(Circulation. 1999;100:129-134.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Nitrate Resistance In Platelets From Patients With Stable Angina Pectoris
From the Department of Cardiology, The Queen Elizabeth Hospital, University of Adelaide, S.A., Australia.
Correspondence to Prof John D. Horowitz, Department of Cardiology, The Queen Elizabeth Hospital, Woodville 5011, S.A., Australia. E-mail jhorowitz{at}medicine.adelaide.edu.au
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Abstract |
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Background—Hemodynamic resistance to nitrates has been previously documented in congestive heart failure. In patients with stable angina pectoris (SAP), we have observed a similar phenomenon: decreased platelet response to disaggregating effects of nitroglycerin (NTG) and sodium nitroprusside (SNP).
Methods and Results—In blood samples from normal subjects (n=32) and patients with SAP (n=56), we studied effects of NO donors (NTG and SNP) on ADP-induced platelet aggregation and on intraplatelet cGMP. NTG and SNP inhibited platelet aggregation in patients to lesser extents than in normal subjects (P<0.01). The cGMP-elevating efficacy of NTG and SNP was diminished in platelets from patients in comparison with those from normals (P<0.001). Inhibition of the anti-aggregatory effects of NTG and SNP by ODQ, a selective inhibitor of NO-stimulated guanylate cyclase, was significantly less pronounced in patients than in normal subjects. Content of O2- was higher in blood samples from patients than in those from normal subjects (P<0.01). In blood samples from patients with SAP, but not in normal subjects, the O2- scavenger superoxide dismutase (combined with catalase) suppressed platelet aggregation (P<0.01) and increased the extent of anti-aggregatory effect of SNP (P<0.01).
Conclusions—In patients with SAP, platelets are less responsive to the anti-aggregating and cGMP-stimulating effects of NO donors; this may reflect both reduction in guanylate cyclase sensitivity to NO and inactivation of the released NO by O2-. The implied impairment of anti-platelet efficacy of endogenous NO (in the form of EDRF) may contribute to platelet hyperaggregability associated with angina pectoris.
Key Words: angina • platelet aggregation inhibitors • nitroglycerin
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Introduction |
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The organic nitrates such as nitroglycerin (NTG) are in widespread use for the treatment of both acute and chronic myocardial ischemia, as well as congestive heart failure. Until recently, it was assumed that their therapeutic efficacy was entirely secondary to relaxation of vascular smooth muscle.1 2 However, findings of several studies have indicated that pharmacological effects of nitrates include inhibition and reversal of platelet aggregation.3 4 The major limiting factor to the clinical utility of nitrates has been the induction of nitrate tolerance by virtue of chronic continuous nitrate therapy. This phenomenon has been documented at vascular5 and platelet levels.6 However, poor hemodynamic responsiveness to nitrates may also occur on a de novo basis (ie, independent of any prior nitrate therapy), particularly in patients with heart failure; this has been termed nitrate resistance.7 We have previously documented the occurrence of diminished anti-aggregatory effects of NTG and sodium nitroprusside (SNP) in platelet-rich plasma from patients with stable angina pectoris (SAP).8 9 However the extent and mechanism(s) of this phenomenon, which is apparently analogous to that of nitrate resistance in the vasculature, have not been investigated. Furthermore, we have documented increased platelet aggregability in platelet-rich plasma obtained from patients with SAP.8 9 The relation between this hyperaggregability and reduced platelet response to NTG and other NO donors has not been examined.
