The peptide angiotensin II (Ang II) plays an important role in cardiac and vascular disorders. AT-1 Ang II receptor stimulation has been associated with most of the known biologic effects of Ang II. In 1982, the chemical compound N-(biphenylyl-methyl) imidazole (losartan) was described as the first nonpeptide AT-1-receptor antagonist with high specific affinity for type-1 receptors, and it became the prototype of a new therapeutic class of antihypertensive drugs (1,2). At present, Ang II AT-1-receptor antagonists represent a new alternative therapeutic approach to hypertension treatment, with the expectation of a lower incidence of adverse events than that seen with angiotensin I converting enzyme inhibitors (3)(Fig. 1).
Thromboxane A2 (TxA2) acts as an amplifying signal for several agonists of platelet activation by being synthesized and released in response to a variety of agonists, including adenosine diphosphate (ADP) (4). It was shown that losartan dose-dependently reduces TxA2-receptor-mediated pulmonary hypertension (5). This was further confirmed by Li et al. (6), demonstrating that losartan antagonized TxA2/prostaglandin H2 (PGH2) receptors in isolated canine coronary arteries.
A recent study from our laboratory has shown that losartan interacts with TxA2/PGH2 receptors in human platelets (7), an effect that was not elicited by the angiotensin I converting enzyme inhibitor captopril (7).
In light of this new information concerning the interaction of losartan with TxA2/PGH2 receptors and its antiplatelet effects, the aim of our study was to analyze the ability of different Ang II AT-1-receptor antagonists to inhibit TxA2-dependent human platelet activation.
Platelet-rich plasma and platelet aggregation
Platelet-rich plasma (PRP) was isolated from whole blood obtained from peripheral blood of healthy men (age between 26 and 40 years). The protocol was approved by the Institutional Ethics Committee. Volunteer donors had not received drugs for ≥20 days before the experiments. The blood was collected in 10% (vol/vol) acid-citrate-dextrose and centrifuged at 1,000 rev/min for 10 min. PRP was collected, and the platelet number was adjusted with platelet-poor plasma, obtained from the same donor, to 2.5 × 108 cells/ml plasma. As previously reported (8,9), platelet activation was evaluated in a lumiaggregometer (two-channel Aggregometer; Chrono-Log Corporation Hovertown, PA, U.S.A.) by the change in light transmission. A platelet-poor sample was used as control for 100% light transmission.
PRP (500 μl) was incubated at 37°C for 3 min in the aggregometer with continuous stirring (1,000 rev/min) and then stimulated with the TxA2 agonist, U46619 (10−6M) or ADP (10−5M).
To standardize the measurements, only the values of turbidimetry at 6 min were used for the calculations. This time period corresponds to the plateau of the maximal value of the first wave of platelet aggregation. This primary wave represents platelet activation rather than platelet aggregation and is partially reversible. The different Ang II AT-1-receptor antagonists were added to the platelet suspension 5 min before platelets were stimulated with the agonists. When a combination of drugs was used (i.e., aspirin or AT-2 antagonist plus AT-1-receptor antagonist), the drugs were preincubated 5 min with platelet suspension before platelets were stimulated with the agonists.
In all cases, the comparative baseline measurements were done in the presence of the solvent of the drugs, 0.8% sodium bicarbonate.
Western blot detection of AT-1 receptors
The presence of Ang II AT-1-type receptors in platelets was detected by Western blotting. The isolated PRP was centrifuged at 2,500 rev/min for 10 min, and the pellet resuspended in Laemmli buffer containing 2-mercaptoethanol (10). The proteins obtained were separated in denaturing sodium dodecyl sulfate (SDS)-10% polyacrylamide gels (10 μg/lane). Proteins were then blotted into nitrocellulose (Immobilon-P; Millipore Ibérica S.A). Blots were blocked overnight at 4°C with 5% nonfat dry milk in TBS-T (20 mM Tris (hydroxymethyl) aminomethane (Tris-HCl), 137 mM NaCl, 0.1% Tween 20). As previously described (11), Western blot analysis was performed with a polyclonal antibody against the AT-1-type receptor. Blots were incubated with the first antibody (1:1,250) for 1 h at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody) at a dilution of 1:1,500 for a further 1 h. Specific AT-1-type receptors were detected by enhanced chemoluminescence (ECL; Amersham Iberica, Madrid, Spain). Prestained protein markers were used for molecular mass determinations.
