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Journal of Human (2008) 22, 303–310 & 2008 Nature Publishing Group All rights reserved 0950-9240/08 $30.00 www.nature.com/jhh ORIGINAL ARTICLE Effects of , a inhibitor, on biomarkers of activity, and fibrinolysis in subjects with multiple risk factors for vascular disease

VL Serebruany1, A Malinin1, G Barsness2, J Vahabi3 and D Atar3 1HeartDrugt Research Laboratories, Johns Hopkins University, Baltimore, MD, USA; 2Mayo Clinic, Rochester, MN, USA and 3Division of Cardiology, Faculty of Medicine, Aker University Hospital, University of Oslo, Oslo, Norway

Aliskiren, an octanamide, is nonpeptide, low molecular significantly affected. Spiking whole with the weight, orally active renin inhibitor effectively prevent- higher aliskiren doses was associated with various ing and release. This has trends in biomarker activity, where 1000 ng mlÀ1 con- been recently approved for the treatment of hyperten- centration mostly decreased (7/33), and 2000 ng mlÀ1 sion. Considering potential links between hypertension, mostly increased (6/33) some biomarkers. In the ther- , the coagulation cascade and fibrinolysis we apeutic of 500 ng mlÀ1 aliskiren does not sought to evaluate the effect of aliskiren on human affect hemostatic biomarkers, except for a moderate but biomarkers of hemostasis. In vitro effects of whole highly significant (P ¼ 0.003) increase of AT-III activity. blood preincubation with escalating of Higher aliskiren doses were associated with more aliskiren (500, 1000 and 2000 ng mlÀ1) were assessed in profound biomarker changes, but they are likely not to 20 aspirin-naive volunteers with multiple risk factors for be clinically relevant since they show diverging (that is, vascular disease. A total of 33 biomarkers were both mild antiplatelet and platelet-activating) trends, measured, of which 18 are related to platelet function, and considering the 2- to 4-fold safety margin. It is 12 to coagulation and 3 to fibrinolysis. Pretreatment of suggested that properties of aliskiren be blood samples with aliskiren 500 ng mlÀ1 resulted in a explored further in an ex vivo clinical setting. significant increase of antithrombin-III (AT-III) activity Journal of Human Hypertension (2008) 22, 303–310; (P ¼ 0.003). All other tested biomarkers were not doi:10.1038/jhh.2008.2; published online 14 February 2008

Keywords: aliskiren; renin inhibitor; platelets; coagulation; fibrinolysis; hypertension; cardiovascular risk factors