The anti-aggregating effect of NTG and other nitrovasodilators is mediated via formation of NO, which activates platelet guanylate cyclase, leading to generation of cGMP [for review see 10]. Although the effects of NTG are mediated primarily by enzymatic thiol-dependent bioconversion to NO, SNP is a more direct NO donor.11 12 Therefore, reduced sensitivity to both NTG and SNP suggests reduction in responsiveness to NO. Furthermore, in our previous studies8 9 we observed a strong interrelationship between cGMP-stimulating and anti-aggregating effects of NTG and SNP: a decreased platelet sensitivity to the anti-aggregatory effects of NTG and SNP was associated with a decrease in intraplatelet cGMP accumulation in response to these NO donors. As the intracellular cGMP level reflects both generation of cGMP by guanylate cyclase and hydrolysis of cGMP by cyclic nucleotide phosphodiesterases (PDE), the input of both enzymes in the observed phenomenon needs to be investigated. Possible impairment of platelet guanylate cyclase activity in patients with SAP has been tested in our previous study9 ; there were no indications of any dysfunction of the enzyme. However, the interaction of guanylate cyclase with NO and availability of NO for enzyme activation have not been examined. Regarding the latter issue, the decreased responsiveness of the platelet cGMP-system to NTG and SNP could be due to increased clearance of NO, by superoxide anion radical (O2-), the concentration of which is elevated in some cardiovascular disease states.13 14 15
This study was designed to investigate further the phenomenon of nitrate resistance in platelets. In blood samples obtained from normal subjects and patients with SAP, we studied the anti-aggregating and cGMP-elevating effects of NTG and SNP. We also assessed the influence of a PDE inhibitor (3-isobutyl-1-methyl-xanthine, IBMX), a selective inhibitor of NO-stimulated guanylate cyclase activity (1H-[1,2,4]oxadiazolo[4,3,-a]-quinoxalin-1-one, ODQ). Possible interactions between O2- and responses to NO donors were studied via measurement of O2- content and by examination of effects of an O2- scavenger superoxide dismutase (SOD) on platelet responsiveness to NO donors.
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Methods |
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Subjects
Studies were performed in the following groups: 1) normal subjects (n=32; 21 men and 11 women) aged 23 to 75 years, mean 48 years) not taking any medication affecting platelet aggregation; and 2) patients with SAP (n=56; 37 men and 19 women aged 34 to 76 years, mean 60 years) undergoing diagnostic cardiac catheterization and coronary angiography. In all cases at least one hemodynamically significant (
50%) stenosis was present in a major coronary
artery; a background aspirin and nitrate medication profile was
recorded at recruitment. Numbers of subjects used in individual experiments are indicated below (see Results). In all cases, blood samples were withdrawn for in vitro platelet aggregation and intraplatelet cGMP assay. The protocol was approved by the Ethics of Research Committee of The Queen Elizabeth Hospital; written informed consent was obtained before study entry.
Blood Sampling and Preparation of Platelets
Blood samples from patients undergoing cardiac
catheterization were withdrawn during the procedure via
a femoral arterial sheath; blood was drawn from other
patients and normal volunteers by venesection from an antecubital vein.
It has been shown8 16 that there is no arteriovenous
difference in platelet function. Blood was collected in 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);
acidified citrate was used in order to minimize deterioration of
platelet function during experiments.17 Blood was
centrifuged at 250g for 10 minutes at room
temperature to obtain platelet-rich plasma. Platelet-poor
plasma was prepared by further centrifugation of the
remaining blood at 2500g for 20 minutes. Platelet counts
were performed on the STKS Coulter Counter (Coulter Electronics
Inc) and the platelet-rich plasma was adjusted with
platelet-poor plasma to a constant count of 250 000/µL.
Platelet Aggregation Studies
Aggregation in whole blood and platelet-rich plasma was
examined using a dual-channel impedance aggregometer (Model 560,
Chrono-Log). Tests were performed at 37°C and stirring speed of 900
rpm. Samples of blood or platelet-rich plasma were diluted 2-fold
with normal saline (final volume 1 mL) and prewarmed for 5 minutes at
37°C. Aggregation was induced with adenosine 5'-diphosphate
(ADP) (final concentration of 1 µmol/L) in experiments with
whole blood and 0.5 µmol/L ADP with platelet-rich plasma.
Aggregation was monitored continually for 7 minutes, and responses were
recorded (RO-3 Rikadenki chart recorder) for electrical
impedance, in ohms. SNP and NTG (final concentration of 10 and 100
µmol/L, respectively) were added to samples 1 minute before ADP. SOD
and catalase (final concentration of 300 U/mL for both enzymes) were
added immediately before NTG or SNP.
1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one
(ODQ) (1 µmol/L) was added 5 minutes before NTG or SNP. The
duration of incubations were estimated as those optimal in preliminary
experiments (data not shown). In control tests,
physiological saline was added in appropriate
volumes. Inhibition of aggregation was evaluated as a percentage
comparing the extent of maximal aggregation in the presence and absence
of the anti-aggregatory agent studied. Representative
aggregograms are shown in Figure 1
.
