Randomized trials have shown that some of
the ACE-Is when given in specific doses, increase survival in specific populations
of patients with heart disease (1-4). In 2004 Pilote and coworkers (5) published
the results of the retrospective head - to - head studies in which they showed
that among 7 ACE-Is used, only ramipril and perindopril reduced 1-year mortality
in patients after acute myocardial infarction. It is generally accepted, that
inhibiting of
Tissue angiotensin converting enzyme (ACE) are an effective
target for preventing premature death, myocardial infarction and stroke. Thus
beneficial cardiovascular effects of some ACE-Is may result from inhibition
of
Tissue ACE caused by their unique pharmacokinetic properties. It may
be related to their chemical structure and bioavaibility (functional group,
dissociation constants from the enzyme, lipophilicity) (6-10). Therefore ACE-Is
are now categorized into 2 groups depending on their relative affinity to
Tissue
ACE (11). Higher - affinity ACE-I (
Tissue ACE-Is) consisted of quinapril,
perindopril, benazepril, ramipril and lower - affinity ACE-Is (
non-Tissue,
plasma ACE-Is) consisted of lisinopril, enalapril and captopril.
Since 90% of the enzyme is found locally as
Tissue-bound ACE, presumably
Tissue ACE-Is could improve more effectively the endothelium function
(8, 9). Vascular endothelial cells constitute an important source of ACE where
circulating or locally produced Ang I serves as available substrate for local
production of Ang II (12).
Tissue ACE-Is through their high affinity
to endothelium significantly prevent the local synthesis of Ang II. Inhibition
of ACE, being the kininase II, cause the subsequent increase of bradykinin level
and mediated by BK
2 receptor release of nitric
oxide (NO), prostacycline (PGI
2) and
Tissue
plasminogen activator (t-PA) (13, 14).
In our previous study we observed NO and PGI
2
dependent antithrombotic effect of
plasma ACE-Is: captopril and enalapril
in thrombosis models in rats (15). Since
Tissue ACE-Is have higher affinity
to endothelium we assumed that
Tissue ACE-Is could exert stronger antithrombotic
effect than
plasma ACE-Is. No clinical and experimental studies comparing
the antithrombotic effect of
Tissue and
plasma ACE-Is have been
carried so far. Therefore the aim of our study has been to compare the antithrombotic
effect of the
Tissue and
plasma ACE-Is and the influence on haemostasis
in the same experimental conditions.
MATERIAL AND METHODS
Chemicals
The following drugs were used in the study: captopril (Research Biochemicals International, USA), enalapril (KRKA, Slovenia), perindopril (Servier, Poland), quinapril (Pfizer, Poland), collagen (Chronolog, USA), trisodium citrate, calcium chloride, Tris Buffer (Polish Chemical Reagents, Poland), Thrombin (Polfa, Poland) and gummi arabici (Polish Chemical Reagents, Poland).
Animals
Male, normotensive Wistar rats, weighing 300-400 g were used in our study. They were housed ten per cage in air-conditioned colony room at the natural light-dark cycle, with food and water continuously available. Procedures involving the animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and Guidelines for the Use of Animals in Biomedical Research (16).
Experimental protocol
Animals received
per os the following drugs for ten days. Group I - captopril:
75 mg/kg/day (twice daily) (CAP), Group II - enalapril: 20 mg/kg/day (ENA),
Group III - perindopril: 2 mg/kg/day (PER), Group IV- quinapril: 3 mg/kg/day
(QUIN). Group V - control (CON) rats received 5% aqueous gummi arabici solution
(VEH). The last dose of drugs was given 12 hours (CAP) and 24 hours (ENA, PER
and QUIN) before the experiments. The dosage of drugs and sampling time were
chosen basing on the range of doses used in other (17, 18, 19) and our previous
studies (15, 20, 21) as well as on the results of SBP measurement, in which
ACE-Is exerted similar hypotensive effect. Experiments were done on the 11
th
day after starting this treatment. Twelve hours before experiments rats were
deprived of food but had free access to water.
Blood pressure measurement
The systolic blood pressure (SBP) was measured in conscious rats, by the "tail cuff" method (Harvard Indirect Rat Tail Blood Pressure Monitor) before and after 10 days of treatment (22). Each value was the average of three consecutive readings.
Venous thrombosis induction
The venous thrombosis was induced as previously described by Reyers et al. (23).
The rat abdomen was opened under pentobarbital anesthesia (40 mg/ kg,
i.p.)
and the vena cava was carefully separated from the surrounding
Tissues
and then ligated tightly with a cotton thread just below the left renal vein.
Subsequently, the abdomen was closed with a double layer of sutures (peritoneum
with muscles and the skin separately). After 2 hours the animals were reanaesthetized,
the abdomen was reopened; the vena cava was carefully dissected and inspected
for the presence of a thrombus. The thrombus was air-dried at 37° C and after
24 hours its weight was measured.
