Valsartan inhibits platelet activity at different doses in mild to moderate hypertensives: Valsartan Inhibits Platelets (VIP) trial

Hypertension

Valsartan inhibits platelet activity at different doses in mild to moderate hypertensives: Valsartan Inhibits Platelets (VIP) trial Victor L. Serebruany, MD,a Alex N. Pokov, MD,a Alex I. Malinin, MD,a Christopher O’Connor, MD,b Deepak L. Bhatt, MD,c Jean-Francois Tanguay, MD,d David C. Sane, MD,e and Charles H. Hennekens, MDf Baltimore, MD; Durham and Winston-Salem, NC; Cleveland, OH; Montreal, Quebec, Canada; and Miami, FL

Background Previous in vitro studies have suggested that valsartan produces significant inhibition of human platelets, probably targeting angiotensin I platelet receptors. To test whether valsartan inhibits platelet activity in mild to moderate hypertensives we conducted the randomized Valsartan Inhibits Platelets (VIP) trial. Methods and Results Seventy-five patients with mild to moderate hypertension were randomized to valsartan 80 (n = 25), valsartan 160 (n = 29), or valsartan 320 mg/d (n = 21) for 9 weeks. Platelet function was assessed at baseline, week 5, and week 9 by aggregometry, flow cytometry, and cartridge-based analyzer. Independently of dose and duration, valsartan provided early sustained significant inhibition of adenosine diphosphate–induced platelet aggregation, decreased shear-induced activation measured with PFA-100 analyzer, and diminished expression of GP IIb/IIIa activity measured by PAC-1 antibody, GPIb (CD42b), vitronectin receptor (CD51/61), P-selectin (CD62p), lysosome-associated membrane protein (CD107a), and CD40-ligand (CD154). The antiplatelet properties of valsartan were more profound in patients with diabetes (n = 28) when compared with the nondiabetic group (n = 47). In subgroup analyses of patients with diabetes there appeared to be stronger inhibition of the platelet receptors, a significant decrease of adenosine diphosphate– and collagen-induced platelet aggregation, and more profound inhibition of GP IIb/IIIa activity. Conclusions In the randomized VIP trial, valsartan produced sustained inhibition of platelet aggregation and major platelet receptors. The antiplatelet properties of valsartan were not dose or time dependent. In subgroup analyses patients with diabetes with mild to moderate hypertension tended to have greater platelet inhibition, a finding which, if confirmed in future studies suggests possible additional advantages for using valsartan in this high-risk population. (Am Heart J 2006;151:92- 9.) Hypertension and its complications, including stroke, heart failure, and end-stage renal disease are major clinical and major public health concerns throughout the world.1 Worldwide hypertension is emerging as the leading avoidable cause of premature mortality.2 In the United States, despite significant progress in the diagnosis, treatment, and prevention of hypertension-related complications, the prevalence of high blood pressure has risen to 28.3% whereas JNC VII goals are achieved in only about one third.3,4 Several recently reported randomized trials of angiotensin receptor blockers (ARBs) in patients From the aHeartDrug Research Laboratories, Johns Hopkins University, Baltimore, MD, b Duke Clinical Research Institute, Durham, NC, cCleveland Clinic, Cleveland, OH, d Montreal Heart Institute, Montreal, Quebec, Canada, eWake Forest University, WinstonSalem, NC, and fUniversity of Miami and Agatston Research Institute, Miami, FL. Submitted January 4, 2005; accepted March 2, 2005. Reprint requests: Victor L. Serebruany, MD, HeartDrug Research Laboratories, Johns Hopkins University, 7600 Osler Drive, Suite 307, Towson, MD 21204. E-mail: [email protected] 0002-8703/$ - see front matter n 2005, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2005.03.001

with hypertension,5,6 heart failure,7,8 and diabetes9,10 have demonstrated beneficial effects on cardiovascular disease morbidity and mortality. These trials have raised the possibility that ARBs may offer additional advantages over other antihypertensive drugs in some patients. In most developed countries cerebrovascular complications of hypertension are largely thrombotic. Hypertension leads to endothelial dysfunction, enhanced coagulation, depressed fibrinolysis, and platelet activation promoting the prothrombotic state.11 The platelet angiotensin receptors were described a decade ago,12,13 and the platelet model has been extensively used in many of in vitro and ex vivo studies.14,15 Activation of the platelet angiotensin receptors potentially could contribute to further progression of thrombotic events. However, whether blockade of these platelet receptors has any clinical relevance, or can be down regulated by ARBs is unclear. Valsartan (Diovan, Novartis Pharmaceutical Corp, East Hanover, NJ) is a nonpeptide, orally active ARB acting on the AT1 receptor subtype.16 In addition to vasodilatation