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cGMP Studies
Platelet-rich plasma (0.5 mL) was incubated at 37°C with
SNP (10 µmol/L) for 2.5 minutes or with NTG (100 µmol/L)
for 5 minutes. ODQ (1 µmol/L) and IBMX (0.5 mmol/L) were
added to plasma 5 minutes before NTG or SNP. Intraplatelet cGMP
content was assayed as described previously.8 Briefly,
after incubation plasma was filtered through GF/C Glass Microfibre
Filters (Whatman) for harvesting the platelets. Filters with
absorbed platelets were rinsed with
physiological saline and placed into 0.5 mL of
4 mmol/L EDTA for further extraction of cGMP in a boiling water
bath for 5 minutes. After centrifugation of samples at
3000g for 10 minutes, cGMP concentration in supernatant was
estimated using "cGMP [125I] assay system"
(Amersham). Results were expressed as pmol
cGMP/109 platelets.
Chemiluminescence Assay of O2
Detection of O2- in
whole blood was performed using a chemiluminescence
technique,18 with lucigenin as a probe for
O2-. Blood samples were diluted
2-fold with normal saline (final volume 1 mL) and prewarmed for 5
minutes at 37°C before the addition of lucigenin (final concentration
125 µmol/L). Chemiluminescence was monitored using a
photoluminometer component of a dual-channel lumi-aggregometer (Model
560, Chrono-Log) equipped with a computer interface system (Aggro/link,
Chrono-Log) and 486DX IBM computer. Intensity of lucigenin
chemiluminescence was expressed in millivolts. Specificity of the
O2- detection was verified with
SOD; addition of SOD (300 U/mL) instantly cancelled lucigenin signal.
Coefficients of variation for replicate estimates were <15%.
Chemicals
ADP sodium salt, SNP, SOD (from bovine erythrocytes), catalase
(from bovine liver), IBMX, and bis-N-methylacridinium
nitrate (lucigenin) were obtained from Sigma (St.Louis, Mo). ODQ was
obtained from Tocris Cookson Inc (St.Louis, Mo). NTG was purchased from
Fisons (Thornleigh, NSW, Australia).
Data Analysis
Responses of platelets to anti-aggregating and
cGMP-elevating effects of NTG and SNP were quantified on the basis of
paired comparison as described previously.8 9
Inhibitory effects (percent) of anti-aggregating agents
were normalized relative to extents of ADP-induced aggregation.
Comparisons between normals and patients with SAP were made using ANOVA
followed by 2-sided Dunnett's test (for multiple comparisons) or
Student's non-paired t test as appropriate. Statistically
significant difference was limited to P<0.05. Results are
expressed as mean±SEM.
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Results |
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Platelet Responsiveness to ADP
Comparison of platelet responses to ADP in the various groups of individuals was potentially complicated by the gender-related differences and variable aspirin intake in patients group. Table 1
summarizes ADP responses from all of
these subjects, with data expressed separately according to gender.
Platelet aggregability toward ADP was greater in women than in men
(ANOVA: P<0.01 for normal subjects; P<0.05 for
all subjects). Platelet responses to ADP (1 µmol/L) were
significantly greater in patients with SAP than in normals (ANOVA:
P<0.01), irrespective of subject gender; concomitant
aspirin therapy was not a significant determinant of response to this
concentration of ADP.
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Analogous results were obtained with platelet-rich plasma. For
example, in male subjects, extent of aggregation was 15.9±0.9 
in
the control group (n=8), 20.8±2.8 in patients with SAP who did not
receive aspirin (n=6) and 18.4±2.4 in patients who received
aspirin (n=8); ANOVA: P<0.05 for both groups of patients
versus normals.
Inhibition of Platelet Aggregation by NTG and SNP
NTG and SNP inhibited platelet aggregation in whole blood
samples from both normals and patients but to different extents.
Representative aggregograms are shown in Figure 1
. There were no differences between sexes and between patients
receiving and not receiving aspirin regarding platelet responses to
anti-aggregatory effects of NTG and SNP. These results were therefore
pooled (Figure 2). There was a
significant attenuation of platelet response to NTG (ANOVA:
P<0.001) and also to SNP (ANOVA: P<0.001) in
patients. Prior therapy with prophylactic nitrates was not
a significant determinant of responsiveness to NTG or SNP in patients.
There was no significant correlation between extent of fixed
coronary artery disease and platelet responsiveness to
NTG or SNP.
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In platelet-rich plasma, anti-aggregatory effects of NTG and SNP in samples from patients with SAP were also less pronounced than in those from normal subjects, although this difference did not reach statistical significance. Specifically, NTG (100 µmol/L) and SNP (10 µmol/L) produced 77±8% and 81±9% inhibition of platelet aggregation, respectively, in samples from normal subjects (n=8), and 68±6% and 69±5% inhibition in patients (n=9).