Introduction blockers,4,5 and a concomitant improvement in hypertension control. As long as hypertension has Being a disease with a worldwide prevalence of over been known as a major risk factor for vascular 1 billion patients and contributing to 7.1 million disease,6 platelet activation and thrombus formation deaths per year, arterial hypertension is a major risk have also been identified as critical elements in the factor for cardiovascular mortality throughout the 1 and natural course of cardiovascular world. Arterial hypertension has been known for disease and stroke. Conceptually, it may seem centuries; however, effective approaches of its somewhat surprising that a condition characterized prevention and treatment do not date back more by hemodynamic vascular stress and abnormal than 50 years. During these last five decades the blood flow under high pressure is associated with medical community has witnessed a tremendous complications that are most often thrombotic rather evolution in antihypertensive pharmacotherapy, 7,8 2 3 than hemorrhagic. In this context, it is noteworthy starting off with and b-blockers the that several non-antiplatelet diminish platelet subsequent discovery of angiotensin converting function and effectively yield additional clinical (ACE) inhibitors and angiotensin receptor benefits in patients with vascular disease,9,10 whereas other agents with a pro-thrombotic capacity Correspondence: Dr VL Serebruany, Department of Neurology, could be harmful for such patients.11 Therefore, it is Osler Medical Center, 7600 Osler Drive, Suit 307, Towson, MD critical that novel antihypertensive drugs not only 21204, USA. provide effective control of , but also E-mail: [email protected] Received 4 October 2007; revised 28 November 2007; accepted 9 exhibit at least a neutral effect on platelets, coagula- January 2008; published online 14 February 2008 tion and fibrinolysis. Aliskiren and biomarkers V Serebruany et al 304 Aliskiren (SPP100, Tekturna and Rasilez) is a variations. All specimens were obtained by veni- novel blood pressure lowering acting as puncture after a 4.5-ml discard sample. an oral renin inhibitor with an antihypertensive Fresh solutions of aliskiren were prepared ex potential that exceeds angiotensin receptor block- tempore on the morning during which biomarker ers.12 Its antithrombotic properties remain largely studies were performed. Since aliskiren is known to unknown. Considering the above mentioned estab- provide a direct effect on renin in plasma, we did lished links between hypertension and hemostasis, not test aliskiren metabolites in our study. Being a the purpose of this study was to evaluate the effects small-molecule long-acting renin inhibitor, over of aliskiren on biomarkers of platelet function, 80% of the drug do not bind to plasma proteins, coagulation and fibrinolysis in vitro in healthy and is eliminated unchanged in urine and feces. volunteers with multiple cardiovascular risk factors. Tree tubes were incubated with aliskiren for 60 min Moreover, renin per se may directly enhance indices at 37 1C to achieve final concentrations of 500, 1000 of hemostasis by increasing platelet activity via and 2000 ng mlÀ1. Another tube was incubated with increase of intraplatelet calcium content, coagula- buffer solution to serve as an internal control or tion biomarkers by activating kallikrein–kinin sys- baseline. The concentration of 500 ng mlÀ1 is tem and blocking plasminogen activator inhibitors achieved after oral administration 300 mg of aliski- À1 (PAIs); therefore, diminishing fibrinolytic capacity. ren (steady state Cmax of 321 ng ml ), whereas the The primary objective of the index study was to concentration of 2000 ng mlÀ1 has been selected to indirectly assess potential worsening of vascular test drug safety in a fourfold margin (data on file, outcomes, which are known to be associated with Novartis PK&PD division). To avoid possible ob- the activation of platelet/coagulation biomarkers server bias, blood samples were coded and blinded. and depression of fibrinolysis. On the other hand, Sampling procedures and biomarker measurements we do not want subjects in our study with the pre- were performed by individuals unaware of the existing platelet activation, and/or those receiving protocol or drug concentration assignment. antithrombotic including aspirin. Therefore, healthy subjects with multiple risk factors for the vascular disease represent the best Platelet biomarkers available cohort. Optical aggregation. The whole blood–citrate mix- ture was centrifuged at 1200 g for 5 min to obtain platelet-rich plasma, which was kept at room Materials and methods temperature for use within 1 h. Platelet counts were determined for each sample with a Coulter Counter Subjects ZM (Coulter Co., Hialeah, FL, USA). Platelet Twenty volunteers donated blood for the study. All numbers were adjusted to 3.50 Â 108 mlÀ1 with of them underwent pre-employment drug screening. homologous platelet-poor plasma. Platelet aggrega- Apparently healthy men and women X21 years of tion was induced by 5 mM adenosine diphosphate age with more than two of the following risk factors and 5 mgmlÀ1 collagen obtained from Chronolog such as family history of coronary disease, sedentary Corporation (Havertown, PA, USA). Aggregation lifestyle, mellitus, hypertension, obesity, studies were performed using a 4-channel Chron- hypercholesterolemia, postmenopausal or post-oo- olog Lumi-Aggregometer (model 560—Ca). Aggrega- phorectomied women, or current or recent smoking. tion was expressed as the maximum percentage of Exclusion criteria were a history of bleeding light transmittance change (% max) from baseline at diathesis, stroke, major surgery or significant trauma the end of the recording time, using platelet-poor in the past 6 months; blood pressure 4200/ plasma as a reference. Aggregation curves were 110 mm Hg; receiving aspirin or any medications recorded for 6 min and analyzed according to the containing aspirin, or any antiplatelet therapy internationally established standards.13 including GPIIb/IIIa inhibitors or thienopyridines within the past 2 weeks; history of platelet count Impedance aggregation. The whole blood–citrate o100 Â 109 lÀ1 or blood dyscrasia. None of these mixture was diluted 1:1 with 0.5 ml phosphate- subjects were treated with . buffered saline and gently swirled. The sample was allowed to warm to 37 1C for 5 min then transferred to the assay well. The electrode was then placed in Samples the cuvette, and platelet aggregation was stimulated Blood samples were obtained for platelet aggrega- with 1 mgmlÀ1 collagen. The change in electrical tion, coagulation, fibrinolytic and flow cytometric impedance was recorded as described previously, studies. All subjects underwent blood sampling that is, utilizing a Chronolog whole blood aggreg- after at least 30 min of rest and 2 h of fasting. Eight ometer.14 tubes (4.5 ml and containing 3.8% sodium citrate) of blood for a total of 36 ml of whole blood–citrate The ultegra analyzer. The device Accumetrics (San mixture were collected from each participant be- Diego, CA, USA) is a cartridge-based rapid analyzer tween 0800 and 1000 hours to avoid diurnal using adenosine diphosphate as agonist is an optical