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and aldosterone-secreting blockade, valsartan is known to inhibit binding to the AT1 receptor in many tissues, including vascular smooth muscle, the adrenal gland, and probably platelets. Previous findings from our laboratory17 and the others18 suggest that valsartan exhibits potent antiplatelet properties. Importantly, platelet inhibition by valsartan appears to be unique and differs from the properties of other antiplatelet drugs. We designed and conducted the randomized Valsartan Inhibits Platelets (VIP) trial to compare how different doses of Diovan affect various platelet activation markers over the initial treatment period of 5 and 9 weeks in patients with mild to moderate hypertension. We performed serial measurements of multiple platelet characteristics including conventional aggregometry, a rapid cartridge-based platelet function analyzer, and also assessed 10 receptors by whole blood flow cytometry.

Methods Population Valsartan Inhibits Platelets was a randomized, double-blind trial conducted in 2 outpatient clinics in the Northern Baltimore area. The trial has been approved by Western Institutional Review Board (Olympia, WA, Protocol # 20030068). Written informed consent was obtained from all patients, including the need for multiple venipunctures, high compliance, and compensation for visits. Men and nonpregnant, nonlactating women 21 to 75 years of age were eligible for screening. Patients also had to have documented evidence of stage 1 (mild) or stage 2 (moderate) hypertension (systolic blood pressure 140-179 mm Hg, diastolic blood pressure 90 - 109 mm Hg; World Health Organization recommendations, 1999). Use of aspirin and Aggrenox was permitted but more aggressive antiplatelet strategies (eg, GP IIb/IIIa inhibitors, clopidogrel, or ticlopidine) for 1 month as well as ARBs or angiotensin-converting enzyme (ACE) inhibitors for 3 months were exclusion criteria. In addition, patients with low platelet counts (b100 000); hematocrit b30, serum creatinine z3 mg/dL, liver impairment defined as alanine aminotransferase/aspartate aminotransferase N3 times upper limit of normal; history of gastrointestinal bleeding, hematochezia, or melena within 30 days, active participation in other investigational drug or device trials within the last 30 days, or allergy or intolerance to any of the study medications were excluded. Using a table of random numbers by an independent statistical center, 75 willing and eligible patients were assigned at random to valsartan 80 mg/d (n = 25), valsartan 160 mg/d (n = 29), or valsartan 320 mg once daily (n = 21). The follow-up visits were scheduled at weeks 5 and 9 after randomization.

Samples Blood samples were obtained with a 19-gauge needle by direct venipuncture and drawn into two 7-mL vacutainer tubes at room temperature containing 3.8% trisodium citrate. The vacutainer tubes were filled to capacity and gently inverted 3 to 5 times to ensure complete mixing of the anticoagulant. The first 4 to 5 mL of blood was used for lipid profile analysis or was

Serebruany et al 93

discharged. All samples were labeled with coded number and analyzed by blinded technicians. Platelet studies were performed at baseline as well as at week 5 and week 9 after randomization. All samples have been analyzed within 15- to 45 - minute intervals after blood collection.