To determine the specificity of the AT-1 polyclonal antibody, we used a homogenate of cultured bovine vascular smooth muscle cells obtained as described (12).
TxB2, the major metabolite of TxA2, was determined by radioimmunoassay. After the platelet-aggregation assay was performed, the medium was recovered from the cuvette and centrifuged at 1,800 rev/min at 4°C for 5 min. The supernatant was recovered and, after adding 10−6M indomethacin, it was quick-frozen at −80°C until the assay for TxB2. The measurement of TxB2 was performed using a commercial kit (Amersham International, Buckinghamshire, U.K.). The intra- and interassay variabilities of the kit were 1.1% and 4.7%, respectively.
TxA2 receptor-ligand binding
Binding of [3H]-U46619 (Dupont NEN, Boston, MA, U.S.A,) to platelets was performed essentially as described by Kattelman et al. (13). PRP was incubated with 1 mM acetylsalicylic acid for ≥30 min at 37°C to inhibit endogenous TxA2 formation. Then platelets were centrifuged at 1,100 rev/min for 10 min and resuspended in HEPES/Tyrode's buffer [134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES, 5 mM dextrose, and 0.3% bovine serum albumin (BSA), pH 7.4] at a final concentration of 3 × 108 platelets/ml. As reported (7), aliquots of platelet suspension were incubated with 4 nM [3H]-U46619 in the presence and in the absence of increasing concentrations of the different Ang II AT-1-receptor antagonists for 30 min at 37°C. Specific binding was ensured using incubations containing a 1,000-fold excess of unlabeled U46619. The reaction was stopped by centrifugation at 2,500 rev/min for 10 min at 4°C. The pellet was washed twice and then transferred to a scintillation vial. Duplicate samples were counted in a liquid scintillation counter (Beckman Instruments Inc., Fullerton, CA, U.S.A.).
Losartan and EXP3174 were obtained from Merck, Sharp and Dohme, S.A. Irbesartan was obtained from Bristol-Myers. Valsartan was obtained from Novartis Laboratories. The active form of candesartan cilexetil (candesartan, CV-11974) was obtained from Astra Laboratories. Telmisartan was obtained from Boehringer Ingelheim. The polyclonal antibody against AT-1-type receptors was purchased from Santa Cruz Biotechnology. The TxB2 radioimmunoassay kit was purchased from Amersham International (Buckinghamshire, U.K.). All other chemical compounds were of the highest commercially available quality from Sigma Chemical Co.
Results are expressed as mean ± SEM. Unless otherwise stated, each value corresponds to a minimum of six different experiments. To determine the statistical significance of our results, we performed analysis of variance (ANOVA) with Bonferroni's correction of multiple comparisons or Student's t test (paired or unpaired). A p value <0.5 was considered statistically significant.
Effect of AT-1-receptor antagonists on TxA2-activated platelets
On activation of platelets with the TxA2 agonist U46619 (10−6M), a 72 ± 4% light transmission was observed. As we previously reported (7), losartan reduced U46619-induced platelet activation in a concentration-dependent manner (Fig. 2A). This effect also was observed with irbesartan (Fig. 2B). Both losartan and irbesartan inhibited platelet activation at 5 × 10−7M, with a maximal effect at 5 × 10−5M(Fig. 2A and B). The data shown in Fig. 2A and B were obtained 6 min after the stimulus and represent maximal platelet activation. However, the turbidimetry curves in the presence of losartan and irbesartan differed as early as 1 min after the experiments began (Fig. 3).
