications in the plasma levels of PAI-1 and vWF. Clinical and laboratory data suggest that essential hypertension is associated with platelet hyper-aggregability 14-16, reduced fibrinolytic activ-ity 17, and increased plasma concentrations of vWF 18. In keeping with these findings, baseline levels of PAI-1 were 66% higher in hypertensive subjects than those found in normotensive subjects. The absence of a significant difference in baseline concentrations of PAI-1 and vWF between healthy volunteers and hypertensive subjects may be related to the small size of the sample and the inclusion of subjects with newly diagnosed hypertension and no associated clinical car-diovascular disease. Platelets play a major role in the induction of a «pro-thrombotic state» associated with high blood pressure 6. Thrombin and TxA2 are potent endogenous agonists of platelet aggregability, in part because thrombin stimulates the release of TxA2 through activation of calcium ion-sensitive phospholipase A2. In addition, Ang II stimulates the release of TxA2 from vascular tissues via amplification of a G-protein­linked arachidonic acid pathway 15. Li et al8 showed that losartan and its active metabolite EXP 3174 competed with the TxA2/prostaglandin endoper-oxide H2 receptor in isolated canine coronary arteries, in the circulation of the rat with spontaneous hyper-tension 9, and in human platelet-rich plasma in vitro 13. The competitive inhibitory effects of losartan on the TxA2 receptor are not a class effect because neither candesartan 8 nor valsartan 10 displayed these effects. On the other hand, irbesartan may share the effects of losartan on inhibiting the TxA2 receptor 10. The concept that losartan may have a beneficial antithrombotic effect, independent of its antihypertensive property, has not been widely accepted because it occurs at doses 1,000-fold higher than those required for binding to vascular AT1 receptors 8, 9, 13. Although the argument has been posed that chronic therapy with losartan (80 to 120 mg oral dose) may expose platelets to circulating drug concentrations amounting to 1 µmol/L 8, 13 this study now shows directly that losartan induced a statistically significant reduction in the extent of thrombin-mediated platelet aggregation within 4 weeks after initiation of therapy at a dose of 50 mg. These findings suggest that inhibition of platelet aggregability may be a component of the therapeutic response associated with current dosing of the Ang II antagonist. We took the precaution of determining the dose-response curve of the selective thrombin receptor agonist through a large range of concen-trations to ensure an accurate quantification of the EC50 dose. In this study, losartan elicited a 25% increase in the dose of thrombin receptor agonist required to produce a 50% response in the extent of aggre-gation compared with the values obtained in the same hypertensive subjects while taking placebo. The in-clusion of similar measurements of platelet aggregability in a group of normal volunteers, spaced 30 days apart, was an added precaution to exclude random variations in the measurements related to time. It is intriguing that the significant increase in the EC50 of aggregation could be demonstrated at the initial therapeutic dose of losartan and at a time at which the reduction in systolic but not diastolic blood pressure had attained statistical significance. Previous studies have emphasized that > 4 weeks are required to reach the nadir of the antihypertensive response 19, 20. Analysis of variance incorporating potential confounders such as gender, blood pressure, and age excluded that the losartaninduced reduction in platelet aggregability was influenced by these variables. Because it was not possible to obtain large quantities of platelet-rich plasma, we only evaluated the effects of losartan on thrombin receptor-mediated 27 J. PAVEL LEVY y cols. aggregability. Therefore, the specific mechanism mediating the antithrombotic effects of the Ang II antago-nist remains to be determined. Thrombin may trigger platelet aggregability by a direct action on platelets 21 or by increased release of TxA27, 22. Losartan may have prevented binding of Ang II to platelets, thus preventing thrombin release. Alternatively, the antiaggregation effects of losartan may be related to blockade of TxA2 receptors 8, 9. Antiplatelet effects of antihypertensive drugs are a topic of intense investigation. Diltiazem at high doses, 23 amlodipine 24, and isradipine22 have been reported to inhibit platelet aggregation. Conflicting results are reported for the effects of angiotensin-converting enzyme inhibitors on platelet function. Whereas captopril therapy may reduce platelet aggregation in response to adenosine phosphate, epinephrine, and collagen 3, a prospective, randomized, double-blind, crossover study did not confirm these findings in hypertensive subjects treated with captopril, enalapril, or fosinopril 15. Keidar et al25 reported a 31% reduction of adenosine phosphate-induced platelet aggregation after fosinopril therapy, whereas Gupta et al26 showed no effects of quinapril in essential hyper-tensive subjects. In contrast, a significant increase in the EC50 of thrombin-mediated platelet aggregability was demonstrated in nonhuman primates only when given combined treatment with fosinopril and pravastatin11. These findings suggest that inhibition of platelet aggregation may be another factor differenti-ating the mode of action of angiotensin-converting enzyme inhibitors from Ang II antagonists. REFERENCES 1. Lip GYH, Beevers DG: Abnormalities of rheology and coagulation in hypertension. J Hum Hypertens 8: 693-702, 1994. 