Platelet assessment A. Conventional optical plasma aggregometry. The blood-citrate mixture was centrifuged at 1200g for 2.5 minutes. The resulting platelet-rich plasma was kept at room temperature for use within 1 hour. The platelet count was determined in the platelet-rich plasma sample and adjusted to 3.5
108/mL with homologous platelet-poor plasma. Platelets were stimulated with 5-Amol adenosine diphosphate (ADP) and 5-Ag/mL collagen (Chronolog, Havertown, PA) and aggregation was assessed as previously described using a Chronolog LumiAggregometer (model 560-Ca) with the AggroLink software package. Aggregation was expressed as the maximal percent change in light transmittance from baseline, using platelet-poor plasma as a reference. Curves were analyzed according to international standards.19 B. Cartridge-based platelet function analyzer (PFA-100). Using the PFA-100 instrument (Dade Behring, Miami, FL), the blood-citrate mixture was aspirated under a constant negative pressure and contacts an epinephrine and collagen-coated membrane. The blood then passes through an aperture that induces high shear stress and simulates primary hemostasis after injury to a small blood vessel under flow condition. The time to aperture occlusion (the closure time) is recorded in seconds and is inversely related to the degree of shear-induced platelet activation.20 C. Whole blood flow cytometry. The surface expression of platelet receptors was determined by flow cytometry using the following monoclonal antibodies: CD41 antigen (GP IIb/IIIa); CD42b (GPIb), CD62p (P-selectin) (DAKO Corporation, Carpenteria, CA); PAC-1 (GP IIb/IIIa activity), CD31 (PECAM-1), CD51/CD61 (vitronectin receptor), CD107a (lysosome-associated membrane protein [LAMP]-1), CD154 (CD40-ligand) (PharMingen, San Diego, CA); cleaved (WEDE15) and intact (SPAN12) platelet thrombin receptors (Beckman Coulter, Brea, CA). The blood-citrate mixture (50 AL) was diluted with 450-AL Tris-buffered saline (10 mmol/L Tris, 0.15 mol/L sodium chloride) and mixed by inverting an Eppendorf tube gently 2 times. The appropriate primary antibody was then added (5 AL) and incubated at 378C for 30 minutes, and then a secondary antibody was applied if needed. After incubation, 400 AL of 2% buffered paraformaldehyde was added for fixation. The samples were analyzed on the FACScan flow cytometer (Becton Dickinson, San Diego, CA) calibrated to measure fluorescent light scatter as previously described.21 All parameters were collected using 4 - decade logarithmic amplification. The data were collected in list mode files and then analyzed. P-selectin was expressed as percent positive cells as previously described.22 Other antigens were expressed as log mean fluorescence intensity.

Statistical analysis The significance of differences between treatments arms was calculated by m2 and Fisher exact tests for discrete variables and Wilcoxon rank sum test for continuous

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Table I. Baseline characteristics in the VIP trialT Treatment group

Characteristics Age (y) Male sex Race Caucasian African American History and risk factors Coronary artery disease Stroke or TIA history Previous MI Unstable angina CHF CABG PTCA Diabetes Peripheral vascular disease Family history of CAD Current or past smoking Sedentary life style Obesity Hypercholesterolemia Concomitant medications Aspirin Aggrenox NSAIDs h-Blockers Diuretics Ca – channel blockers Oral hypoglycemic drugs Statins SSRIs Changes of blood pressure (mm Hg) Baseline Week 5 Week 9

Valsartan 80 mg (n = 25)

Valsartan 160 mg (n = 29)

Valsartan 320 mg (n = 21)

68.1 F 5.9 7 (28)

66.9 F 7.6 11 (38)

67.2 F 6.1 7 (33)

24 (96) 1 (4)

28 (97) 1 (3)

21 (100) -

11 3 6 6 3 1 8 5 4 4 4 14 7 7

(44) (12) (24) (24) (12) (4) (32) (20) (16) (16) (16) (56) (28) (28)

13 (44.8) 2 (7) 4 (14) 5 (17) 1 (4) 5 (17) 8 (27) 13 (45) 1 (4) 8 (27) 8 (27) 16 (55) 7 (24) 14 (48)

8 2 4 2 5 4 3 10 1 6 1 9 3 9

(38) (10) (19) (10) (24) (19) (14) (47) (5) (29) (5) (43) (14) (43)

14 1 12 6 13 6 5 6 4

(56) (4) (48) (24) (52) (24) (20) (24) (16)

19 2 10 7 14 10 13 13 11

8 1 5 2 11 7 7 7 7

(38) (5) (24) (10) (52) (33) (33) (33) (33)