Mechanisms of Nitrate Resistance
Taking into consideration the fundamental involvement of the cGMP
system in the anti-aggregatory effects of nitrovasodilators, we assayed
intraplatelet cGMP content after incubation of platelet-rich
plasma with NTG and SNP (Figure 3). Basal
cGMP concentrations in platelets from normal subjects and patients
with SAP did not differ: 0.38±0.03 and 0.37±0.04 pmol
cGMP/109 platelets, respectively. There was,
however, a significant attenuation of cGMP response to both NO donors
in patients relative to normals (ANOVA: P<0.001 for both
NTG and SNP). For example, in platelets from normal subjects
10 µmol/L SNP increased intraplatelet cGMP content 5.2-fold,
whereas in platelets from anginal patients, this concentration of
SNP produced only a 2.3-fold increase in cGMP. Prior NTG therapy was
not a significant determinant of cGMP response in patients. We
investigated whether the reduced accumulation of cGMP in response to NO
donors in patients' platelets was a result of increased activity
of PDE. Incubation of platelet-rich plasma with IBMX alone led to a
significant increase (210±24% of baseline) in intraplatelet cGMP.
However, when IBMX was added in combination with SNP, the SNP-dependent
component of the total cGMP increase (201±35% of control) did not
differ from the cGMP-elevating effect of SNP alone (230±20% of
control). Thus, inhibition of PDE did not restore the impaired cGMP
response to NO donor in platelets from anginal patients.
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We explored the phenomenon of nitrate resistance further, examining the
interaction of platelet guanylate cyclase with NO. We
used ODQ, a compound that potently and selectively inhibits
NO-stimulated guanylate cyclase activity.19 In
our experiments, ODQ in a concentration of 1 µmol/L abolished
SNP-induced elevation of intraplatelet cGMP content with both
normal subjects and patients (Figure 3). In this concentration,
ODQ alone did not affect platelet aggregation response to ADP but
reduced the anti-aggregatory effects of NTG and SNP in whole blood by
51±8% and 58±9%, respectively (P<0.05), in normals, but
not in patients (7±7% and 2±7%, respectively). Similar results were
observed in platelet-rich plasma. Thus, with both NO donors,
effects of ODQ were significantly less pronounced in patients than in
normal subjects. These results might possibly reflect a decreased
NO-sensitivity of platelet guanylate cyclase in
patients with SAP, or reduced availability of NO for activation of
guanylate cyclase because of a clearance of NO by
O2- in
blood.20 To examine the possible involvement of
O2- in nitrate resistance at
platelet level, we used SOD, a scavenger of
O2-. In order to prevent any
interference from H2O2
generated during the SOD-catalyzed dismutation of
O2-, catalase was added. Equal
amounts of both enzymes, 300 U/mL each, were used.21 As
shown in Figure 4, SOD/catalase did not
affect the extent of platelet aggregation in blood samples from
normal subjects (extent of aggregation was 97±8% of control, n=18)
but inhibited the increased aggregation in patients' samples
(aggregation was 74±4%, n=20, P<0.01 versus control).
There were no statistically significant effects of SOD/catalase on
SNP-dependent inhibition of aggregation in normal subjects. However, in
blood samples from patients with SAP, addition of SOD/catalase
increased the extent of anti-aggregatory effect of SNP; platelet
aggregation decreased from 69±4% of control to 55±4%
(P<0.01). Although these results implied that
O2- concentrations were
increased in patients with SAP, in our further experiments we examined
this possibility directly utilizing a chemiluminescence technique;
lucigenin was used as a specific probe for
O2-.18 There
was a significantly (P<0.01) higher
O2- chemiluminescence signal in
blood samples from patients (174±37 mV, n=15) than in those from
normal subjects (45±11 mV, n=6).
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Discussion |
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In the current study, platelets obtained from patients with SAP manifested increased aggregability with respect to normal subjects. Furthermore, the anti-aggregating and cGMP-stimulating effects of NTG and SNP were reduced in platelets from these patients relative to normal subjects, thus representing the phenomenon of nitrate resistance at the platelet level. This decrease in platelet responsiveness to NO donors may be attributed to reduction in platelet guanylate cyclase sensitivity to NO and to inactivation of the released NO by O2-.