2. Oikawa T, Freeman M, Lo W, Vaughan DE, Fogo A: Modulation of plasmin-ogen activator inhibitor-1 in vivo: a new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition. Kidney Int 51: 164-172, 1997. 3. Swartz SL, Moore TJ: Effect of angiotensin II on collagen-induced platelet activation in normotensive subjects. Thromb Haemost 63: 87-90, 1990. 4. Ware JA, Heistad DD: Seminars in medicine of the Beth Israel Hospital, Boston. Platelet-endothelium interactions. N Engl J Med 328: 628-635, 1993. 5. Strawn WB, Chappell MC, Dean RH, Kivlighn S, Ferrario CM: Inhibition of early atherogenesis by losartan in monkeys with dietinduced hypercholesterolemia. Circulation 101: 1586-1593, 2000. 6. Lip GY, Blann AD: Does hypertension confer a prothrombotic state? Virchow's triad revisited. Circulation 101: 218-220, 2000. 7. Brown NJ, Vaughan DE. The renin-angiotensin and fibrinolytic systems: co-conspirators in the pathogenesis of ischemic cardiovascular disease. Trends Cardiovasc Med 6: 239-243, 1996. 8. Li P, Ferrario CM, Brosnihan KB: Non peptide angiotensin II antagonist losartan inhibits thromboxane A2-induced contractions in canine coronary arteries. J Pharmacol Exp Ther 281: 1065-1070, 1997. 9. Li P, Ferrario CM, Brosnihan KB: Losartan inhibits thromboxane A2 -induced platelet aggregation and vascular constriction in spontaneously hypertensive rats. J Cardiovasc Pharmacol 32: 198-205, 1998. 10. Li P, Fukuhara M, Diz DI, Ferrario CM, Brosnihan KB. Novel angiotensin II AT1 receptor antagonist irbesartan prevents thromboxane A2-induced vasocon-striction in canine coronary arteries and human platelet aggregation. J Pharmacol Exp Ther 292: 238-246, 2000. 11. Hale LP, Craver KT, Berrier AM, Sheffield MV, Case LD, Owen J: Combination of fosinopril and pravastatin decreases platelet response to thrombin receptor agonist in monkeys. Arterioscler Thromb Vasc Biol 18: 1643-1646, 1998. 12. Larsson PR, Schwieler JH, Wallen NH, Hjemdahl P: Acute effects of angiotensin II on fibrinolysis in healthy volunteers. Blood Coagul Fibrinolysis 10: 19-24, 1999. 13. Liu ECK, Hedberg A, Goldenberg HJ, Harris DN, Webb ML: Dup 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/ prostaglandin H2 (TP) receptors. Prostaglandins 44: 89-99, 1992. 14. Andrioli G, Ortolani R, Fontana L, Gaino S, Bellavite P, Lechi C, Minuz P, Manzato F, Tridente G, Lechi A: Study of platelet adhesion in patients with uncomplicated hypertension. J Hypertens 14: 1215-1221, 1996. 15. Moser L, Callahan KS, Cheung AK, Stoddard GJ, Munger MA: ACE inhibitor effects on platelet function in stages I-II hypertension. J Cardiovasc Pharmacol 30: 461-467, 1998. 16. Brass LF, Ahuja M, Belmonte E, Pizarro S, Tarver A, Hoxie JA: The human platelet thrombin receptor. Turning it on and turning it off. Ann NY Acad Sci 714: 1-12, 1994. 17. Ridker PM, Gaboury CL, Conlin PR, Seely EW, Williams GH, Vaughan DE: Stimulation of plasminogen activator inhibitor in vivo by infusion of angiotensin II. Evidence of a potential interaction between the renin-angiotensin system and fibrinolytic function. Circulation 87: 1969-1973, 1993. 18. Blann AD, Waite MA: von Willebrand factor and soluble Eselectin in hypertension: influence of treatment and value in predicting the progression of atherosclerosis. Coron Artery Disease 7: 143-147, 1996. 19. Goldberg AI, Dunlay MC, Sweet CS: Safety and tolerability of losartan potassium, an angiotensin II receptor antagonist, compared with hydrochlorothiazide, atenolol, felodipine ER, and angiotensin-converting enzyme inhibitors for the treatment of systemic hypertension. Am J Cardiol 75: 793-795, 1995. 20. Ferrario CM, Flack JM. Pathologic consequences of increased angiotensin II activity. Cardiovasc Drugs Ther 10: 511-518, 1996. 21. Moore TJ, Williams GH: Angiotensin II receptors on human platelets. Circ Res 51: 314-320, 1981. 22. FitzGerald GA: Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists. Am J Cardiol 68: 11B-15B, 1991. 23. Altman R, Scazziola A, Dujovne C: Diltiazem potentiates the inhibitory effect of aspirin on platelet aggregation. Clin Pharmacol Ther 44: 320-325, 1988. 24. Hernández R, Carvajal AR, Armas-Padilla, MC, ArmasHernández MJ, Guerro J, Barragán O: The effects of oncedaily amlodipine on platelet aggrega-tion and blood pressure in patients with essential hypertension (abstr). Am J Hypertens 5 (pt. 2): 111A, 1992. 25. Keidar S, Oiknine J, Leiba A, Shapira C, Leiba M, Aviram M: Fosinopril reduces ADP-induced platelet aggregation in hypertensive patients. J Cardiovasc Pharmacol 27: 183-186, 1996. 26. Gupta RK, Kjeldsen SE, Motley E, Weder AB, Zweifler AJ, Julius S: Platelet function during antihypertensive treatment with quinapril, a novel angiotensin converting enzyme inhibitor. J Cardiovasc Pharmacol 17: 13-19, 1991. 28