A reduction of U46619-induced platelet activation also was observed in the presence of both valsartan and the main active metabolite of losartan, EXP 3174 (Fig. 4A and B). However, only a maximal dose of valsartan (5 × 10−5M) and EXP3174 (5 × 10−5M) achieved a significant reduction of U46619-stimulated platelet activation (Fig. 4A and B).
Conversely, whereas the active form of candesartan cilexetil, CV-11974, failed to modify U46619-induced platelet activation, telmisartan attenuated the activation of human platelets produced by the TxA2 agonist (Fig. 5A and B). The level of reduction achieved by telmisartan was of lesser magnitude than that obtained by losartan and irbesartan but significantly higher than EXP3174 and valsartan (Figs. 2A and B, 4A and B, and 5B).
Spontaneous platelet activation (<5% light transmission) was not changed by the incubation of the PRP with any of the AT-1-type receptor antagonists (percentage light transmission: losartan, 3 ± 2; irbesartan, 4 ± 1; EXP 3174, 4 ± 2; valsartan, 3 ± 1; CV 11974, 3 ± 2; telmisartan, 3 ± 2, NS).
Effect of AT-1-receptor antagonists on ADP-induced platelet activation
We determined the effect of the different Ang II AT-1-type receptor antagonists on a partly TxA2-dependent platelet activator, ADP (10−5M). ADP induced a 62 ± 3% light transmission. An inhibitory effect of 32 ± 3% was detected with 3 × 10−3M aspirin.
No additional effect on ADP-related activation was obtained with aspirin concentrations >3 × 10−3M (data not shown). Losartan (5 × 10−5M) reduced ADP-induced platelet activation to a similar degree as aspirin (Table 1). Moreover, no further inhibitory effects on ADP-stimulated platelet activation were observed by adding aspirin to either losartan or irbesartan (Table 1).
Conversely, only with the highest concentration of EXP3174 (5 × 10−5M) and valsartan (5 × 10−5M), a slightly although statistically significant effect on ADP-stimulated platelet activation was achieved (Table 1). Telmisartan (5 × 10−5M) also reduced ADP-induced platelet activation, although to a lesser degree than losartan and irbesartan and with higher ability than EXP3174 and valsartan (Table 1). CV11974 (5 × 10−5M) failed to modify ADP-induced platelet activation (Table 1). In addition, in platelets preincubated with these AT-1-blockers, aspirin can still exert its effects (Table 1).
Under basal conditions, the level of TxB2 released by human platelets was 0.4 ± 0.03 ng, which was markedly increased by ADP to 4.2 ± 0.3 ng (p < 0.05). Aspirin blunted the release of TxB2 by ADP-stimulated platelets (Table 2). Neither losartan, irbesartan, telmisartan, EXP3174, valsartan, nor CV-11974 modified TxB2 release by ADP-stimulated platelets (Table 2).
Role of exogenous ANG II and AT-2 receptors on platelet activation
Western blot analysis using a polyclonal antibody against AT-1-type receptors demonstrated the presence of Ang II AT-1-type receptors in human platelets (Fig. 6). The polyclonal anti-AT-1 antibody used in our study recognized a single band of 60 kDa in homogenates of bovine vascular smooth muscle cells, indicating the specificity of the antibody (Fig. 6).
No significant effect on U46619 (10−6M)-induced platelet activation was observed by the presence of 10−7M Ang II (Fig. 6). However, a higher dose of U46619 (5 × 10−5M) enhanced the level of platelet activation that had been obtained with 10−6M U46619 (percentage light transmission: U46619 at 10−6M, 72 ± 4; U46619 at 5 × 10−5M, 83 ± 2). These results suggested that 5 × 10−6M U46619 did not exert a maximal platelet activation, discarding that the absence of effect of Ang II on U46619-induced platelet activation accounted for that fact. Moreover, in the presence of an angiotensinase inhibitor, 1,10-O-phenantholine (5 × 10−6M), Ang II (10−7M) also failed to modify U46619-induced platelet activation (percentage light transmission: U46619, 72 ± 4; U46619 + Ang II, 77 ± 3; NS).
