Recent studies have suggested that both symptomatic ischemic heart disease and some coronary risk factors are associated not only with endothelial dysfunction,1,2 but also with decreased platelet responsiveness to the anti-aggregatory effects of nitric oxide (NO).3-8 Utilizing nitroglycerin, which releases NO via enzymatic bioconversion, and a more direct NO donor, sodium nitroprusside (SNP), we demonstrated “NO resistance” at the platelet level in patients with stable angina pectoris,5 acute coronary syndromes (ACS),6 aortic stenosis7 and heart failure.8 The mechanisms underlying this phenomenon reflect a combination of reduced soluble guanylate cyclase sensitivity to NO and decreased bioavailability of NO as a result of increased blood levels of superoxide anion radical (O2−).5
We recently showed that angiotensin converting enzyme (ACE) inhibition decreases blood O2− levels and ameliorates platelet NO resistance.8 Angiotensin II (Ang II), a key product of ACE, increases the expression and activity of endothelial NAD(P)H oxidase, a major source of O2−, resulting in oxidative stress in the vessel wall.9,10 Platelets also possess receptors for Ang II, and Ang II potentiates platelet aggregation11,12 and platelet activation (shape change).13,14 Recently, angiotensin-(1-7) [Ang-(1-7)] was identified as a vasodilator and possible physiological antagonist of Ang II.15-17 However, the effect of Ang-(1-7) on platelet aggregation has not been examined. Ang-(1-7) can be formed from either Ang I or Ang II by endopeptidase or ACE-2, respectively, and Ang-(1-7) is further metabolized by ACE [for review see Ref.18]. Ang-(1-7) plasma levels increase during therapy with ACE-inhibitors and may contribute to the therapeutic effects of ACE-inhibition.18 We therefore tested the hypothesis that Ang-(1-7) and Ang II exert opposite effects on platelet homeostasis, whereby Ang-(1-7) counteracts platelet NO resistance.
The effects of Ang-(1-7) on platelet function were examined in blood samples obtained from: normal subjects (n = 17; 10 men and 7 women; aged 33 ± 11 [SD] years) with no history or risk factor of cardiovascular disease; and from patients with acute coronary syndromes (ACS): unstable angina pectoris or non-ST elevation myocardial infarction (n = 17; 13 men and 4 women; aged 59 ± 15 [SD] years). The clinical details of the ACS patients are summarized in Table 1. Patients who were on ACE-inhibitors or angiotensin type 1 receptor antagonists were excluded. The effects of Ang II on platelet function were studied in a separate group of normal subjects (n = 8; age 29 ± 8 [SD] years). The protocol was approved by the Ethics of Research Committee of the Queen Elizabeth Hospital; written informed consent was obtained before study entry.
Venesection via an antecubital vein was performed after 30 minutes of supine rest. Blood was collected into plastic tubes containing 1:10 volume of acid citrate anticoagulant (2 parts of 0.1 mol/L citric acid to 3 parts of 0.1 mol/L trisodium citrate).5
Platelet Aggregation Studies
Aggregation in whole blood was studied using a dual-channel impedance aggregometer (Model 560, Chrono-Log, Havertown, PA, USA) connected to Aggro-Link computer interface (Model 810, Chrono-Log, Havertown, PA, USA) as previously described.6 Samples of whole blood were diluted 2-fold with normal saline (final volume 1 mL) and prewarmed for 5 minutes at 37°C. Aggregation studies were performed at this temperature with a stirring speed of 900 rpm. Aggregation was induced by the stable thromboxane A2 mimetic U46619 at final concentrations of 1, 3 or 5 μmol/L. SNP (10 μmol/L) alone, or followed immediately by Ang-(1-7) or Ang II (final concentrations of 1 nmol/L to 1 μmol/L), was added to samples one minute before the induction of aggregation with U46619. Both the extent of aggregation (Ohms) and rate of aggregation (Ohms/min) were monitored for 7 minutes. Typical aggregation tracings are shown in (Fig. 1)
U46619 and SNP were obtained from Sigma (St. Louis, Missouri, USA). Ang-(1-7) and Ang II were purchased from Auspep (Parkville, Victoria, Australia).
Inhibition of U46619-induced platelet aggregation by SNP was expressed as percentage reduction in aggregation from that seen in the absence of SNP. The effects of Ang-(1-7) were examined with U46619 concentrations associated with approximately 20% inhibitory effect of SNP, the 20% response being selected to facilitate detection of any potentiation.