163 F 13/89 F 11 152 F 11/86 F 8T 147 F 13/84 F 7T

(66) (7) (35) (24) (48) (35) (45) (45) (38)

160 F 18/91 F 9 149 F 11/84 F 8T 145 F 12/83 F 7T

162 F 11/91 F 11 146 F 12/82 F 9T 142 F 13/82 F 7T

Values are given as number (percentage) or mean F SD. CABG, Coronary artery bypass grafting; CAD, coronary artery disease; CHF, congestive heart failure; MI, myocardial infarction; NSAIDs, nonsteroid antiinflammatory drugs; PTCA, percutaneous transluminal coronary angioplasty; SSRI, nonselective serotonin reuptake inhibitors; TIA, transient ischemic attack. TP value b.05 versus baseline.

variables. The significance of differences between individual flow cytometric histograms was calculated using the Kolmogorov-Smirnov test incorporated in the CELLQuest’ (Becton Dickinson, San Diego, CA) software. Statistical analyses were performed using SPSS/E11.5 (SPSS, Inc, Chicago, IL). To control for any baseline differences analysis of variance was used. All P values are 2 sided.

Results Patients Among the 75 randomized patients treated and followed up for 9 weeks there were no deaths or serious adverse events, including symptoms attributable to hypertension. Two patients from the valsartan 160 mg group and one from the valsartan 320 mg group had

headaches or/and mild dizziness during the first several days of therapy. Table I shows baseline pretreatment distribution of demographics, risk factors, clinical characteristics, concomitant medications, and changes of blood pressure in 3 arms. At baseline there were no statistically significant differences between the treatment groups. Some small differences observed are not unexpected because of the relatively small sample size of particular note, aspirin was used by 56%, 66%, and 38% of the 80 -, 160 -, and 320 - mg groups, respectively. As expected, valsartan produced large and significant decreases in systolic and diastolic blood pressure within each group but the between group differences were small and not significant.

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Table II. Changes of platelet characteristics in the VIP Trial Baseline Conventional aggregometry ADP (5 Amol/L)-induced platelet aggregation Valsartan 80 mg/d 68.2 F Valsartan 160 mg/d 63.0 F Valsartan 320 mg/d 67.0 F Collagen (5 Ag/mL)-induced platelet aggregation Valsartan 80 mg/d 61.5 F Valsartan 160 mg/d 67.1 F Valsartan 320 mg/d 65.2 F PFA-100 (epinephrine/collagen cartridge) Valsartan 80 mg/d 233.4 F Valsartan 160 mg/d 225.2 F Valsartan 320 mg/d 234.4 F Flow cytometryy CD31 Valsartan 80 mg/d 62.3 F Valsartan 160 mg/d 61.3 F Valsartan 320 mg/d 60.1 F CD41 Valsartan 80 mg/d 416.1 F Valsartan 160 mg/d 413.7 F Valsartan 320 mg/d 408.5 F PAC-1 Valsartan 80 mg/d 7.2 F Valsartan 160 mg/d 6.9 F Valsartan 320 mg/d 7.1 F CD42 Valsartan 80 mg/d 252.5 F Valsartan 160 mg/d 245.5 F Valsartan 320 mg/d 247.9 F CD51/61 Valsartan 80 mg/d 9.0 F Valsartan 160 mg/d 8.6 F Valsartan 320 mg/d 9.3 F CD62p, % of positive cells Valsartan 80 mg/d 9.3 F Valsartan 160 mg/d 10.3 F Valsartan 320 mg/d 10.2 F CD107a Valsartan 80 mg/d 6.1 F Valsartan 160 mg/d 5.9 F Valsartan 320 mg/d 6.4 F CD154 Valsartan 80 mg/d 5.4 F Valsartan 160 mg/d 5.5 F Valsartan 320 mg/d 5.7 F PAR-1 (WEDE) thrombin receptor Valsartan 80 mg/d 28.6 F Valsartan 160 mg/d 29.9 F Valsartan 320 mg/d 32.4 F PAR-1 (SPAN-12) thrombin receptor Valsartan 80 mg/d 18.4 F Valsartan 160 mg/d 18.2 F Valsartan 320 mg/d 17.2 F