Arterial thrombosis induction
Thrombosis was induced by an electric stimulation according to Schumacher, et
al. as follows (24). Rats were anesthetized (pentobarbital, 40 mg/kg i.p.),
fixed on an operation table and kept warm by radiant heat from a light bulb.
The left common carotid artery was exposed at the minimum length of 15 mm and
carefully freed from the surrounding
Tissue. A piece of parafilm "M"
was placed under the exposed vessel for electrical isolation. A stainless steel
L-shaped wire was placed on the dorsal surface of the artery (anode). The cathode
was a subcutaneous metal clip attached to the hindlimb. The electrodes were
connected into a circuit with a miliamperometer and potentiometer. A Doppler
flow probe (1 mm-diameter) was connected to the exposed artery upstream from
the electrode and connected to a blood flowmeter (Transonic System Inc.). After
5 min stabilization, the baseline blood flow was determined. Electrical current
of 1 mA was delivered by a constant current stimulator for 10 minutes. To demonstrate
evidence of thrombus formation, carotid blood flow (CBF) was continuously measured
before, during and 45 minutes after electrical stimulation (
Fig.1). At
the end of the experiment a segment of common carotid artery was removed, the
thrombus was isolated, dried at 37° C for 24 hours and weighted. Immediately
after removal of the thrombus the blood was collected from the heart for the
measurement of haemostatic parameters.
Fibrin generation
For fibrin generation assay we used previously described method (25) modified
and adapted to the use of laboratory animals (26). Blood samples were drawn
immediately after arterial thrombus removal from carotid artery of animals treated
for 10 days with ACE-Is or VEH. Blood was added to 3.13% trisodium citrate (9:1),
then centrifuged at 2000 g in 4°C for 20 minutes and
plasma was stored
deep-frozen in aliquots of 1 ml at -70°C until further assays were performed.
Fibrin time curve was made by adding CaCl
2 (36
mM) and thrombin (0.09 IU/ml) to the Tris buffer (66 mM Tris and 130 mM NaCl,
pH=7.4) and mixing this with rat
plasma. Optical density (OD) was measured
via microplate reader (Dynex Tech., USA) at 1 minute intervals for 10 minutes.
Based on the principle of integrals the area under the curve expressed by summation
of OD values reflected the intensity of fibrin generation.
Haemostatic parameters
Prothrombin time (PT), and activated partial thromboplastin time (APTT) were automatically determined by optical method (Coag-A-mate XM; Organon Teknika, Belgium) adding routine laboratory reagents (Organon Teknika) to collected rat
plasma. Euglobulin clot lysis time (ECLT) was evaluated according to Lidbury et al. (27).
Preparation of washed platelets
Blood samples were taken from the heart on anticoagulant (170mM trisodium citrate,
130mM citric acid and 101mM glucose) in volume ratio 9:1. Platelets washing
was carried out as described previously (28). In brief, platelet rich
plasma
(PRP) was obtained by centrifugation of blood at 180g for 20 minutes at room
temperature. PRP was then centrifuged at 400g for 15 minutes and obtained platelets
were washed with calcium-free Tyrode's buffer (137mM NaCl, 2.6mM KCl, 12mM NaHCO
3,
0.9mM MgCl
2, 5.5mM glucose, 0.35% albumin, apyrase
0.5U/ml, pH 6.5) by a centrifugation at 400 g for 15 min. The washed platelets
were finally suspended in a calcium - free Tyrod - Hepes buffer (137mM NaCl,
2.6mM KCl, 12mM NaHCO
3, 0.9mM MgCl
2,
5.5mM glucose, 0.35% albumin, pH 7,4) The final concentration of platelets was
3×10
5 platelets/µl.
Platelet adhesion to fibrillar collagen
Platelet adhesion was assayed according to Mant (29). The 250µl of washed platelet samples were incubated in an Elvi 840 aggregometer at 37°C and stirred at 900 rpm with EDTA (5mM) to prevent platelet aggregation. After 5 minutes preincubation, collagen (50µl/ml) was added and platelets were further incubated for 10 minutes. Samples of the suspension were obtained before and 15 minutes after adding the collagen, then platelets were counted in a haemocytometer after dilution with Unopette system. Index of adhering platelets was calculated using a formula [(platelet count before adding the collagen - platelet count after adding the collagen)/platelet count before adding the collagen] × 100%.
Platelet aggregation in the whole blood
Platelet aggregation in the whole blood was monitored by measuring electric
impedance using a Chronolog aggregometer (Chrono Log, Havertown, PA) according
to Cardinal et al. (30). 1 ml samples (0.5 ml of citrated blood and 0.5 ml of
0.9% NaCl) were left to equilibrate at 37°C for 5 minutes before the agonist
(collagen 5 mg/ml) addition. The platelet aggregation was evaluated by measuring
of the electric impedance (
)
5 minutes after the agonist addition.