We investigated whether the decreased platelet response to NTG and
SNP in patients with SAP is associated with a defect in the NO/cGMP
pathway (Figure 3). The intracellular cGMP system includes the
enzymes responsible for cGMP generation (guanylate
cyclase), decomposition (cyclic nucleotide
phosphodiesterases), and signal transduction (cGMP-stimulated protein
kinases).22 Previously, using the lipophilic analog of
cGMP (db-cGMP), we have shown9 that the NO/cGMP pathway is
intact distal to cGMP formation; the amount of cGMP generated in
response to NO donor ultimately predetermines the extent of
anti-aggregatory effect. As the current results show no evidence of
phosphodiesterase dysfunction, attenuated platelet cGMP response to
NTG and SNP suggests impairment at the site of guanylate
cyclase. Our previous experiments have not detected any dysfunction
of the enzyme; tests were performed in platelet cytosol
fraction.9 However, dithiothreitol, a strong
sulfhydryl-reducing agent, normally used for the preparation of
guanylate cyclase to prevent the oxidation of the
enzyme,22 could obscure any preexisting impairment in
SH-dependent enzyme sensitivity to NO induced by oxidative
stress.23 Therefore, in the current study, we examined the
interaction of guanylate cyclase with NO donors in intact
platelets. We used ODQ, a compound that inhibits activation of
guanylate cyclase by NO, but does not affect basal activity
of the enzyme.19 ODQ completely suppressed the
cGMP-elevating effects of SNP in both normals and patients (Figure 3). In aggregation studies, the inhibition of the
anti-aggregatory effects of NTG and SNP by ODQ was significantly less
pronounced in patients than in normal subjects. These results imply a
decrease in sensitivity of guanylate cyclase to NO in
aggregating platelets of patients with SAP. It is possible that
this impairment in the enzyme function could be caused by
O2-. Indeed,
O2- inhibits human platelet
guanylate cyclase24 and enhances platelet
aggregation in vitro21 and in vivo, in the animal
model.25 Furthermore, increased
O2- generation by neutrophils
has been reported in patients with ischemic heart disease
(stable and unstable angina)13 and myocardial
infarction.14 15
Inactivation of NO, both endogenous (EDRF) and exogenous
(released from NO donors) by increased concentrations of
O2- could be another
detrimental factor. In the current study, we detected a 4-fold higher
level of O2- in blood samples
from patients with SAP, as compared with normal subjects. We attempted
to reduce the concentration of
O2- with SOD (in combination
with catalase). Whereas in blood samples from normal subjects, addition
of SOD did not affect aggregation, in samples from anginal patients SOD
inhibited aggregation and enhanced anti-aggregatory efficacy of SNP
(Figure 4). Although kinetics of
O2- turnover and peroxynitrite
formation were not measured in the current study, our findings imply
that O2- can diminish
platelet responsiveness to NO donors and, probably, contributes to
the phenomenon of nitrate resistance at the platelet level.
Incomplete suppression of the anti-aggregating effects of NTG by ODQ observed even in blood samples from normal subjects suggests the existence of an additional, cGMP-independent component for the mechanism of NTG effect. This interesting observation is consistent with previous claims26 27 that the cellular effects of organic nitrates are not restricted to cGMP-dependent pathways. However, the precise mechanism(s) of the implied cGMP-independent effects are peripheral to the thrust of the current work.
The current study has several limitations. The results do not necessarily reflect accurately the extent of platelet resistance to NO (and NTG) in vivo. However, it is interesting to view these findings relative to the previous report by Folts and coworkers28 that the anti-oxidant N-acetylcysteine potentiated responsiveness to NTG in reversing in situ platelet aggregation in the canine stenosed coronary artery. No precise correlation can yet be drawn between the currently defined phenomenon of NO resistance in platelets and either the originally designated condition of vasomotor resistance to NTG in patients with chronic cardiac failure or the phenomenon of impaired endothelial function, with its associated reduction in NO-mediated responses to vasomotor stimuli.
Decreased platelet responsiveness to exogenous sources of NO implies diminution of responsiveness to endogenous NO (EDRF). This provides a potential basis not only for local or generalized increases in platelet aggregability associated with acute myocardial ischemia and/or acute redox stress; such changes could also be associated with further diminution in platelet responsiveness to organic nitrate therapy.