In different cells, the Ang II AT-2 receptors appear to antagonize actions mediated by activated AT-1 receptors as they relate to cell growth and cell proliferation (14). Moreover, by selectively blocking AT-1 receptors, the levels of Ang II available to act on AT-2-type receptors could be increased. Therefore despite the lack of information about the presence of Ang II AT-2-type receptors in human platelets, we further determined the involvement of AT-2 receptors in the antiplatelet effects of losartan and irbesartan. In the presence of PD123319 (10−8M), an AT-2-receptor antagonist (15), the inhibitory effect of both losartan (5 × 10−5M) and irbesartan (5 × 10−5M) on U46619-induced platelet activation was of similar levels as in the absence of the AT-2 antagonist (Table 3). The dose of 10−8M PD123319 was chosen based in a previous kinetic study of AT-2 receptors in bovine endothelial cells in which this concentration of PD123319 demonstrated the maximal effect related to AT-2 blockade (16). Preincubation of platelets with EXP3174 (5 × 10−6M) or exogenous Ang II (10−7M) also failed to change the effect of losartan (5 × 10−5M) and irbesartan (5 × 10−5M) on U46619-stimulated platelet activation (Table 3).
Interaction of AT-1-receptor antagonists with TxA2 receptors in human platelets
Both losartan and irbesartan competed with radiolabeled U46619 and showed 50% binding inhibition at 7 × 10−7 and 3 × 10−7M, respectively (Fig. 7). Valsartan and the in vivo metabolite of losartan, EXP3174, also inhibited the binding of [3H]-U46619 to human platelets (Fig. 8). However, this effect was obtained with a dose of 5 × 10−5M of both EXP3174 and valsartan (Fig. 8). A dose of 5 × 10−6M of either EXP3174 or valsartan tended to diminish the binding of [3H]-U46619 to human platelets, but it did not reach statistical significance (Fig. 8). Telmisartan also reduced the binding of [3H]-U46619 at a dose of 5 × 10−6M, showing a higher relative potency than EXP3174 and valsartan (Fig. 7). However, the concentration of telmisartan needed to obtain a significant reduction of the binding of [3H]-U46619 to human platelets was 10-fold higher than either losartan or irbesartan (Fig. 7). No dose of CV-11974 modified the binding of [3H]-U46619 to human platelets (Fig. 8). The antagonist of AT-2 receptors, PD123319 (10−8M), also failed to modify the binding of [3H]-U46619 to platelets (data not shown).
These results provide new evidence of the antiaggregating effects of some AT-1-receptor antagonists. Our findings support the hypothesis that some AT-1-receptor antagonists, particularly losartan and irbesartan, could act as antiplatelet-activating agents independent of their effects on the AT-1 receptor.
Li et al. (6) showed that losartan inhibited TxA2-induced vasoconstriction of canine coronary arteries. Furthermore, it has been also reported that losartan reduced the pulmonary hypertension induced by the TxA2 agonist U46619 and acted as a competitive antagonist of TxA2 receptors in endothelium-denuded rat aortic rings (5,17). Moreover, we recently demonstrated that losartan reduced platelet activation produced by TxA2 by competing with its platelet receptor (7), an effect that was recently confirmed by Li et al. (18) for irbesartan on TxA2-induced vasoconstriction and platelet aggregation. In our study, we analyzed whether this is a common characteristic of the pharmacologic class of AT-1 antagonists or is specific of certain AT-1-receptor inhibitors.
Both losartan and irbesartan dose-dependently reduced U46619-induced platelet activation and attenuated the binding of TxA2 to its human platelet receptor. This effect also was shown with lesser potency by telmisartan, valsartan, and the hepatic metabolite of losartan, EXP3174. However, CV-11974, the active form of candesartan cilexetil, was ineffective. These results suggest for the first time that the antiplatelet effect of the AT-1-receptor antagonists could be a differentiating property between them.