Statistical analysis
The data are shown as mean ± SEM. While calculating thrombus weight, the lack of the thrombus was marked as 0 mg (23). Incidence of occlusion in carotid artery was calculated by Fisher's exact test. Multiple group comparisons were performed by Kruskal-Wallis nonparametric ANOVA, followed by Dunn's multiple comparisons test. Values of p < 0.05 were considered significant.
RESULTS
Blood pressure
plasma and
Tissue ACE-Is in the applied doses had similar hypotensive effect after 10 days of treatment. SBP decreased to 113±3 mmHg, 111±3 mmHg, 113±2 mmHg and 112±3 mmHg vs 127±2 mmHg in CON (n=25), for CAP (n=15), ENA (n=15), PER (n=15) and QUIN (n=15), respectively (p<0.001).
Carotid Blood Flow
The mean Initial Carotid Blood Flow (I-CBF) was similar in CON and ACE-Is-treated
groups (
Fig. 1,
Tab. 1). Electrical stimulation led to gradual
flow fall caused by the increasing arterial thrombus formation in the lumen
of carotid artery. Whereas CBF decreased to zero in most of the VEH treated
rats, vascular occlusion was prevented by ACE-Is. Mean CBF over the duration
of experiment was significantly higher in rats treated with QUIN in comparison
to other groups of animals (
Fig. 1). As shown in
Fig. 1 and
Tab.
1 the final flow was significantly higher in OUIN, PER and CAP treated groups
than in CON. The incidence of occlusion was reduced only in OUIN treated group
(p<0.05), (
Tab. 1)
|
Fig. 1. Carotid blood flow (CBF) during the arterial thrombus
formation.
CON (control, n=25) CAP (captopril, n=15), ENA (enalapril, n=15), PER
(perindopril, n=15), QUIN (quinapril, n=15).
|
Tab. 1.
Flow parameters in the carotid artery of rats during arterial thrombus
formation |
|
** p<0.01,
*** p<0.001 vs initial flow,
#p<0.05, ##p<0.01, ###p<0.001 vs control. |
Arterial and Venous thrombus weight
Administration of PER and QUIN caused marked decrease in arterial thrombus weight
0.17±0.05 mg; (p<0.01) and 0.32±0.1 mg; (p<0.001) vs CON (1.06±0.16 mg) respectively
(
Fig. 2A). CAP and ENA did not influence arterial thrombus weight.
In venous thrombosis model PER, QUIN and CAP significantly reduced the thrombus
weight vs CON (0.90±0.2mg, 0.54±0.06mg and 1.20±0.3 mg vs 2.25±0.16mg; p<0.01,
p<0.01, p<0.05; respectively) (
Fig. 2B).
|
Fig. 2. The columns represent the thrombus weight in arterial
thrombosis (A) and venous thrombosis (B) in rats treated with ACE-Is.
CON (control, n=25), CAP (captopril, n=15), ENA (enalapril, n=15), PER
(perindopril, n=15), QUIN (quinapril, n=15). *p<0.05, **p<0.01,*** p<0.001
vs CON, #p<0.05 vs CAP, p<0.05,
p<0.01,
p<0.001
vs ENA.
|
When comparing the differences between
Tissue and
plasma ACE-Is, in arterial thrombosis PER was more effective in reducing thrombus weight vs CAP (p<0.05) and ENA (p<0.001) while QUIN only vs ENA (p<0.01). Both PER and QUIN reduced the thrombus weight stronger in comparison to ENA both in arterial (p<0.01; p<0.01) as well as venous thrombosis (p<0.01; p<0.001).
Fibrin Generation
ENA, PER and QUIN decreased the fibrin generation (
Fig. 3) and the optical
density value in the 10
th minute was significantly
lower in comparison to VEH treated animals (79±5, 63±9 and 54±5 vs 89±4 in CON
group; (ns, p<0.05, p<0.001) respectively. ENA failed to influence fibrin generation.
|
Fig. 3. The curves represent optical density (OD) changes during
fibrin generation in plasma of rats treated with ACE-Is.
CON (control, n=15), CAP (captopril, n=8), ENA (enalapril, n=8), PER
(perindopril, n=8), QUIN (quinapril, n=8). *p<0.05, **p<0.01 vs CON
in 10th minute.
|
Haemostatic parameters
Neither CAP nor ENA changed PT and APTT (
Tab. 2). QUIN and PER significantly
prolonged PT (p<0.05, p<0.01) as well as APTT (p<0.01) vs CON. CAP slightly
shortened ECLT (
Tab. 2). ENA had no influence on this parameter. QUIN
(p<0.01) and PER (p<0.01) shortened ECLT in comparison to CON.
Tab. 2.