Week 5

Week 9

7.6 11.5 13.9

59.2 F 11.5T 57.2 F 12.1T 57.3 F 10.6T

61.7 F 8.6T 60.1 F 7.5 61.0 F 15.0T

12.6 8.9 11.2

56.0 F 11.6T 61.3 F 13.2T 61.5 F 14.1

58.0 F 10.2 63.5 F 12.5 60.3 F 12.7

49.3 49.3 49.6

256.5 F 48.1T 275.2 F 32.6T 268.3 F 44.3T

262.7 F 48.2T 251.0 F 63.3 275.3 F 48.4T

9.4 8.8 8.2

60.7 F 8.7 59.6 F 9.9 63.1 F 11.0

64.7 F 9.3 61.5 F 10.6 62.0 F 10.6

71.5 72.0 81.7

391.5 F 71.9 362.1 F 74.9T 403.9 F 78.7

384.8 F 63.1 354.2 F 60.2T 389.2 F 115.3

2.4 1.9 2.2 69.0 59.2 63.1

6.9 F 1.5 5.6 F 1.9Tz 6.8 F 1.4 232.3 F 70.6 261.3 F 59.8 230 F 78.4

6.6 F 2.4 5.7 F 2.3T 6.1 F 2.3 262.0 F 105.1 254.3 F 59.0 239.1 F 61.0

1.8 1.7 2.2

6.9 F 1.5T 6.6 F 2.0T 6.3 F 1.3T

7.5 F 1.7T 6.8 F 1.7T 6.7 F 2.1T

3.7 2.7 3.4

8.3 F 2.9 7.7 F 3.2T 7.6 F 2.5T

7.6 F 3.6T 6.9 F 2.9T 7.4 F 3.1T

1.8 2.2 1.9

3.5 F 1.2T 3.4 F 1.4T 3.6 F 1.5T

4.1 F 1.8T 3.4 F 1.4T 2.7 F 1.2Tz

1.1 1.7 1.1

4.6 F 1.4T 4.4 F 1.8T 4.6 F 1.4T

4.6 F 1.0T 4.9 F 1.4 4.6 F 1.3T

11.4 11.0 10.8

25.0 F 9.8 24.9 F 9.8T 28.9 F 11.9

26.5 F 7.8 25.2 F 10.5 29 F 10.4

5.5 4.8 3.9

18.2 F 5.7 17.2 F 5.1 16.7 F 5.7

17.5 F 4.2 18.2 F 6.2 17.3 F 4.6

Aggregation was expressed as the maximal percentage change in the light transmittance from baseline, using platelet-poor plasma as reference. TP value b.05 versus baseline. yValues are given as mean log of fluorescence intensity unless otherwise indicated. zP value b.05 versus valsartan 80 mg group.

Platelet findings The data on platelet characteristics in the 3 treatment groups are presented in Table II.

At baseline, platelet activity was remarkably elevated in most patients. Therapy with valsartan resulted in substantial inhibition of platelet activity as reflected by

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Table III. Changes of platelet characteristics in patients with diabetes versus patients without diabetes

Conventional aggregometry ADP (5 Amol/L)-induced platelet aggregation Patients with diabetes Patients without diabetes Collagen (5 Ag/mL)-induced platelet aggregation Patients with diabetes Patients without diabetes PFA-100 (epinephrine/collagen cartridge) Patients with diabetes Patients without diabetes Flow cytometryz CD31 (PECAM) Patients with diabetes Patients without diabetes CD41 Patients with diabetes Patients without diabetes PAC-1 Patients with diabetes Patients without diabetes CD42 Patients with diabetes Patients without diabetes CD51/61 Patients with diabetes Patients without diabetes CD62p, % positive cells Patients with diabetes Patients without diabetes CD107a Patients with diabetes Patients without diabetes CD154 Patients with diabetes Patients without diabetes PAR-1 (WEDE) thrombin receptor Patients with diabetes Patients without diabetes PAR-1 (SPAN-12) thrombin receptor Patients with diabetes Patients without diabetes

