An increasing number of clinical and experimental studies has confirmed the role of angiotensin II (Ang II) as an important etiologic factor for the development of thrombotic diseases. The idea of the link between the renin-angiotensin system (RAS) and thrombosis originally emerged from the observation that an infusion of Ang II to normotensive and hypertensive subjects results in an increase of plasminogen activator inhibitor-1 (PAI-1) plasma level (1). Then it was demonstrated that Ang II regulates the expression of PAI-1 in cultured endothelial cells (2). Moreover, a significant increase in tissue factor activity after Ang II stimulation also was demonstrated recently (3), suggesting that not only fibrinolysis but also coagulation may be regulated by the RAS.
Thus, the RAS blockade should prevent thrombus formation. Besides numerous profitable effects of angiotensin-converting enzyme inhibitors (ACE-Is) including plaque stabilization, antiischemic, and antiremodeling action, Pfeffer et al. (4) have shown that long-term administration of captopril, an ACE-I containing the thiol group, to patients with left ventricular dysfunction after myocardial infarction reduces the rate of recurrent coronary thrombosis. Other studies have demonstrated beneficial effect of captopril on the fibrinolytic balance (5,6), which supports the previous finding. A fibrinolytic effect also was demonstrated for ACE-Is not containing the thiol group (7).
However, in our previous study we demonstrated that captopril exerted a more pronounced antithrombotic effect than enalapril when given in an equipotent dose in a venous thrombosis model in normotensive rats (8). This antithrombotic action of captopril was nitric oxide and prostacyclin dependent (8). It is conceivable, therefore, that the more pronounced antithrombotic effect of captopril is dependent on its chemical structure and related to the presence of the thiol group in the molecule. Therefore, in our study we examined the effect of thiol repletion on the development of venous thrombosis in rats and its relation to coagulation and fibrinolysis. We show here for the first time that thiol repletion can prevent the development of venous thrombosis by the enhancement of the fibrinolytic processes, independent of ACE-inhibitory activity of the drugs. Moreover, we demonstrate here that the antithrombotic action of the thiol compounds is nitric oxide and prostacyclin dependent.
MATERIALS AND METHODS
Male Wistar rats 200-280 g in weight were used in this study. The animals were housed in a room with a 12-h light/dark cycle, in group cages as appropriate, were given tap water to drink, and were fed a standard rat chow. Twenty-four hours before venous thrombosis induction, rats were deprived of food and had free access to water only.
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 (Thromb Haemost 1987;58:1078-84).
Drugs and substances
Captopril (RBI, Natick, U.S.A.), epicaptopril (SQ 14534, kindly provided by Dr. Piotr Oblakowski, Bristol-Myers Squibb, Warsaw, Poland), N-acetylcysteine (RBI), enalapril (Sigma, Steinheim, Germany), indomethacin (RBI), L-NAME (NG-nitro-L-arginine methyl ester; RBI), 3.13% trisodium citrate (Polish Chemical Reagents, Gliwice, Poland), and pentobarbital (Vetbutal, Polfa, Pulawy, Poland) were used in the experiments. To estimate hemostatic parameters, appropriate reagents (Organon Teknika Corp., North Carolina, U.S.A.) were used.
The animals received captopril (5 mg/kg twice daily; CAP), epicaptopril (SQ 14534)-the thiol-containing stereoisomer of captopril with essentially no ACE-I properties (5 mg/kg twice daily; EPI), N-acetylcysteine (3.75 mg/kg twice daily; ACC), or enalapril (3 mg/kg once daily; ENA) in aqueous solutions for 10 days, per os, or distilled water (the same volume and route; VEH; see figure legends for the numbers of experiments). The administered amounts of the thiol compounds were calculated to contain the same total number of thiol groups. The dose of enalapril was equipotent to that of captopril with respect to ACE inhibition. On the day 11, mean arterial blood pressure (the tail-cuff method; Student Oscillograph, Harvard Rat Tail Blood Pressure Monitor) (9) was measured 5 min before the venous thrombosis induction and calculated as the average of at least three consistent determinations. "Template" bleeding time was also measured just before operation by longitudinal incision of a rat tail, according to the method of Dejana et al. (10). Then the animals were anesthetized with pentobarbital (45 mg/kg, i.p.), and venous thrombosis was performed as previously described by others (11). In brief, the abdomen was opened, 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 h the animals were reanesthetized, the abdomen was reopened, and blood was collected for hemostatic estimations. Then the vena cava was carefully dissected and inspected for the presence of a thrombus. The thrombus was kept at 37°C, and after 24 h, its dry weight was measured.