Coagulation parameters, platelet adhesion to fibrillar collagen and whole
blood platelet aggregation in rats with arterial thrombosis. |
|
*p<0.05, ** p<0.01 vs CON |
Platelet adhesion to fibrillar collagen
When the platelets were incubated with collagen, 34.7±0.8% of the platelets
adhered. Pretreatment with CAP, ENA, PER and QUIN resulted in a significant
reduction of platelet adhesion (
Tab. 2).
Whole blood platelet aggregation
The collagen induced platelet aggregation was inhibited significantly after
ENA (p<0.05), PER (p<0.05) and QUIN (p<0.05) treatment (
Tab. 2), while
CAP did not significantly affect the whole blood platelet aggregation.
DISCUSSION
In the present study we have presented that given in equipotent hypotensive doses,
Tissue ACE-Is (quinapril and perindopril) exerted more pronounced antithrombotic effect than
plasma ACE-Is (captopril and enalapril) in experimental thrombosis. This effect was related to the more distinct activation of fibrinolysis and, inhibition of coagulation by
Tissue ACE-Is.
We have applied here experimental model of stasis induced venous thrombosis that not only resembles pathological conditions of thrombus formation in human, but also separates the hemodynamic and haemostatic effects of the studied substances, due to the minor relationship between arterial and venous blood pressure. Moreover, in this model further activation of renin-angiotensin-system (RAS) occurs due to false information about hypovolemia as a result of the vena cava ligation (23), which enables studying the effects of various compounds affecting RAS. Using this model we have previously demonstrated that captopril and AT1-antagonist (losartan) or Ang-(1-7) exert strong antithrombotic effect (15, 20, 21). The drugs preventing venous thrombosis interfere with coagulation and act on endothelium to increase naturally occurring fibrinolytic activity.
We found that QUIN and PER reduced venous thrombus weight to a greater extent than CAP and ENA. CAP showed also stronger antithrombotic effect than ENA on venous thrombus formation. We have previously shown that more distinct antithrombotic effect of CAP was related to the presence of sulfhydryl group in the molecule (31). Thus it should be emphasized the role of thiol group which is responsible for the stabilization and prolongation of NO half-life potentiating its profibrinolytic and antithrombotic effect. This observation is in good agreement with a study which demonstrated that pre-treatment of endothelial cells with N-acetylcysteine abolished the strain-induced PAI-1 release and activated fibrinolysis (32).
We also used platelet dependent arterial thrombosis model in which electric current is a factor, causing damage of endothelium and deeper layers of vessel wall. Changes in blood flow are a reliable marker of platelet-rich thrombus formation (33). Although platelets initiate arterial thrombosis the activation of blood coagulation and fibrinolysis is the next critical step (34). We observed significant reduction of arterial thrombus weight in rats treated with PER and QUIN. Others have also shown inhibition of arterial thrombotic process by quinapril, imidapril and captopril in rats (18, 19, 35, 36, 37).
In our study we have observed more pronounced effect of OUIN in maintenance
of the carotid blood flow over the period of experiment in comparison to PER,
even though similar effect of both drugs on thrombus weight occurred. It could
be related to the differences between QUIN and PER in the affinity with ACE.
In
in vitro and experimental animals studies it was shown, that the potency
against
plasma as well as
Tissue ACE was greater for QUIN than
for PER and
plasma ACE-Is (6-8). Moreover, on the basis of the earlier
data related to the dynamics of arterial thrombus formation we could suggest,
that only drugs with relatively strong antithrombotic potency (thrombin inhibitors,
aspirin) maintain CBF on the initial level and markedly reduce the thrombus
weight (24). For drugs with weaker antithrombotic potency the strong stimulus
such as electrical current causing severe endothelial damages, leads finally
to closing the lumen of the vessel by growing thrombotic material.
It is possible, that differences between
Tissue and
plasma ACE-Is
in terms of their more pronounced inhibition of
Tissue RAS and in consequence
in stronger impact on thrombosis occurred. Since the positive correlation between
arterial thrombus weight reduction and inhibition of ACE activity has already
been shown (36) and QUIN was also more effective than ENA in ACE inhibition
and reducing
Tissue Ang II in rat (38, 39), in the present study we have
not measured the
Tissue ACE activity. We thought that at least two mechanisms
may account for stronger antithrombotic effect of
Tissue ACE-Is. First,
Tissue ACE-Is through their high affinity to endothelium considerably
prevent the local synthesis of Ang II. This may result in attenuation of prothrombotic
action of this peptide. Ang II is well known to contribute to endothelial dysfunction
by inducing oxidative stress (40), inhibiting NO synthesis (41) and enhancing
leukocytes infiltration and adhesion to vascular wall (42). Few observations
indicate that Ang II may activate the coagulation cascade by increasing
Tissue
factor (TF) expression (43). Moreover, Ang II inhibits fibrinolysis by increasing
plasminogen activator inhibitor type 1 (PAI-1) expression (44). What's more,
we proved prothrombotic effect of this peptide in
in vivo model of venous
and arterial thrombosis in hypertensive rats (45, 46). Secondly, ACE-Is by increasing
bradykinin concentration enhances the release of NO, PGI
2
and t-PA-strong antithrombotic agents (47, 48). The fact that the reduction
of thrombus weight was more pronounced after QUIN and PER administration may
be linked to their greater ability to release these autacoids from endothelium.