Baseline

Week 5

Week 9

68.6 F 14.3 64.3 F 8.6

53.9 F 11.8Ty 60.1 F 10.5T

58.4 F 10.1T 62.4 F 10.0

66 F 9.2 64 F 11.9

58.0 F 15.3T 60.6 F 11.3T

57.3 F 12.1Ty 62. 8 F 11.3

219.4 F 51.4 236.2 F 46.8

274.7 F 36.1T 262.2 F 44.4T

265.3 F 51.0T 258.4 F 57.1T

60.7 F 8.4 61.7 F 8.9

61.3 F 8.6 60.4 F 10.5

59.2 F 9.6 64.4 F 10.3

410.8 F 69.2 415.4 F 76.2

358.8 F 67.2Ty 397.2 F 80.9

356.3 F 73.2T 384.1 F 82.3T

6.8 F 2.0 7.2 F 2.1

6.1 F 1.8 6.5 F 1.7

255.7 F 61.8 244.3 F 63.2

256.4 F 62.8 235.4 F 72.4

253.8 F 52.9 252.9 F 88.5

8.7 F 2.0 9.0 F 1.9

6.2 F 1.5T 6.9 F 1.7T

6.6 F 1.5T 7.2 F 1.9T

10.9 F 3.3 9.3 F 3.1

8.2 F 2.9T 7.7 F 2.9T

7.4 F 2.9T 7.2 F 3.4T

6.0 F 1.9 6.2 F 2.0

3.8 F 1.8T 3.3 F 1.4T

3.1 F 1.5T 3.6 F 1.6T

5.5 F 1.4 5.5 F 1.3

4.2 F 1.3T 4.8 F 1.3

4.4 F 1.1T 4.9 F 1.3

32.6 F 10.7 28.5 F 10.9

26.8 F 8.4T 25.5 F 11.6

24.4 F 8.6T 27.9 F 9.9

18.6 F 3.8 17.6 F 5.3

16.1 F 5.5 18.2 F 5.2

16.0 F 5.5 18.5 F 4.7

5.2 F 1.9Ty 6.7 F 2.5

Aggregation was expressed as the maximal percentage change in the light transmittance from baseline, using platelet-poor plasma as reference. PECAM, Platelet/endothelial cell adhesion molecule. TP value b.05 versus baseline. yP value b.05 versus nondiabetic group. zValues are given as mean log of fluorescence intensity unless otherwise indicated.

conventional aggregometry, cartridge-based platelet analyzer, and the whole blood flow cytometry. Such inhibition of platelet biomarkers in general was not dependent on valsartan dose or treatment duration. Treatment with valsartan 80 to 320 mg/d in patients with mild to moderate hypertension resulted in a consistent inhibition of ADP-induced conventional platelet aggregation, with the strong earlier trend towards decreased collagen-induced aggregation. Rapid platelet analyzers revealed consistently prolonged closure time by PFA-100 instrument at week 5 after treatment began. There were numerous down

regulations of the platelet surface receptor expression found after initiation of valsartan therapy, such as blockade of GPIb and GP IIb/IIIa activity measured with PAC-1 antibody (for valsartan 160 mg/d only), inhibition of vitronectin receptor, P-selectin, LAMP-1, and CD40ligand. Expression of PECAM-1, GP IIb/IIIa antigen level, and both intact and cleaved thrombin receptor expressions were not affected by valsartan.

Subgroup analyses of patients with diabetes There were 28 patients with type 2 diabetes mellitus and 47 patients without diabetes enrolled in the trial.

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Differences in the platelet characteristics between patients with and without diabetes before and after treatment with valsartan are presented in Table III. Overall, baseline platelet characteristics in patients with diabetes suggest more activation when compared with the nondiabetic cohort as assessed by higher platelet aggregability in response to ADP and collagen, shorter closure time with PFA-100 analyzer, and increased GP IIb/IIIa receptor expression. Treatment with valsartan resulted in a significant inhibition of ADPinduced conventional platelet aggregation at week 5 and significant decrease of collagen-induced aggregation at week 9. Therapy with valsartan was associated with additional inhibition of GP IIb/IIIa antigen expression at week 5 and GP IIb/IIIa activity at week 9 when compared with those changes observed in patients without diabetes.