To investigate the involvement of nitric oxide and prostacyclin in the antithrombotic action of epicaptopril, the animals were additionally administered either nitric oxide synthase inhibitor L-NAME (30 mg/kg s.c., in distilled water, 20 min before the venous thrombosis induction) or indomethacin (2.5 mg/kg s.c., 2 h before the venous thrombosis was induced), respectively. Indomethacin was dissolved in distilled water by adding minute amounts of NaOH, adjusting the pH to 7.4. Indomethacin or L-NAME does not influence the development of the venous thrombosis when administered alone (8).
The blood for hemostatic evaluations was collected 2 h after the induction of venous thrombosis. Blood samples of 1 ml each were carefully taken from the site of the thrombus formation, by a puncture of the vena cava just below the ligature, to estimate local changes in coagulation and fibrinolysis. Thereafter samples of 2 ml of blood were collected from the heart to assess hemostatic changes in systemic circulation. All samples were mixed with 3.13% trisodium citrate in a volume ratio of 9:1. The platelet-poor plasma was obtained by centrifugation of the blood at 450 g for 20 min at 4°C. Samples of 0.15 ml were immediately frozen to −20°C. Then prothrombin time (PT) and activated partial thromboplastin time (APTT) were automatically determined by optical method (Coag-A-Mate XM; Organon Teknika, Belgium) using routine laboratory reagents (Organon Teknika) and euglobulin clot lysis time (ECLT) according to Lidbury et al. (12).
The data are shown as mean ± SEM. In calculating thrombus weight, lack of a thrombus was regarded as 0 mg. The incidence of thrombus formation was compared between groups using the Fisher Exact test. All other parameters were compared between groups by means of the Mann-Whitney U test. The p values <0.05 were considered significant.
Incidence of thrombosis and thrombus weight
The incidence of venous thrombosis was significantly reduced by CAP (72%; p < 0.05), EPI (62%; p < 0.001), ACC (64%; p < 0.01), but not ENA (80%, NS) in comparison with VEH rats (97%, see Fig. 1A).
In VEH rats, the dry thrombus weight amounted to 1.16 ± 0.18 mg. CAP and EPI administration resulted in a marked decrease in this parameter by 64% (p < 0.01) and 71% (p < 0.001), respectively. Slight thrombus weight reduction also was seen in ACC-treated (by 26%) and ENA-treated (by 19%) groups, but it did not reach statistical significance (Fig. 1B).
The antithrombotic effect of EPI was blunted by administration of INDO or L-NAME. The incidence of venous thrombosis in EPI+INDO and EPI+L-NAME-treated animals amounted to 90% in both groups and was indistinguishable from control (NS vs. VEH; Fig. 1A). Similarly the thrombus weight in EPI+INDO and EPI+L-NAME groups returned toward normal (80% and 103% of that observed in VEH animals, respectively; NS vs. VEH; p < 0.01 vs. EPI; Fig. 1B).
Mean blood pressure and bleeding time
Mean blood pressure measured by the tail-cuff method amounted to 95 ± 2 mm Hg in the VEH group and was not altered by 10 days of treatment with either ENA, ACC, CAP, or EPI (see Table 1).
Similarly, no differences in "template" bleeding time were observed between these groups of animals (Table 1).
Euglobulin clot lysis time was reduced in systemic blood in rodents treated with ACC, CAP, or EPI (by 27, 13, and 31%, respectively; p < 0.05; Fig. 2A). When the blood was derived from the place of thrombus formation, significant decrease in this parameter was noted in ACC, CAP, and EPI (by 38, 28, and 35%, respectively; p < 0.05) in comparison with VEH-treated animals (Fig. 2B). ENA did not change this parameter in the blood collected from either the heart or the place of thrombus formation (Fig. 2A and B).