It has recently been shown that
Tissue ACE-Is (quinapril and perindopril)
caused experimental thrombolysis in rats to a greater extent than captopril
by the mechanism which involved bradykinin release from endothelium (49).
ACE-Is are known to affect haemostasis at different levels. As was previously shown antithrombotic effect of ACE-Is may depend on the inhibition of platelet and erythrocyte aggregation (35, 37), elevation of NO release (50), reduction of serum and aortic ACE activity, reduction of aortic PAI-1 protein level (36), down regulation of glycoprotein IIb/IIIa complex on platelet surface (37) and attenuation of
Tissue factor expression (47). This clearly suggests that various mechanisms are involved in the antithrombotic effect of ACE-Is.
In the second part of our study we compared the impact of studied drugs on haemostasis
with respect to their antithrombotic activity. Since the studies evaluating
the effect of ACE-Is on clinical outcomes were done in patients with artery
disease (1-4), we compared the influence of
Tissue and
plasma
ACE-Is on haemostasis in rats developing arterial thrombosis. We found superiority
of PER and QUIN over CAP and ENA in fibrinolytic system activation in rats.
Fibrinolysis is largely regulated by endothelial expression of t-PA and PAI-1
and ACE is the crucial mediator of the interaction between PAI-1 and t-PA (48).
Strong profibrinolytic effect of ACE-Is - as a result of bradykinin-depend release
of PGI
2 and t-PA from endothelium in rats was
also shown by others (49,51,52). Clinical studies have also shown profibrinolytic
effect of
Tissue ACE-Is, but large comparative studies have not been
done yet (53,54,55).
We have also showed that
Tissue ACE-Is inhibited coagulation cascade.
Both QUIN and PER and to a lesser extent ENA inhibited fibrin generation, prolonged
PT and APTT pointing to the impact on extrinsic and intrinsic pathways of coagulation
cascade. It has been previously shown, that fibrin generation assay depends
on
plasma levels of various coagulation factors, like V, VII, VIII, IX,
X or endogenous thrombin. Moreover, fibrin generation increases after adding
exogenous TF or von Willebrand factor but decreases when exogenous t-PA is added,
indicating that the method is also sensitive to agents affecting vascular wall
(25). The expression of TF on endothelial cell membrane is critical for the
initiation of extrinsic coagulation cascade and platelet activation after vessel
injury. Therefore a possibility that ACE-Is could prevent thrombus formation
through reduction of TF expression in endothelium cannot be excluded. It was
shown that ENA reduced
plasma level of TF in patients after myocardial
infarction (56). We assume that stronger inhibitory effect of QUIN and PER on
coagulation cascade may depend on NO and PGI
2
release from endothelium. These mediators markedly decreased expression of TF
(47). On the other hand it was demonstrated that
Tissue ACE-I - ramipril
did not change the coagulation parameters in rats, rabbits or dogs but reduced
the level of thrombin - antithrombin (TAT) complexes in patients with arterial
hypertension (57, 58).
The first step of a haemostatic plug formation is adhesion of circulating thrombocytes to the subendothelial matrix of damaged vessel wall. Adhered platelets recruit additional platelets within developing aggregate (59). Among the components of the wall collagen is considered to be the most important element involved in that process since it is a unique ligand for platelet adhesion causing also platelet activation (60). ACE-Is are believed to inhibit platelet activity (61), thus a question arises whether this action could contribute to the effective prevention of arterial thrombosis. Our results show similar antiadhesive and antiaggregative effects of all studied ACE-Is. This suggest that platelets inhibition is not responsible for the stronger antithrombotic effect of
Tissue ACE-Is in our experimental condition.
We have recently showed that
plasma and
Tissue ACE-Is have comparable effects on coagulation and fibrinolysis in nonthrombogenic rats (62). In the present study, when endothelium denudation is followed by thrombin generation, platelet activation and impairment of fibrinolysis, the differences in the effect of
Tissue ACE-Is are clearly demonstrated. Also in clinical studies cardioprotective benefits of ACE-Is was particularly apparent in high-risk patients (63, 64) Thus our observation may have clinical significance since thrombotic disorders often accompany cardiovascular disease.
CONCLUSION
In conclusion, we have demonstrated the more pronounced antithrombotic effect of
Tissue ACE-Is (quinapril and perindopril) in comparison to
plasma ACE-Is (captopril and enalapril) in normotensive rats both in venous and arterial thrombosis models. The stronger antithrombotic effect of
Tissue ACE-Is seems to be complex and involves the activation of fibrinolytic pathway and inhibition of coagulation system. The demonstration that ACE-Is do not influence haemostasis in the same way could have an important clinical implication.