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Acknowledgments |
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This work was supported by a grant from the National Health and Medical Research Council of Australia. We gratefully acknowledge the assistance of the staff of the Cardiac Catheterization Laboratory and Coronary Care Unit, The Queen Elizabeth Hospital.
Received December 14, 1998; revision received April 7, 1999; accepted April 22, 1999.
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Circulation. 1991;83:2122–2127.In patients with stable
angina pectoris, platelets are more aggregable and less responsive
to the anti-aggregating and cGMP-stimulating effects of NTG and SNP,
thus representing nitrate resistance at the platelet
level. Decreased platelet responsiveness to NO donors may reflect
both reduction in platelet guanylate cyclase
sensitivity to NO and inactivation of the released NO by
O2-. The implied impairment of
anti-platelet efficacy of endogenous NO (in the form of
EDRF) may contribute to platelet hyperaggregability associated with
angina pectoris.
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 R. A Anderson, G. R Ellis, L. M. Evans, K. Morris, Y. Y Chirkov, J. D Horowitz, S. K Jackson, A. Rees, M. J Lewis, and M. P Frenneaux Platelet nitrate responsiveness in fasting and postprandial type 2 diabetes Diabetes and Vascular Disease Research, May 1, 2005; 2(2): 88 - 93. [Abstract] [PDF]
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F. Krotz, H.-Y. Sohn, and U. Pohl Reactive Oxygen Species: Players in the Platelet Game Arterioscler Thromb Vasc Biol, November 1, 2004; 24(11): 1988 - 1996. [Abstract] [Full Text] [PDF]
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 T. Heitzer, I. Ollmann, K. Koke, T. Meinertz, and T. Munzel Platelet Glycoprotein IIb/IIIa Receptor Blockade Improves Vascular Nitric Oxide Bioavailability in Patients With Coronary Artery Disease Circulation, August 5, 2003; 108(5): 536 - 541. [Abstract] [Full Text] [PDF]
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 J. D. Horowitz Amelioration of nitrate tolerance: matching strategies with mechanisms J. Am. Coll. Cardiol., June 4, 2003; 41(11): 2001 - 2003. [Full Text] [PDF]
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 M. Frenneaux New tricks for an old drug Eur. Heart J., December 2, 2002; 23(24): 1898 - 1899. [PDF]
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S.R. Willoughby, S. Stewart, Y.Y. Chirkov, J.A. Kennedy, A.S. Holmes, and J.D. Horowitz Beneficial clinical effects of perhexiline in patients with stable angina pectoris and acute coronary syndromes are associated with potentiation of platelet responsiveness to nitric oxide Eur. Heart J., December 2, 2002; 23(24): 1946 - 1954. [Abstract] [PDF]
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N. Haramaki, H. Ikeda, Y. Takajo, A. Katoh, S. Kanaya, S. Shintani, R. Haramaki, T. Murohara, and T. Imaizumi Long-Term Smoking Causes Nitroglycerin Resistance in Platelets by Depletion of Intraplatelet Glutathione Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1852 - 1856. [Abstract] [Full Text] [PDF]
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Y. Y. Chirkov, A. S. Holmes, S. R. Willoughby, S. Stewart, R. D. Wuttke, P. R. Sage, and J. D. Horowitz Stable angina and acute coronary syndromes are associated with nitric oxide resistance in platelets J. Am. Coll. Cardiol., June 1, 2001; 37(7): 1851 - 1857. [Abstract] [Full Text] [PDF]
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 A. K. Nightingale, P. P. James, J. Morris-Thurgood, F. Harrold, R. Tong, S. K. Jackson, J. R. Cockcroft, and M. P. Frenneaux Evidence against oxidative stress as mechanism of endothelial dysfunction in methionine loading model Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1334 - H1339. [Abstract] [Full Text] [PDF]
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P. R. Sage, I. S. de la Lande, I. Stafford, C. L. Bennett, G. Phillipov, J. Stubberfield, and J. D. Horowitz Nitroglycerin Tolerance in Human Vessels : Evidence for Impaired Nitroglycerin Bioconversion Circulation, December 5, 2000; 102(23): 2810 - 2815. [Abstract] [Full Text] [PDF]
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I. L. Megson, D. J. Webb, Y. Y. Chirkov, A. S. Holmes, L. P. Chirkova, and J. D. Horowitz Nitrate Resistance in Platelets From Patients With Stable Angina Pectoris Response Circulation, September 12, 2000; 102 (11): e87 - e87. [Full Text] [PDF]
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