Administration of INDO or L-NAME reversed the influence of EPI on the euglobulin clot lysis time in both systemic blood (111% and 106% of control for EPI+INDO and EPI+L-NAME, respectively; NS vs. VEH; p < 0.05 vs. EPI; Fig. 2A) and in the blood obtained by the puncture of the vena cava (94 and 91% of control for EPI+INDO and EPI+L-NAME, respectively; NS vs. VEH; p < 0.05 vs. EPI; Fig. 2B).
No changes in prothrombin time were observed in the blood collected from the heart between VEH-, ENA-, ACC-, CAP-, and EPI-treated rodents (Fig. 3A). However, when the blood was obtained by a puncture of the vena cava, a small but significant increase in this parameter could be noticed in both CAP-treated (by 9%; p < 0.05) and EPI-treated (by 14%; p < 0.05), but not ACC-or ENA-treated animals in comparison with rats administered VEH (Fig. 3B). In the EPI-treated group, INDO and L-NAME attenuated the changes in prothrombin time observed in the blood collected from the place of thrombus formation (NS vs. VEH; p < 0.001 and p < 0.05 vs. EPI for EPI+INDO and EPI+L-NAME, respectively; Fig. 3B).
No differences in activated partial thromboplastin time were perceived either in the systemic blood or in the blood collected from the place of thrombus formation between any groups of rats (Fig. 4A and B). Neither INDO nor L-NAME influenced this parameter when administered to animals treated with EPI (Fig. 4A and B).
The most striking feature of this study is that captopril, epicaptopril (the thiol-containing stereoisomer of captopril with essentially no ACE-I properties), and N-acetylcysteine, but not enalapril, after their long-term administration, reduced the incidence of thrombosis and/or the thrombus weight in normotensive rats. Thus, we demonstrated that thiol compounds can exert an anti-thrombotic effect by themselves, regardless of their ACE-inhibitory properties. This observation is in excellent agreement with our previous studies demonstrating superiority of captopril over enalapril (8) and AT1 angiotensin-receptor antagonist losartan (13) in preventing thrombus formation in the same experimental model. It has been demonstrated recently that the release of PAI-1 from endothelium can be augmented by an excess of angiotensin II (1,2). Consequently, many clinical studies have shown a decrease in PAI-1 and an increase in tPA after treatment with various ACE inhibitors (5,7). In this study we investigated the role of the thiol group in the moiety of captopril on the fibrinolytic effect of the drug. Because studies on ACE inhibitors failed to demonstrate any changes in coagulation and fibrinolysis parameters in peripheral blood (15,16), we decided to estimate them also in the plasma obtained directly from the site of the thrombus formation. We demonstrated that antithrombotic effect of captopril, epicaptopril, and N-acetylcysteine was accompanied by a significant increase in fibrinolysis in the blood taken from both the ligated vena cava and from the heart. On the contrary, enalapril in the dose equipotent to that of captopril with respect to ACE inhibition failed to produce any antithrombotic effect, as measured by the incidence of thrombosis, the thrombus weight, and the fibrinolytic status of the animals. Thus, we not only proved that the presence of the thiol group in the moiety may substantially contribute to fibrinolytic effect of captopril, but also that thiol agents increase fibrinolysis regardless of their ACE-inhibitory properties. This conclusion is in line with the study by Cheng et al. (17), who demonstrated in vitro that PAI-1 release from endothelium is modulated by reactive oxygen species, which can be scavenged by thiol-containing agents. In their study (17), pretreatment of endothelial cells with antioxidant N-acetylcysteine abolished the strain-induced PAI-1 release.