Discussion The VIP trial provides, to our knowledge, the first randomized data of the magnitude and time course of different doses of valsartan in patients with mild to moderate hypertension on inhibition of platelet bio marker activity after 9 weeks. The findings are consistent regardless of the method used for assessing platelet function. Applying a wide panel of techniques minimizes the error by measuring different parameters indicative of various platelet characteristics. In the present trial, the antiplatelet activity of valsartan was documented by conventional optical aggregometry induced by several agonists and by the PFA-100 platelet analyzer designed specifically to assess shear-induced activation. In addition, we used whole blood flow cytometry techniques measuring expression of multiple receptors located on the platelet surface. Considering the marked heterogeneity of platelet activity among and within the groups, we used multiple tests to comprehensively assess platelet function to ensure adequate evaluation of platelets. Based on the totality of currently available evidence, it is reasonable to expect valsartan to exhibit antiplatelet properties. Interestingly, ATIII platelet receptors appear to be regulated differently than ATIII receptors found in mononuclear leukocytes.23 Platelet receptor messenger RNA levels were inversely correlated with plasma angiotensin II levels, whereas mononuclear leukocyte receptor mRNA levels were positively correlated with plasma angiotensin II levels in patients with primary hypertension. In contrast, in secondary hypertension both platelets and mononuclear leukocytes ATIII receptor mRNA, which were elevated, then decreased after removal of the adrenal tumor or correction of the renal artery stenosis.24 Angiotensin II stimulation significantly increases platelet-free calcium concentrations, intracellular pH,

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and thrombin-induced platelet aggregation in the untreated hypertensive and normotensive patients.25 The release of angiotensin II after aggregant stimulation suggests that this may be one of the mechanisms through which platelets can locally modulate vascular tone and promote atherogenesis.26 The infusion of angiotensin II increased plasma h-thromboglobulin levels, reflecting a-granule secretion, and expression of P-selectin.27 Not surprisingly, ARBs are capable of reducing or even blocking such harmful effects. For instance, ARBs may act as weak but competitive antagonists at human platelet thromboxane A2/prostaglandin H2 receptors, therefore, diminishing platelet aggregation induced by thromboxane.28 The antiplatelet properties of the earlier generation ARBs such as losartan were investigated as well. Losartan in vitro is capable of diminishing thromboxane- and ADP-stimulated platelet aggregation in a dose-dependent manner.29 Interestingly, these data are supported by our previous in vitro17 and index ex vivo results; however, platelet inhibition with valsartan was not dose dependent nor time dependent. Indeed, the lowest valsartan dose (80 mg/d) provided a similar degree of platelet inhibition as the higher (160-320 mg/d) doses. Such effects were already observed at week 5, and most platelet biomarkers were not inhibited more strongly at week 9. Our randomized data in human beings are consistent with those from the previous animal study suggesting that the antiplatelet effects of ARBs do not appear to be related to the level of blood pressure reduction.30 Another ARB, irbesartan, affects platelets in a manner similar to losartan and valsartan,31,32 whereas candesartan and its active metabolite failed to reduce platelet activation.33 Another attractive theory links antiplatelet properties of ARBs with the modulation of nitric oxide release.18,34,35 However, these data are based on experimental in vitro work, and their potential clinical significance remains unclear. The index data also is in agreement with the predominantly favorable effects of ACE inhibitors on the platelet function.36 However, there are no data with regard to the comparative antiplatelet properties of ARBs and ACE inhibitors, which may be similar, and warrant further investigation.37 It is known that therapy with valsartan is associated with the reduction of plasma levels of CRP.38 Therefore, it seems important in the future to correlate platelet biomarkers with those reflective of cardiovascular prognosis. The data from the VIP trial suggest that the antiplatelet benefits of valsartan may extend beyond simply diminishing platelet aggregation and modulating specific receptors. Inhibition of GPIb, P-selectin, and platelet vitronectin receptor strongly suggest that valsartan specifically targets platelet-endothelial cross-talk precluding vascular wall-platelet interactions. It is plausible to speculate that based on the VIP data, valsartan