Journal of Human Hypertension Aliskiren and biomarkers V Serebruany et al 305 detection system, which measures platelet-induced previously.17 Other antigens were expressed as log aggregation as an increase in light transmittance. mean fluorescence intensity. When the activated platelets are exposed to the fibrinogen-coated microparticles, agglutination oc- curs in proportion to the number of available Coagulation biomarkers platelet receptors. The whole blood–citrate mixture Von Willebrand factor was measured by an Asser- is being added to the cartridge, and agglutination achrom enzyme-linked immunosorbent assay between platelets and coated beads is being (Diagnostica Stago Inc., Parsippany, NJ, USA). recorded,15 and the results are reported as platelet Antithrombin-III (AT-III) level was determined by activation units. The data mirror turbidometric the Sta Stachrom-AT III chromogenic assay platelet aggregation and reflect the degree of platelet (Stago). The level of AT-III antigen and total protein activity. S was measured by an immuno-turbidimetric assay (Liatest, Stago); reptilase time, functional protein C and functional protein S was measured by Sta and PFA-100 analyzer. Using the PFA-100 instrument Staclot clot-based assays (Stago). The quantitative (Dade Behring Inc., Miami, FL, USA), the blood– determination of fibrinogen was performed by the citrate mixture is aspirated under a constant nega- Clauss method applying the Sta fibrinogen reagent tive pressure and contacts an epinephrine- and and Sta analyzers. collagen-coated membrane. The blood then passes through an aperture that induces high shear stress and simulates primary hemostasis after injury Biomarkers of fibrinolysis to a small blood vessel under flow condition. The Antiplasmin and plasminogen levels were deter- time to aperture occlusion (the closure time) is mined by the Sta Stachrom antiplasmin and recorded in seconds and is inversely related to Stachrom plasminogen chromogenic assays, and 16 the degree of shear-induced platelet activation. fibrinogen degradation products were measured by All assays were performed in duplicate. An electro- FDP plasma latex agglutination assays (All Stago). nic quality control test was performed on each instrument every day before performing any patient samples. Statistical analysis Analysis of variance was used to define significance Flow cytometry. The surface expression of platelet of differences in biomarkers between baseline receptors was determined by the whole blood flow and post-aliskiren incubation. The Mann–Whitney cytometry using the following monoclonal antibo- U-test was used to analyze nonparametric data. dies: CD 41 antigen (GP IIb/IIIa), CD 42b (GP Ib), CD Normally distributed data were expressed as ± ± 62p (P-selectin), PAC-1 (GP IIb/IIIa activity), CD 31 mean s.e. (m s.e.), and skewed data as median (platelet/endothelial cell adhesion molecule (PE- (range). All P-values were two sided. All analyses CAM)-1), CD 51/CD 61 (vitronectin receptor), CD 63 were performed using the SPSS v9.0 program (SPSS (LIMP or LAMP-3), CD 107a (LAMP-1), CD154 CD40- Inc., Chicago, IL, USA). ), CD 165 (GP37) (PharMingen, San Diego, CA, USA); CD36 (thrombospondin, GPIV), WEDE15 Results and SPAN12 thrombin receptor epitopes (Beckman Coulter, Brea, CA, USA). Formation of platelet– Subjects leukocyte aggregates was assessed by dual labeling Demographic data and risk factors are presented in with pan-platelet marker (CD151), and then with Table 1. Participants were predominantly Caucasian CD14, the macrophage receptor for endotoxin young- or middle-aged men. Most of the volunteers lipopolysaccharides. The blood–citrate mixture had a history of smoking and attained to sedentary (50 ml) was diluted with 450 ml Tris-buffered saline life style, whereas obesity, disorders of lipid meta- (10 mmol lÀ1 Tris, 0.15 mol lÀ1 sodium chloride) and bolism and family history of vascular disease were mixed by inverting an Eppendorf tube gently two less frequent. times. The appropriate primary antibody was then added (5 ml) and incubated at 37 1C for 30 min, and then a secondary antibody was applied if needed. Biomarker data After incubation, 400 ml of 2% buffered paraformal- The combined results of platelet, coagulation and dehyde was added for fixation. The samples were fibrinolytic biomarkers when compared with the analyzed on a Becton Dickinson FACScan flow reference values are summarized in Tables 2 and 3. cytometer (San Diego, CA, USA) measuring fluor- Pretreatment of blood samples with the therapeutic escent light scatter as described previously.8 All aliskiren dose of 500 ng mlÀ1 resulted in a significant parameters were collected using four-decade loga- increase of AT-III activity (P ¼ 0.003). All other rithmic amplification. The data were collected in platelet-coagulation biomarkers were unaffected. list mode files and then analyzed. P-selectin was Spiking samples with the higher aliskiren concentra- expressed as percent positive cells as described tions was associated with various counter directed