Concentration-response curves were constructed for the interaction of Ang-(1-7) in concentrations of 1 nmol/L-1 μmol/L with SNP inhibition of U46619-induced aggregation. Concentration-response relationships were bidirectional, exhibiting local maxima with lack of effect at higher Ang-(1-7) concentrations. The local maximum was designated as Cmax; this varied between 10 and 100 nmol/L in different subjects. Therefore, individual concentration-response curves were re-aligned to superimpose Cmax for each curve on the same point on the x-axis. The same approach was applied for the assessment of the interaction of Ang II with U46619-induced aggregation.
Comparison of the effects of different concentrations of Ang-(1-7) or Ang II on platelet aggregation and the net effect of Ang-(1-7) or Ang II on the inhibitory effects of SNP within each group were made using ANOVA and Student's paired t test as appropriate. Comparisons between normal subjects and patients with ACS were made using Student's non-paired t test. All P values were two-sided and P < 0.05 was considered to indicate statistical significance. Results are expressed as mean ± SEM.
U46619-Induced Platelet Aggregation and Its Inhibition With SNP
With both parameters of platelet aggregation, rate (Ohms/min) and extent of aggregation (Ohms), there was a trend for ACS patients to show greater platelet aggregation than normal subjects at 1 μmol/L U46619 (Fig. 2). A similar trend was seen for 3 or 5 μmol/L U46619 (data not shown).
SNP at 10 μmol/L inhibited U46619-induced platelet aggregation, whether measured via rate or extent of aggregation (Figs. 1 and 2). There was also a trend for less inhibition of platelet aggregation by SNP in blood samples obtained from patients than in those from normal subjects. For example, at 1 μmol/L U46619, the inhibitory effects of SNP were 81 ± 6% and 57 ± 11% (P = 0.06) in normal subjects and patients, respectively, when assessed via rate of aggregation, and were 78 ± 8% and 49 ± 12% (P = 0.07) when assessed via extent of aggregation (Fig. 2).
As illustrated in the representative experiment (Fig. 1, upper panel), Ang-(1-7) did not induce platelet aggregation and did not affect U46619-induced aggregation. In both normal subjects and patients, there were no significant direct effects of any concentration of Ang-(1-7) on platelet aggregation (data not shown). However, as shown in Fig. 1 (upper panel), Ang-(1-7) potentiated the inhibition of aggregation produced by SNP. In this representative experiment, the extent of platelet aggregation was reduced by 27% in the presence of SNP (in comparison with U46619 alone), and by 53% in the presence of SNP plus Ang-(1-7), constituting 26% (incremental) potentiation effect of Ang-(1-7). In both normal subjects and patients, these effects of Ang-(1-7) were bimodal, with marked potentiation of SNP response at 10-100 nmol/L Ang-(1-7) and lack of effect at higher concentrations. In blood samples obtained from normal subjects, maximum incremental effects of Ang-(1-7) on inhibition of aggregation were 25 ± 4% and 28 ± 5%, for rate and extent of aggregation respectively (P < 0.01 for both; Fig. 3). This effect of Ang-(1-7) corresponded to a 2.3-fold potentiation of anti-aggregatory effect of SNP (set at ≈20%; see Methods, Data analysis). Ang-(1-7) similarly potentiated the anti-aggregatory effects of SNP in ACS subjects (Fig. 3).
Angiotensin II Studies
Ang II alone did not induce platelet aggregation, but augmented aggregation induced by U46619 (see Figure 1, lower panel for a representative experiment). In this experiment, Ang II (10 nmol/L) increased the extent of platelet aggregation by 14%, both in the absence and in the presence of SNP. In the entire cohort of 8 normal subjects, concentration response curves for Ang II potentiation of U46619-induced platelet aggregation were bimodal, with peak effect at 10-100 nmol/L. Mean potentiation at Cmax for Ang II was of a similar magnitude in the absence and presence of SNP (Fig. 4). Maximum potentiation of the rate of platelet aggregation by Ang II was 21 ± 6% in the absence of SNP and 26 ± 9% in the presence of SNP (P < 0.01 for both).
The major finding of this study was that Ang-(1-7), whereas lacking intrinsic effects, markedly potentiated the anti-aggregatory effects of the NO donor SNP. We also found that Ang II potentiated U46619-induced platelet aggregation, but did not interact with platelet NO responsiveness. The effects of both Ang-(1-7) and Ang II exhibited bimodal concentration-response characteristics.