Acknowledgment: The authors would like to thank
Pfizer Poland for a supply of quinapril.
This work was supported by Grant No. 3 PO5B 198 22 from the Polish Committee
for Scientific Research.
REFERENCES
- Pffefer MA, Braunwald E, Moye LA, et al. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and verticular enlargement trial. The SAVE Investigators. N Engl J Med 1992; 327: 669-77.
- Yusuf S, Pepine CJ, Garces C, et al. Effect of enalapril on myocardial infarction and unstable angina in patients with low ejection fractions (SOLVD). Lancet 1992; 340: 1173-1178.
- Yusuf S, Sleight P, Pogue J, et al. Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in hig-risk patietnts. The HOPE (Heart Outcomes Prevention Evaluation) Study Investigators. N Engl J Med 2000; 342: 145-153.
- The EURopean trial On reduction of cardial events with Perindopril in stable coronary Artery diesease Investigators. Efficacy of perindopril in reduction of cardiovascular events among patients with stable coronary artery diesease: randomised, double-blind, placebo-controlled, multicentre trial (the Europa study). Lancet 2003; 362: 782-788.
- Pilote L, Abrahamowicz M, Rodrigues E, Eisenberg MJ, Rahme E. Mortality rates in elderly patients who take different angiotensin-converting enzyme inhibitors after acute myocardial infarction: a class effect? Ann Intern Med. 2004; 141: 102-112.
- Johnston CI, Fabris B, Yamada H, et al.Comparative studies of Tissue inhibition by angiotensin converting enzyme inhibitors. J Hypertens Suppl. 1989; 7: S11-16.
- Fabris B, Jackson B, Cubela R, Mendelsohn FA, Johnston CI. Angiotensin converting enzyme in the rat heart: studies of its inhibition in vitro and ex vivo. Clin Exp Pharmacol Physiol. 1989; 16: 309-313.
- Fabris B, Chen BZ, Pupic V, Perich R, Johnston CI. Inhibition of angiotensin converting enzyme (ACE) in plasma and Tissue. J Cardiovasc Pharmacol 1990; 15: S6-S13.
- Cushman DW, Chenug HS. Concentrations of angiotensin converting enzyme in Tissues of the rat. Biochim Biophys Acta 1971; 250: 261-265.
- Nakajima T, Yamada T, Setoguchi MP. Prolonged inhibition of local angiotensin-converting enzyme after single or repeated treatment with quinapril in spontaneously hypertensive rats. J Cardiocasc Pharmacol 1992; 19: 102-107.
- Dzau VJ, Bernstein K, Celermajer D, et al. Working Group on Tissue Angiotensin-converting enzyme, International Society of Cardiovascular Pharmacotherapy. The relevance of Tissue angiotensin-converting enzyme: manifestations in mechanistic and endpoint data. Am J Cardiol 2001; 88: 1L-20L.
- Danser AHJ, van Kats JP, Admiraal PJ, et al. Cardiac renin and angiotensins. Uptake from plasma versus in situ synthesis. Hypertension 1994; 24: 37-48.
- Pretorius M, Rosenbaum D, Vaughan DE, Brown NJ. Angiotensin-converting enzyme inhibition increases human vascular Tissue-type plasminogen activator release though endogenous bradykinin. Circulation 2003; 107: 579-585.
- Swies J, Chlopicki S, Gryglewski RJ. Kinins and thrombolysis. J Physiol Pharmacol 1993; 44: 171-177.
- Pawlak R, Chabielska E, Golatowski J, Azzadin A, Buczko W. Nitric oxide and prostacyclin are involved in antithrombotic activity of captopril in venous thrombosis in rats. Thromb Haemost 1998; 79: 1208-1212.
- Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost 1987; 58: 1078-1084.
- Richer C, Doussau MP, Giudicelli JF. Systemic and regional hemodynamic profile of five angiotensin I converting enzyme inhibitors in the spontaneously hypertensive rat. Am J Cardiol 1987; 59: 12D-17D.
- Mehta JL. Modulation of arterial thrombosis by angiotensin-converting enzyme inhibition and angiotensin II type 1-receptor blockade. Am J Cardiol 1988; 82: 53S-56S.
- Moriyama S, Ishigai Y, Mori T, Fukuzawa A, Shibano T. Evaluation of high through-to-peak ratio of perindopril in SHR. Clin Exp Hypertens 1999; 21: 1223-1238.
- Kucharewicz I, Pawlak R, Matys T, Pawlak D, Buczko W. Antithrombotic effect of captopril and losartan is mediated by angiotensin-(1-7). Hypertension 2002; 40: 774-779.