Journal of Human Hypertension Aliskiren and biomarkers V Serebruany et al 306 Table 1 Demographics and risk factors in participating subjects trends in biomarker activity. The 1000 ng mlÀ1 con- centration was associated with some changes Parameter n ¼ 20 of platelet biomarkers suggestive of mild platelet inhibition as measured by decreased adenosine Age (years) 36.3±8.6 Male 14 (70%) diphosphate- and collagen-induced aggregation, pro- longation of the closure time with the PFA-100 Ethnic origin Platelet Analyzer, diminished platelet activation Caucasian 17 (85%) units as reflected with the Ultegra Rapid Analyzer, African-American 3 (15%) reduction of GP IIb/IIIa activity when measured with Risk factors a specific PAC-1 antibody and decreased formation of Family history 7 (35%) platelet–monocyte aggregates. Incubation of whole Sedentary lifestyle 12 (60%) blood with aliskiren at 1000 ng mlÀ1 was not asso- Obesity 7 (35%) ciated with any changes in coagulation biomarkers, Current or past smoking 12 (60%) Hypercholesterolemia 4 (20%) with the exception of the consistently increased AT-III activity, which was observed for each tested

Table 2 Platelet activity biomarkers

Parameter Aliskiren concentration Reference range

Baseline 500 ng mlÀ1 1000 ng mlÀ1 2000 ng mlÀ1

Conventional plasma aggregometry ADP 5 mM, % 69.1±5.0 66.6±4.1 61.0±7.9 68.6±5.7 66–81 P-value NS 0.001 NS Collagen 5 mgmlÀ1, % 78.6±7.2 79.0±5.9 63.4+5.3 65.6±4.8 65–82 P-value NS o0.0001 o0.0001

Whole blood aggregometry ADP 5 mM, ohms 26.6±8.1 25.9±6.6 27.4±5.7 24.0±6.3 22–28 P-value NS NS NS

Cartridge-based rapid analyzers PFA-100, Closure time CADP Cartridge, s 220±43 231±38 269±44 211±35 185–245 P-value NS 0.002 NS Ultegra, PAU 174±22 159±30 122±26 194±47 150–220 P-value NS 0.006 0.02