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may potentially override insufficient platelet inhibition or/and not adequate antithrombotic properties of mono therapy with aspirin, or newly discovered antiplatelet agents such as selective serotonin reuptake inhibitors or statins. This hypothesis requires further research as data are sparse describing the effects of valsartan on platelet function on top of the above-mentioned medications. Most of these data are derived from in vitro tests, animal experiments, uncontrolled human studies in healthy volunteers, and patients with hypertension. There is no evidence that use of ARBs increases the risk of bleeding events; therefore, they can be safely used in combination with aspirin or/and clopidogrel. Lack of clinically meaningful hemorrhagic episodes associated with ARBs represents a major clinical advantage of this class of agents when used in high-risk patients with vascular disease requiring aggressive antiplatelet strategies. Several intriguing hypotheses may be formulated from VIP. First, the effects of the low-dose valsartan (80 mg/d) were similar to that of the higher (160-320 mg/d) doses. Treating with valsartan for 5 weeks was sufficient to yield the comprehensive antiplatelet benefit as well, with no remarkable enhancement at week 9. In subgroup analyses of patients with diabetes, an antiplatelet advantage of valsartan was apparent and if confirmed in future studies suggests possible additional advantages for using valsartan in this high-risk population. Finally, our previous in vitro study data17 are consistent with the present randomized data in human beings suggesting that valsartan inhibits platelet aggregation, prolongs the closure time with the PFA-100 analyzer, and also down regulates the expression of GPIb, GP IIb/IIIa, P-selectin, vitronectin (CD51/61), and LAMP-1 (CD107a) receptors. Considering the specificity of the receptors blocked, it seems that valsartan is indeed inhibiting the platelet-endothelial cross-talk diminishing the platelet-vascular wall interactions.

Strengths and limitations The strengths of the VIP trial include the randomized design, comprehensive laboratory assessment, long-term monitoring, and serial blood sampling for platelet analyses performed in a single core laboratory facility. Several limitations also merit mention. First, the trial was of a relatively small sample size, so chance remains a plausible alternative explanation for the observed findings. On the other hand, considering high costs and mandatory specialized personnel training to conduct platelet research, such studies rarely are able to enroll N75 patients. High frequency of the use of concomitant medications may have affected the platelet characteristics; however, the use of major drugs was similar between the groups, represents the attractive breal lifeQ clinical setting, except perhaps for aspirin which, when controlled, had no material effect upon the results. In fact, use of aspirin strengthens the study suggesting

additive antiplatelet effects of valsartan on top of those of aspirin; moreover, it will be not ethical to restrict aspirin use in such high-risk population. The study was done predominantly in the Caucasian patients; however, considering racial differences in clinical outcomes in ARB trials, higher prevalence of diabetes and hypertension in the African American population, it will be important to investigate how valsartan affects platelets in black patients. The expression of multiple activationdependent platelet receptors was studied, but their individual roles in patients with hypertension are unknown. Finally, recent advances in platelet function assessment by cartridge-based analyzers and flow cytometry techniques are expanding our knowledge of antiplatelet properties beyond conventional aggregometry. Therefore, the benefits of valsartan in an expanding array of clinical conditions, including hypertension and diabetes, may be indeed related to platelet inhibition via surface receptor blockade, despite small or negligible differences in platelet aggregation. In summary, this randomized trial suggests that valsartan produced sustained platelet inhibition already at a low dose (80 mg/d) starting at week 5. This effect was not dose or time dependent over 9 weeks. Whether valsartan reduces occlusive vascular events via modulation of additional alternative pathways of platelet inhibition in patients with hypertension, diabetes, myocardial infarction, or ischemic stroke requires further research. The study was supported in part by Novartis Pharmaceuticals, (East Hanover, NJ). We thank all the nurses and laboratory personnel for their outstanding assistance. Drs. Serebruany and Malinin are listed as inventors in the U.S. patent application (USN 60/395,014): b Methods for inhibiting platelet activity with valsartan, and valeryl 4-hydroxy-valsartan.Q

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