- Chabielska E, Pawlak R, Golatowski J, Buczko W. The antithrombotic effect of captopril and losartan on experimental arterial thrombosis in rats. J Physiol Pharmacol 1998; 49: 251-60.
- Zatz R. A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab Anim Sci 1990; 40: 198-201.
- Reyers I, De Gaetano G, Donati MB. Venostasis induced thrombosis in rat is not influenced by circulating platelet or leucocyte numbers. Agents Action 1989; 28: 137-140.
- Schumacher WA, Steinbacher TE, Heran CL, Megill JR, Durham SK. Effects of antithrombotic drugs in a rat model of aspirin-insensitive arterial thrombosis. Thromb Haemost 1993; 69: 509-514.
- He S, Bremme K, Blomback M. A laboratory method for determination of overall haemostatic potential in plasma. I. Method design and preliminary results. Thromb Res 1999; 96: 145-156.
- Buczko W, Mogielnicki A, Kramkowski K, et al. Aspirin and the fibrynolytic response. Thromb Res 2003; 110: 331-334.
- Lidbury PS, Korbut R, Vane JR. Sodium nitroprusside modulates the fibrinolytic system in the rabbit. Br J Pharmacol 1990; 101: 527-530.
- Matys T, Chabielska E, Pawlak R, Kucharewicz I, Buczko W. Losartan inhibits the adhesion of rat platelets to fibrillar collagen-a potential role of nitric oxide and prostanoids. J Physiol Pharmacol 2000; 51: 705-713.
- Mant MJ. Platelet adherence to collagen: a simple, reproducible, quantitative method for its measurement. Thromb Res 1977; 11: 729-737.
- Cardinal DC, Flower RJ. The study of platelet aggregation in whole blood. Br J Pharmacol 1979; 66: 94P-95P.
- Pawlak R, Chabielska E, Matys T, Kucharewicz I, Rolkowski R, Buczko W. Thiol repletion prevents venous thrombosis in rats by nitric oxide/prostacyclin-dependent mechanism: relation to the antithrombotic action of captopril. J Cardiovasc Pharmacol 2000; 36: 503-509.
- Cheng JJ, Chao YJ, Wung BS, Wang DL. Cyclic strain-induced plasminogen activator inhibitor-1(PAI-1) release from endothelial cells involves reactive oxygen species. Biochem Biophys Res Commun 1996; 225: 100-105.
- Folts J, Gallagher K, Rowe G. Blood flow reductions in stenosed canine arteries: vasospasm or platelet aggregation? Circulation 1982; 65: 248-255.
- Ofosu FA. The blood platelet as a model for regulating blood coagulation on cell surfaces and its consequences. Biochemistry 2002; 67: 47-55.
- Korbut RA, Madej J, Adamek-Guzik T, Korbut R. Secretory dysfunction of vascular endothelium limits the effect of angiotensin converting enzyme inhibitor quinapril on aggregation of erythrocytes in experimental hypertension. J Physiol Pharmacol 2003; 54: 397-408.
- Mitsui T, Chishima S, Odawara A, Ohtani A. Imidapril, an angiotensin converting enzyme inhibitor, inhibits thrombosis via reduction in aortic plasminogen activator inhibitor type -1 levels in spontaneously hypertensive rats. Biol Pharm Bull 1999; 22: 863-865.
- Zurbano MJ, Anguera I, Heras M, et al. Captopril administration reduces thrombus formation and surface expression of platelet glycoprotein IIb/IIa in early postmyocardial infarction stage. Arterioscler Thromb Vasc Biol 1999; 19: 1791-1795.
- Nakajima T, Yamada T, Setoguchi MP. Prolonged inhibition of local angiotensin- converting enzyme after single or repeated treatment with quinapril in spontaneously hypertensive rats. J Cardiocasc Pharmacol 1992; 19: 102-107.
- Vertes V, Haynie RC. Comparative pharmacokinetics of captopril, enalapril and quinapril. Am J Cardiol 1992; 69: 8C-16C.
- Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 1994; 74: 1141-1148.
- Wolf G, Ziyadeh FN, Schroeder R, Stahl RA. Angiotensin II inhibits inducible nitric oxide synthase in tubular MCT cells by a posttranscriptional mechanism. J Am Soc Nephrol 1997; 8: 551-557.
- Pastore L, Tessitore A, Martinotti S, et al. Angiotensin II stimulates intercellular adhesion molecule-1 (ICAM-1) expression by human vascular endothelial cells and increases soluble ICAM-1 release in vivo. Circulation 1999; 100: 1646-1652.
- Nishimura H, Tsuji H, Masuda H, et al. Angiotensin II increases plasminogen activator inhibitor-1 and Tissue factor mRNA expression without changing that of Tissue type plasminogen activator or Tissue factor pathway inhibitor in cultured rat aortic endothelial cells. Thromb Haemost 1997; 77: 1189-1195.
- 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 1993; 87: 1969-1973.