Flow cytometry CD31 (PECAM), MFI 63.1±7.4 63.0±6.4 62.2±6.3 70.2±6.8 58–88 P-value NS NS 0.01 CD41 (GP IIb/IIIa), MFI 377.7±25.3 366.1±33.2 388.0±47.1 435.5±81.5 270–490 P-value NS NS 0.004 CD42a (GP Ib), MFI 199.1±27.0 212.1±24.7 206.5±41.4 250.9±67.9 160–260 P-value NS NS 0.001 PAC-1, MFI 12.6±2.4 11.9±2.7 9.9±1.9 12.5±3.0 8–14 P-value NS o0.0001 NS CD51/61 (vitronectin), MFI 7.2±1.1 7.0±1.2 7.5±1.0 11.3±2.0 5.6–12.0 P-value NS NS o0.0001 CD62p (P-selectin), %+ 9.8±2.0 9.6±1.9 9.3±2.2 12.3±2.8 8.6–12.9 P-value NS NS 0.0001 CD63, MFI 7.9±1.9 7.7±1.5 8.0±2.0 9.7+3.3 6.5–13.2 P-value NS NS 0.02 CD107a, MFI 5.7±1.6 5.3±1.8 5.4±1.6 5.4±1.4 4.8–6.1 P-value NS NS NS CD151+14, MFI 168.1±30.5 156.0±31.1 121.7±37.0 167.8+29.4 89–150 P-value NS 0.0001 NS CD154, MFI 5.7±2.1 5.9±1.9 5.6±1.7 5.5±2.0 3.8–7.3 P-value NS NS NS CD165, MFI 25.2+5.9 27.3±6.0 28.1±6.0 26.6+5.4 18–31 P-value NS NS NS WEDE-15, MFI 43.8±9.7 42.2±8.9 38.7±7.9 36.1+10.9 29–54 P-value NS NS 0.03 SPAN-12, MFI 27.4±7.1 27.6±5.8 23.9±6.3 27.3±6.6 16–30 P-value NS NS NS

Abbreviations: MFI, log mean fluorescence intensity; NS, nonsignificant when compared with own baseline; PAU, platelet activation units; %+, percent positive cells.

Journal of Human Hypertension Aliskiren and biomarkers V Serebruany et al 307 Table 3 Coagulation/fibrinolytic biomarkers

Parameter SPP100 Reference range

Baseline 500 ng mlÀ1 1000 ng mlÀ1 2000 ng mlÀ1 aPTT, s 30.5±3.3 28.9±3.3 29.8±3.6 30.9±3.7 26–36 P-value NS NS NS Prothrombin time (s) 12.7±0.7 13.1±0.5 12.7±0.7 12.9±0.6 11.6–14.0 P-value NS NS NS Fibrinogen, mg mlÀ1 291.5±57.4 290.9±43.4 292.6±58.1 289.8±49.2 175–425 P-value NS NS NS Thrombin time (s) 17.8±1.4 17.6±1.4 17.6±1.4 18.1±1.3 16–20 P-value NS NS NS Reptilase time (s) 15.8±1.3 15.6±1.4 16.0±1.4 15.8±1.5 14–20 P-value NS NS NS Antithrombin III activity, % activity 83.3±4.2 90.1±6.6 90.3±7.6 90.8±6.4 80–120 P-value 0.003 0.003 0.0001 Protein C activity, % 128.4±11.3 117.4±13.4 119.7±14.6 119.5±10.8 70–180 P-value NS NS NS Protein S, activity, % 88.4±9.2 87.4±9.7 86.5±8.9 83.0±10.3 60–150 P-value NS NS 0.01 Protein S, free, % 78.8±8.4 79.0±9.3 78.5±8.2 82.2±9.8 50–171 P-value NS NS NS vWillebrand Ag, % 75.3±5.1 76.4±5.6 76.6±4.5 74.6±3.3 449 P-value NS NS NS Plasminogen activity, % 98.2±5.3 97.2±4.6 95.7±3.7 97.6±4.4 65–176 P-value NS NS NS D-dimer, mgmlÀ1 FEU 0.3±0.05 0.3±0.05 0.3±0.05 0.3±0.05 o0.55 P-value NS NS NS Antithrombin III antigen, % 101.6±8.2 101.8±6.9 99.8±7.1 100.5±6.8 80–120 P-value NS NS NS Fibrinogen degradation products, mgmlÀ1 o5 o5 o5 o5 o5 P-value NS NS NS a2-antiplasmin, % 115.6±19.1 115.8±16.4 112.9±18.3 116.9±17.5 85–156 P-value NS NS NS