The production of TxA2 through the arachidonic acid pathway acts directly to amplify platelet activation (19). ADP stimulates the arachidonic acid pathway to a further production of TxA2, which potentiates the activating effect of ADP on platelets (19). ADP-induced platelet activation was 30% reduced by the cyclooxygenase inhibitor aspirin. This suggests that cyclooxygenase activity, mainly due to TxA2 release, contributes ∼30% to ADP-induced human platelet activation. Both losartan and irbesartan also diminished ADP-mediated platelet activation by ∼28%, an effect that was not modified by the presence of aspirin. However, the effect of the other AT-1-receptor antagonists on ADP-induced platelet activation was increased by aspirin to the levels found with aspirin alone. These results could suggest that losartan and irbesartan inhibited ADP-induced platelet activation by a similar pathway of action to that of aspirin. However, whereas aspirin prevented the release of TxB2 by ADP-stimulated platelets, it was not modified by losartan and irbesartan.
Receptor studies have demonstrated that human platelets possess specific saturable binding sites for Ang II, which have been characterized as AT-1-type receptors (20,21). Our study demonstrates for first time by Western blotting that human platelets express a 60-kDa protein band corresponding to AT-1-type receptors.
Although human platelets expressed AT-1 receptors, the addition of exogenous Ang II did not affect in vitro platelet activation. Moreover, although the presence of Ang II AT-2-type receptors in platelets remains to be established, addition of an AT-2-receptor antagonist, PD123319 (15,22), to platelets failed to modify the effect of losartan and irbesartan on platelet activation. These findings showed that the antiplatelet effects of losartan and irbesartan were attributable to autocrine effects of endogenous Ang II, which could be present in plasma, on AT-2 receptors due to AT-1 blockade. This was further supported by the fact that the combination of EXP3174, which is a potent AT-1 antagonist, and an excess of exogenous Ang II failed to modify the inhibitory effects of either losartan or irbesartan on U46619-induced platelet activation. Taken together, these results supported that the effect of both losartan and irbesartan on platelet activation was unrelated to an AT-1-dependent or AT-2-dependent activation.
A study by Ding et al. (23,24) has shown that Ang II potentiated platelet activation, whereas, as occurred in our study, other studies could not find an effect of Ang II on platelets. The relatively low number of AT-1 receptors reported in platelets might explain the absence of a direct effect of Ang II on human platelets.
The different ability of the Ang II AT-1-receptor antagonists to inhibit the TxA2/PGH2 receptors suggests that the structural requirements to antagonize the platelet TxA2/PGH2 receptor may be different from those involved in antagonism of the AT-1 receptor. In this regard, and based on the different antiplatelet effects of losartan and its main metabolite, EXP3174, we could hypothesize on the importance of the hydroxylic radical contained in the imidazole ring. Both losartan and EXP3174 have an imidazole ring, and EXP3174 differs from losartan in that EXP3174 has a carboxylic radical whereas losartan possesses a hydroxylic radical (Fig. 1). Like EXP3174, valsartan also contains a carboxylic radical, which could explain its similar ability to inhibit the interaction between TxA2 and platelets (Fig. 1). The chemical structure of irbesartan, the other AT-1-receptor antagonist that demonstrated similar antiplatelet effects to losartan, does not contain the hydroxylic radical. However, irbesartan could undergo a ketoenolic tautomerization from a ketonic form to an enolic form, which could give it the hydroxylic radical in the imidazole ring. This type of chemical conversion is very common in organic compounds that contain keto or enolic moieties. As an example, phosphoenolpyruvate contains a phosphate ester that can undergo hydrolysis to yield the enol form of pyruvate, which immediately tautomerizes to a keto form (25). Pyrimidine and purine bases also may exist in two or more ketoenolic tautomeric forms, which is sometimes the cause of mistakes in which DNA polymerases insert an incorrect base (26).