Despite numerous investigations of the effects of Ang II and Ang-(1-7) on vascular smooth muscle and other tissues, none have investigated the effects of Ang-(1-7) on platelet aggregation, and only three studies to date11,12,19 examined the direct effects of Ang II on platelet aggregation. Two studies11,12 showed Ang II does not induce platelet aggregation, but potentiates the pro-aggregatory effects of adrenaline, the thromboxane A2 mimetic U44069, and thrombin; whereas a third study19 showed that Ang II does not modify either U46619- or ADP-stimulated platelet aggregation. The magnitude of potentiation of aggregation by Ang II documented in the current work with U46619 (Fig. 4) was comparable to that seen by others with U4406911 and thrombin.12 However, Ang II did not modify the anti-aggregatory effects of the NO donor SNP in our in vitro system. This observation implies that the pro-aggregatory activity of Ang II cannot be simply explained by increased clearance of NO by O2−.
This work is the first report on the interaction of Ang-(1-7) with platelet aggregation. The lack of any direct effect of Ang-(1-7) alone on platelet aggregation is perhaps surprising. Ang-(1-7) has been reported to release both NO20,21 and prostacyclin22,23 in vascular smooth muscle. However, it is unlikely that Ang-(1-7) stimulated prostacyclin generation in our in vitro system, although this might occur in vivo.24
The bimodal concentration-response relationships for both Ang-(1-7) and Ang II are consistent with previous findings in other tissues.25-29 For example, potentiation of bradykinin-induced vasodilation by Ang-(1-7) in rat mesenteric arterioles exhibits a local maximum at a dose of 100 pmol of topically applied Ang-(1-7), with evidence of abolition (and possibly reversal) of this potentiation at 1000 pmol of Ang-(1-7).26 Such bimodal effects suggest either release of a secondary mediator within a narrow concentration range, or activation of a second receptor system at higher peptide concentrations. The increase in platelet SNP responsiveness by low Ang-(1-7) concentrations (<1 μmol/L) might have been mediated by a specific Ang-(1-7) receptor,30 although this has yet to be demonstrated in platelets. The lack of effect of higher Ang-(1-7) concentrations (≥1 μmol/L) may have been due to an interaction with the AT1 receptor as has previously been proposed in vascular beds.31 Thus, Ang II and Ang-(1-7) might contribute to proaggregatory/anti-aggregatory platelet homeostasis. Further studies are required to examine the mechanisms of the effects we observed: whether the effects of Ang-(1-7) were receptor-mediated and whether secondary effector mechanisms contribute, as described for the potentiation of the effects of bradykinin on vascular smooth muscle via a specific Ang-(1-7) receptor, followed by secondary release of NO and/or prostanoids.15,20,26
As we have documented previously in a number of cardiovascular disease states, and in ACS in particular, a phenomenon of “NO resistance” is manifested at the platelet level as a decreased (by 20 to 30%) platelet responsiveness to the anti-aggregatory effects of NO donors.5,6 In the present study the potentiation of the SNP responses by Ang-(1-7) was of the same order as the difference we previously described between normal and NO-resistant populations5-8 when ADP was used to induce platelet aggregation. The present study was not designed to identify or quantify NO resistance in the presence of U46619. However, a trend for decreased platelet responses to the inhibitory effect of SNP on U46619-induced platelet aggregation in blood samples from ACS patients (Fig. 2) is consistent with our previous studies using ADP to induce platelet aggregation.5-8 A potentially important issue, not completely addressed in the current study, is whether the NO-potentiating effects of Ang-(1-7) might be accentuated in the presence of platelet NO resistance. A substantially larger population of ACS patients would be needed to evaluate this. The trend toward hyperaggregability in the ACS patients is consistent both with their overt ischemia, and the fact that they were older than normal subjects. However, hyper-aggregability did not interact significantly with the potentiation effects of Ang-(1-7).
We recently demonstrated that short-term therapy with the ACE inhibitor perindopril potentiates platelet responsiveness to NO donor in patients exhibiting NO resistance.8 ACE inhibition augments circulating Ang-(1-7) levels substantially (5- to 25-fold)18 and our present findings suggest that Ang-(1-7) may mediate this effect of perindopril. Thus, strategies that increase Ang-(1-7) levels may have favorable effects both on platelet homeostasis and on frequency of atherothrombotic events.
This work was supported by a grant from the National Health and Medical Research Council of Australia. D. J. Campbell is a recipient of a Career Development Fellowship (Award CR 02M 0829) from the National Health and Medical Research Council of Australia.
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