concentration of the renin inhibitor. When testing the unknown mechanisms of platelet physiology. 2000 ng mlÀ1 concentration of the drug there were Further, they may help achieving a better compre- also some significant changes in platelet-coagulation hension of renin-angiotensin’s role in the modula- biomarkers. In contrast to the 1000 ng mlÀ1 dose, tion of platelet activity, blood coagulation and clot these differences suggested possible mild platelet formation. activation, and reached statistical significance for the The renin–angiotensin–aldosterone system Ultegra Analyzer readings, enhanced expression of (RAAS) has been identified as one of the major PECAM-1, GPIb, vitronectin receptor, P-selectin and factors in the pathogenesis of most known vascular finally CD63 expression. diseases and their complications. A link between RAAS activation and thrombosis formation also definitely exists. It is known that the RAAS releases Discussion its pro-thrombotic potential through the enhanced production of angiotensin II. Appearing within the The present study indicates that the renin-inhibitor vasculature, angiotensin II stimulates the release of aliskiren, independently of the applied dose, con- PAI-1, the major physiological inhibitor of fibrino- sistently activates human AT-III activity in the lysis in vivo,18 thereby effectively reducing fibrino- in vitro setting. In concentrations exceeding twofold lytic activity.19,20 Angiotensin II also increases the therapeutic range, aliskiren provides mild antiplate- expression of PAI-1 in cultured endothelial and let and effects as well; however, these vascular smooth muscle cells.21,22 In endothelial observed changes were random and often divergent, cells, the effect of angiotensin II on PAI-1 expression that is, counter-directed, which suggests that they is mediated through its hexapeptide metabolite, likely do not have any clinical relevance. In angiotensin IV. Thus, the effect of angiotensin II on addition, since the majority of changes in platelet endothelial PAI-1 expression is not blocked by activity and coagulation were observed far beyond either angiotensin II type 1 (AT1), or type 2 (AT2) the expected therapeutic concentrations of aliskiren, receptor antagonists. The RAAS has been implicated the clinical significance of these very high doses of in the proliferative response of the vascular wall aliskiren is limited. Nevertheless, these findings after arterial injury,23 in the synthesis of proto- could provide new insights by further defining oncogenes that are linked to the adaptive process of