Conversely, the active form of candesartan, CV-11974, did not share the antiplatelet effects shown by the rest of AT-1-receptor antagonists. The chemical structure of CV-11974 shows a heterocycle moiety, which hypothetically could confer stiffness to its radicals, making difficult its interaction with TxA2/PGH2 receptors. On the other hand, telmisartan is a biphenyl nontetrazole molecule that, although it has neither carboxylic nor hydroxylic radicals in the imidazole ring, possesses two condensed heterocycles (benzoimidazole) that could produce steric disability for TxA2 to interact with its platelet receptor. However, other chemical properties of the AT-1 antagonists such as the net charge, pucker ability, and their hydrophilic and stereochemistry properties could be limiting factors for their interaction with TxA2/PGH2 receptors. Therefore further studies beyond the scope of our research are needed to elucidate the importance of the chemical properties of each specific AT-1 antagonist to interact with TxA2/PGH2 receptors.
On the basis of the results presented here, the concentration of losartan and irbesartan required to inhibit platelet aggregation (5 × 10−7M) is very high compared with that obtained in humans treated with losartan and irbesartan (∼5 × 10−9M). The focus of this study was only to analyze the direct effect of different AT-1-receptor antagonists on platelets, but thrombosis is a multicellular event in which other cells such as neutrophils and endothelium are involved in the regulation of platelet reactivity (8,9,27,28). In this regard, it has been postulated that short-term in vivo administration of losartan increases the levels of nitric oxide (29), a potent antiplatelet agent (8,28). Therefore, the concentration of losartan and irbesartan that in vivo would be needed to inhibit platelet activation may be significantly lower than the concentration required in our in vitro study. We must keep in mind the importance of Ang II in the in vivo platelet activation. In this regard, not only through its vasoconstrictor properties but also by stimulating the production of plasminogen-activator inhibitor-1 (30), Ang II may favor the in vivo activation of platelets. Both mechanisms are dependent mainly on AT-1-receptor activation (22,30).
In summary, the results of this study demonstrated that losartan and irbesartan reduced TxA2-dependent platelet activation independent of their effects on Ang II AT-1-type receptors. Telmisartan, valsartan, and the main in vivo active metabolite of losartan, EXP3174, also showed an inhibitory effect on TxA2/PGH2 receptors in human platelets, although with lesser ability than losartan and irbesartan. This property was not shared by CV-11974, the active form of candesartan cilexetil. Therefore our experimental study suggests that AT-1-receptor antagonists may not only counteract the pathologic actions of Ang II in arterial hypertension, but also, and particularly in the cases of losartan and irbesartan, may have additional AT-1-independent actions preventing TxA2-related platelet activation. This feature could differentiate these AT-1-receptor antagonists and probably accounts for more beneficial cardiovascular effects, which must be further studied.
Acknowledgment: We thank M. Begoña Ibarra for secretarial assistance. This work was supported by a grant to the School from Laboratorios Merck. M.M. is a postdoctoral fellow of Comunidad Autónoma de Madrid. A. Jiménez and Almudena López-Blaya are fellows of Fundación Conchita Rábago de Jiménez Díaz.