- Mogielnicki A, Chabielska E, Pawlak R, Szemraj J, Buczko W. Angiotensin II enhances thrombosis development in renovascular hypertensive rats. Throm
- Mogielnicki A, Chabielska E, Pawlak R, Szemraj J, Buczko W. Angiotensin II enhances thrombosis development in renovascular hypertensive rats. Thromb Haemost 2005;93:1069-76.
- Kamińska M, Mogielnicki A., Stankiewicz A., K. et al Angiotensin II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rats. J Physiol Pharmacol 2005; 56: 571-585.
- Kubo-Inoue M, Egashira K, Usui M, et al. Long-term inhibition of nitric oxide synthesis increases arterial thrombogenecity in rat carotid artery. Am J Physiol Heart Circ Physiol 2002; 282: H1478-1484.
- Vaughan DE. Angiotensin and vascular fibrinolytic balance. Am J Hypertens 2002; 15: 3S-8S.
- Gryglewski RJ, Swies J, Uracz W, Chlopicki S, Marcinkiewicz E. Mechanisms of angiotensin converting enzyme inhibitor induced thrombolysis in Wistar rats. Thromb Res 2003; 110: 323-329.
- Sasaki Y, Noguchi T, Seki J, Giddings JC, Yamamoto J. Protective effects of imidapril on He-Ne laser-induced thrombosis in cerebral blood vessels of stroke-prone spontaneously hypertensive rats. Thromb Haemost 2000; 83: 722-727.
- Katoh M, Egashira K, Mitsui T, Chishima S, Takeshita A, Narita H. Angiotensin-converting enzyme inhibitor prevents plasminogen activator inhibitor-1 expression in a rat model with cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis. J Mol Cell Cardiol 2000; 32: 73-83.
- Bachetti T, Comini L, Pasini E, Cargnoni A, Curello S, Ferrari R. Ace-inhibition with quinapril modulates the nitric oxide pathway in normotensive rats. J Mol Cell Cardiol 2001; 33: 395-403.
- Labinjoh C, Newby DE, Pellegrini MP, Johnston NR, Boon NA, Webb DJ. Johnston NR, Boon NA, Webb DJ. Potentiation of bradykinin-induced Tissue plasminogen activator release by angiotensin-converting enzyme inhibition. J Am Coll Cardiol 2001; 38: 1402-1408.
- Vaughan DE, Rouleau JL, Ridker PM, Arnold JM, Menapace FJ, Pfeffer MA. Effects of ramipril on plasma fibrinolytic balance in patients with acute anterior myocardial infarction. HEART Study Investigators. Circulation 1997; 96: 442-447.
- Soejima H, Ogawa H, Yasue H, Suefuji H, Kaikita K, Nishiyama K. Effects of imidapril therapy on endogenous fibrinolysis in patients with recent myocardial infarction. Clin Cardiol 1997; 20: 441-445.
- Soejima H, Ogawa H, Yasue H, et al. Effects of enalapril on Tissue factor in patients with uncomplicated acute myocardial infarction. Am J Cardiol 1996; 78: 336-340.
- Omosu M, Komine I, Becker RH, Scholkens BA. General pharmacology of ramipril. Biochem Biophys Res Commun 1988; 38: 1309-1317.
- Ekholm M, Wallen NH, Johnsson H. Long-term angiotensin-converting enzyme inhibition with ramipril reduces thrombin generation in human hypertension. Clin Science 2002; 103: 151-155.
- Andrews RK, Berndt MC. Adhesion-dependent signalling and the initiation of haemostasis and thrombosis. Histol Histopathol 1998; 13: 837-844.
- Sixma JJ, van Zanten GH, Saelman EU et al.: Platelet adhesion to collagen. Thromb Haemost 1995; 74: 454-459.
- Bauriedel G, Skowasch D, Schneider M, Andrie R, Jabs A, Luderitz B. Antiplatelet effects of angiotensin-converting enzyme inhibitors compared with aspirin and clopidogrel: A pilot study with whole-blood aggregometry. Am Heart J 2003; 145: 343-348.
- Kramkowski K, Mogielnicki A, Chabielska E, et al. W The effect of 'Tissue' and 'plasma' angiotensin converting enzyme inhibitors on overall haemostatic potentials in rats. Thromb Res 2006; 25: (in press).
- Tatti P, Pahor M, Byington RP, et al. Outcome results of the Fosiopril versus Amlodipine Cardiovascular Events randomized Trial (FACET) in patients with hypertension and NIDDM. Diabetes Care 1998; 21: 597-603.
- Estacio RO, Jeffers BW, Hiatt WR, Biggerstaff SL, Gifford N, Schrier RW. The effect of nisoldipine as compared with enalapril on cardiovascular outcomes in patients with non- insulin - dependent diabetes and hypertension. N Engl J Med 1998; 338: 645-52.