Journal of Human Hypertension Aliskiren and biomarkers V Serebruany et al 308 cardiovascular hypertrophy,24 and in the synthesis indirectly affect the hemostatic system. Other of tumor necrosis factor and interleukin-1 by mono- possibilities linking aliskiren and modulation of nuclear cells.25 ACE converts angiotensin I to hemostasis may be due to compliment inhibition,44 angiotensin II and inactivates bradykinin. An in- and/or hormonal disturbances.45 Despite the fact, crease in ACE activity may impair endothelial that in the in vitro setting aliskiren exhibited function by increasing angiotensin II and accelerat- no pronounced changes in the hemostatic indices, ing bradykinin degradation. Bradykinin is an it is possible that in the clinical ex vivo setting, endothelium-dependent vasodilator that stimulates chronic RAAS modulation will be associated with the release of nitric oxide (NO), prostacyclin and the mild inhibition of platelet activity,46 coagula- endothelium-dependent hyperpolarizing factors, tion47 and enhanced fibrinolysis by blocking t-PA classical modulators of hemostasis. Bradykinin is and PAI-1.48 also a potent stimulator for tissue plasminogen activator (t-PA) in endothelial cells.26 Numerous reports suggest that ACE inhibitors are capable of Limitations enhancing fibrinolytic activity through the inhibi- There are a few limitations of the study worth 27–29 tion of PAI-1 production. ACE inhibitors aug- mentioning. We conducted this study in the in vitro ment t-PA production by coronary arteries in setting, and are not drawing any definite clinical 30 hypertensive patients, and downregulate tissue conclusions based solely on the index data. 31 factor production in monocytes. Although compatible in size with other similar Besides coagulation hemostasis, the RAAS plays investigations, this study was performed in a small 32 an important role in platelet physiology. Platelet cohort, so chance represents a plausible alternative receptor mRNA levels are known to be inversely explanation. In addition, the expression of multiple correlated with plasma angiotensin II levels, activation-dependent platelet receptors was studied whereas mononuclear leukocyte receptor mRNA but their individual roles are mostly unknown. We levels are positively correlated with plasma angio- are also unaware of any reported data linking tensin II levels in patients with primary hyperten- aliskiren with the AT-III changes, and cannot 33 sion. In secondary hypertension due to renal artery presently explain this finding limiting the clinical stenosis, both platelets and mononuclear leucocytes utility of this study. ATIII receptor mRNA are elevated initially, but Taken together, in the therapeutic concentration of subsequently decreased after correction of the 500 ng mlÀ1 aliskiren in vitro does not affect hemo- underlying cause, that is, successful revasculariza- static biomarkers, except for a moderate increase of 33 tion of the renal artery stenosis. Angiotensin II AT-III activity. Higher aliskiren doses were asso- stimulation significantly increases intracellular pla- ciated with more profound biomarker changes, but telet calcium concentrations, intracellular pH and they are likely not to be clinically relevant since thrombin-induced platelet aggregation in untreated they show diverging (that is, both mild antiplatelet 34 hypertensive as well as normotensive individuals. and platelet-activating) trends, as well as consider- The release of angiotensin II following pro-aggrega- ing the 2- to 4-fold safety margin. Also, for the future tory stimulation suggests that this may be one of the studies, it will be important to expand the research mechanisms through which platelets can locally on fibrinolytic indices such as PAI-1 and t-PA, modulate vascular tone and promote atherogen- which may be affected by aliskiren. 35 esis. The infusion of angiotensin II increased In conclusion, the moderate but consistent in- plasma b-thromboglobulin levels, reflecting a-gran- crease of AT-III activity with aliskiren that was 36 ule and expression of P-selectin. Several observed in our in vitro experiments could be angiotensin receptors blockers demonstrate not attributed to the itself rather 10,37,38 profound, but still evident antiplatelet effect. than being explained by the immediate modulation Aliskiren ((2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2- of the RAAS system. Therefore, it is suggested that methylpropyl)-5-amino-4-hydroxy-2,7-di-isopropyl- antithrombotic properties of aliskiren be explored 8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-octana- further in an ex vivo clinical setting. mid hemifumarate)), an octanamide, is the first representative of a new class of completely nonpep- tide, low molecular weight, orally active transition What is known about the topic state renin inhibitors.39 Aliskiren effectively reduces K Renin inhibitors in general and aliskiren in particular are blood pressure in patients with arterial hyperten- potent agents reducing arterial blood pressure. 40,41 42 K Changes in platelet activity coagulation, and biomarkers sion regardless of their racial origin, and of fibrinolysis may affect or/and predict vascular reduces production of angiotensin I–II in a dose- outcomes. dependent manner.43 On the basis of our findings, there seems to be no overt perturbation of the What this study adds hemostatic system upon exposure of the organism K Aliskiren cause mild activation of antithrombin-III what may be clinically relevant if validated in larger studies. to aliskiren. However, it is well established that K Therapeutic doses of aliskiren do not influence platelet renin inhibition by aliskiren is accompanied by activity and fibrinolysis. decrease in angiotensin II levels what itself may

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