1. Wong PC, Price WA Jr, Chiu AT, et al. Nonpeptide angiotensin II receptor antagonist: studies with EXP9270 and DuP753. Hypertension
2. Siegl PKS. Discovery of losartan, the first specific non-peptide angiotensin II receptor antagonist. J Hypertens
3. Burrell LM, Johnston CI. Angiotensin II receptor antagonists. Potential in elderly patients with cardiovascular disease. Drugs Aging
4. Patrono C, Renda G. Platelet activation and inhibition in unstable coronary syndromes. Am J Cardiol
5. Bertolino F, Valentín JP, Maffre M, Jover B, Bessac AM, John GW. Prevention of thromboxane A2
receptor-mediated pulmonary hypertension by a nonpeptide angiotensin II type 1 receptor antagonist. J Pharmacol Exp Ther
6. Li P, Ferrario CM, Bronsnihan KB. Nonpeptide angiotensin II antagonist losartan inhibits thromboxane A2
-induced contractions in canine coronary arteries. J Pharmacol Exp Ther
7. Guerra JI, Montón M, Rodríguez-Feo JA, et al. Effect of losartan on human platelet activation. J Hypertens
8. López-Farré A, Caramelo C, Esteban A, et al. Effects of aspirin on platelet-neutrophil interactions: role of nitric oxide and endothelin-1. Circulation
9. López-Farré A, Riesco A, Digiuni E, et al. Aspirin-stimulated nitric oxide production by neutrophils after acute myocardial ischemia in rabbits. Circulation
10. Laemmli NK. Change of structural proteins during the assembly of the head of bacteriophage T4. Nature
11. Montón M, López-Farré A, Mosquera JR, et al. Endogenous angiotensin II produced by endothelium regulates interleukin-1β-stimulated nitric oxide generation in rat isolated vessel. Hypertension
12. López-Farré A, Mosquera JR, Sánchez de Miguel L, et al. Endothelial cells inhibits nitric oxide generation by smooth muscle cells: role of transforming growth factor-β. Arterioscler Thromb Vasc Biol
13. Kattelman EJ, Venton DL, Le Breton GC. Characterization of U46619 binding in unactivated intact human platelets and determinations of binding site affinities of four TxA2
receptor antagonist. Thromb Res
14. Bauer JH, Reams GP. The angiotensin II type 1 receptor antagonists: a new class of antihypertensive drugs. Arch Intern Med
15. Lo M, Kin KL, Lantelme P, Sassard J. Subtype 2 of angiotensin II receptors
controls pressure-natriuresis in rats. J Clin Invest
16. Montón M, Castilla MA, Álvarez MV, et al. Effects of angiotensin II on endothelial cell growth: role of AT-1 and AT-2 receptors
. J Am Soc Nephrol
17. Corriu C, Bernard S, Schott C, Stoclet JC. Effects of losartan on contractile responses of conductance and resistance arteries from rats. J Cardiovasc Pharmacol
18. Li P, Fukuhara M, Diz DI, Ferrario CM, Brosnihan KB. Novel angiotensin II AT-1 receptor antagonist irbesartan prevents thromboxane A2
-induced vasoconstriction in canine coronary arteries and human platelet aggregation. J Pharmacol Exp Ther
19. Kroll MH, Schafer AL. Biochemical mechanisms of platelet activation. Blood
20. Crabos M, Bertschin S, Bühler FR, et al. Identification of AT1 receptors
on human platelets and decreased angiotensin II binding in hypertension. J Hypertens
21. Moore TJ, Williams GH. Angiotensin II receptors
on human platelets. Circ Res
22. Timmermans PBMWM, Smith RD. Angiotensin II receptor subtypes: selective antagonists and functional correlates. Eur Heart J
23. Ding YA, MacIntyre DE, Kenyon CJ, Semple PF. Angiotensin II effects on platelet function. J Hypertens
24. Burnier M, Centeno G, Grouzmann E, Walker E, Waeber B, Brunner HR. In vitro effects of DuP 753, a nonpeptide angiotensin II receptor antagonist, on human platelets and rat vascular smooth muscle cells. Am J Hypertens
25. Lehninger AL, Nebon DL, Cox MM. Nucleotides and Nucleic acids. In: Principles of biochemistry.
New York: Worth Publishers, 1997:324-57.
26. Echols H, Goodman MF. Fidelity mechanisms in DNA replication. Annu Rev Biochem
27. Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity in haemostasis and thrombosis. FASEB J
28. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology. Pharmacol Rev
29. Gohlke P, Pees C, Unger T. AT2
stimulation increases aortic cyclic GMPc in SHRSP by a kinin-dependent mechanism. Hypertension
30. 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