Oxidative stress is one of the mechanisms that may activate coagulation, thus promoting thrombosis (18,19). Hence, free-radical scavengers should exert antithrombotic effect. Previous studies demonstrated the ability of thiol-containing agent 2-mercaptopropionylglycine to prolong clotting times of human plasma in vitro (14) and the ability of N-acetylcysteine to prevent intravascular fibrin formation (20) and reduce plasma viscosity in vivo (21). Therefore, in our study we examined the role of extrinsic and intrinsic pathways of fibrinogen activation in antithrombotic effect of captopril and the role of thiol group in this action. Small but significant increases in prothrombin time was noted in the blood collected from the site of thrombus formation of captopril and epicaptopril but not N-acetylcysteine- or enalapril-treated animals, with no changes in systemic circulation. No differences in activated partial thromboplastin time in the blood either collected from the heart or taken from the vena cava were perceived between any groups of rodents. This finding suggests that the suppression of the extrinsic pathway of the coagulation cascade might have contributed to the antithrombotic activity of captopril and epicaptopril, but not N-acetylcysteine. Whether the effect of captopril and epicaptopril on prothrombin time was direct or indirect remains to be determined. Nevertheless, our results are not in line with these obtained by Wegener et al. (20), who demonstrated that N-acetylcysteine counteracts fibrin deposition in the lung in the model of intravascular coagulation, and at the same time, did not increase its elimination. In their study, however, N-acetylcysteine was used in a short-term manner in a relatively high dose of 125 mg/kg, whereas we used a 30-fold smaller dose for 10 days. Moreover, in their study, the potential fibrinolytic effect of N-acetylcysteine was eliminated by a former injection of tranexamic acid.
The experimental data indicate that the presence of the thiol group in the moiety of a drug may potentiate the effect of nitric oxide (NO), the well-known endothelial mediator with strong antithrombotic potential (22). It was demonstrated by Stamler et al. (23) that NO circulates in plasma mainly as an S-nitroso adduct to the thiol groups of serum albumin. This results in the stabilized form of nitric oxide, which can act longer and more extensively than its gaseous form (24). Because NO, prostacyclin, and tissue plasminogen activator (tPA) are liberated from the endothelium in a coupled manner (25), we hypothesized that the antithrombotic activity of thiol compounds may be nitric oxide/prostacyclin dependent. To verify this, we blocked nitric oxide synthase or cyclooxygenase using L-NAME or indomethacin, respectively, in the animals administered long-term epicaptopril. This procedure not only abolished the antithrombotic activity of epicaptopril in our model, but also resulted in the complete reversal of the improvement of fibrinolytic potential, as well as in the cessation of the suppression of the extrinsic pathway of blood coagulation produced by epicaptopril alone. Therefore, one plausible explanation is that the antithrombotic activity of epicaptopril results from the nitric oxide/prostacyclin-dependent release of tPA from the endothelium. Nevertheless, these data, together with our previous study (8), clearly demonstrate that thiol compounds exert antithrombotic activity by the nitric oxide/prostacyclin-dependent mechanism.
Because both ACE inhibitors (26) and thiol-containing agents (27) are believed to inhibit platelet activity, the question arises whether this action could contribute to their beneficial effect against venous thrombosis. We addressed this issue by analysis of the interaction between the vessels and thrombocytes, finding no changes in the bleeding time among studied groups of animals. It must be noted that bleeding time is a rough in vivo method, and it cannot detect mild inhibition of platelet aggregation or adhesion. This result, however, is in concordance with our previous results, which failed to demonstrate any influence of captopril on platelet aggregation at the time of thrombus formation in the same experimental conditions (8).
In summary, we have demonstrated that captopril, epicaptopril, and N-acetylcysteine, unlike enalapril, exerted an antithrombotic effect in a venous thrombosis model in normotensive rats. This effect was accompanied by suppression of the extrinsic pathway of the coagulation cascade and/or an increase in fibrinolysis and was nitric oxide/prostacyclin dependent. Thus, we demonstrated not only the ability of thiol group to potentiate the antithrombotic effect of captopril, but also that thiol compounds can exert antithrombotic action regardless of their ACE-inhibitory properties.
Acknowledgment: This work was supported by grant No. 4 PO5A 085 10 from the State Committee for Scientific Research, Poland. We are most grateful to Bristol-Myers Squibb (Poland and U.S.A.) for the supply of epicaptopril. We thank Aneta Buslowska for excellent technical assistance.
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