LOSARTAN METABOLITE EXP3174 REDUCES THE WEIGHT OF FORMED THROMBUS IN 2K1C HYPERTENSIVE RATS

Losartan (LOS), angiotensin II (Ang II) receptor antagonist (AT1A), is widely used in the treatment of hypertension and heart failure. There is also evidence that LOS may influence hemostasis. LOS administration, long-term and acute, has been associated with significant improvement in plasma fibrinolytic parameters in patients with hypertension and heart failure (1). The antiadhesive and anti-aggregatory action of LOS as well as its beneficial effect on endothelial function and coagulation has also been reported (2, 3). The antithrombotic activity of LOS was confirmed in a preventive model (LOS administered before thrombosis induction) of venous thrombosis in renovascular hypertensive rats, in a thrombotic challenge in mice as well as in the mouse arterial thrombosis in NO/PGI2- and platelet-dependent mechanism (4-6).

LOS is metabolized by cytochrome P450 enzymes, first to the aldehyde intermediate (EXP3179) and then to an active 5-carboxylic acid derivative EXP3174, which has a higher affinity to AT1 and a longer half-life than either LOS or EXP3179 (7, 8). Thus, LOS hypotensive activity is predominantly mediated by EXP3174 (8). LOS is mainly metabolized by cytochrome P450 enzymes CYP3A4 and CYP2C9, and therefore, modulation of these enzymes activities may cause significant changes in the pharmacokinetic profiles of losartan and its active metabolite EXP3174 (9-11). It was shown that the allelic variant CYP2C9*30 is associated to diminished response to the antihypertensive effect of losartan (12). Moreover, the interaction with herbs (e.g. Ginkgo biloba, Rhodiola rosea) and other drugs (e.g. ketoconazole) may affect losartan metabolism leading to significant inter-individual differences among individuals in response to LOS treatment concerning its hypotensive efficacy and toxicity (13, 14).

Although, the hypotensive effect of EXP3174 is well documented, its antithrombotic effect is not fully elucidated. It has been found that EXP3174 inhibited human platelet aggregation induced in vitro, although EXP3174 is less potent than LOS in reducing thromboxane A2-dependent platelet activation (15). It was also shown that EXP3174 in in vitro studies (30-min exposition) and in an ex vivo study (given 1 hour before blood sampling) caused inhibition of rat platelet adhesion to collagen and platelet aggregation stimulated by the thromboxane A2 analog U46619 (3). These effects were closely correlated with NO release from rat platelets and human umbilical endothelial cells. We have also found that EXP3174 inhibited platelet aggregation in vivo, reducing mice mortality, and this effect was abolished when animals were pretreated with L-NAME, which suggests an NO-involved mechanism (5). The involvement of NO in the mechanism of EXP3174 action was also shown in bovine aortic endothelial, cells, since EXP3174-induced and AT1-independent activation of endothelial NO synthase was observed (16). Therefore, it should be taken under consideration that EXP3174 might possess an individual pharmacological potency dependent or not on AT1, which may contribute to the beneficial effects of LOS on hemostasis during antihypertensive therapy.

Bearing in mind the extensive influence of LOS on thrombosis, we hypothesized that EXP3174 may potentially affect the process of thrombus resolution. Thus, we evaluated the ability of EXP3174 to reduce a stabilized venous thrombus in in vivo conditions. Changes in plasma hemostatic parameters and blood platelet reactivity were also evaluated. Since there is an imbalance in the hemostatic system in hypertension, leading to hypercoagulable state that may participate in clinical events such as arterial thrombosis and venous thromboembolism, our study was performed on renovascular hypertensive rats. This model mimics the pathological conditions of renin-angiotensin-aldosterone system (RAAS) activation and is particularly useful when studying the various effects of RAAS blockers.

MATERIALS AND METHODS

Animals and hypertension induction

All the procedures were approved by the Local Ethical Committee on Animal Testing at the Medical University of Bialystok. The animals’ health status was observed during the experiments by a health surveillance program according to the Guidelines for the Care and Use of Animals in Biomedical Research.

Male Wistar rats (150 – 170 g) were housed in a room with a 12-h light/dark cycle and were given tap water and fed standard rat chow. Rats were anesthetized with pentobarbital (40 mg/kg, intraperitoneally). Two-kidney one-clip (2K-1C) renovascular hypertension was induced by a standardized clipping of the left renal artery as previously described (17). After 6 weeks, hypertension was confirmed by systolic blood pressure (SBP) measurement using the “tail cuff” method (Non-Invasive Blood Pressure System, Harvard, Germany). To minimize the stress during procedure of SBP measurement, five days prior to the experiment, rats were acclimatized to tail cuff procedure. Rats were placed in a restraint holder and set on top of heating platform to maintain body temperature to 37ºC, the cuff was placed on rat tail, rats were allowed to calm down for at least 5 minutes then sample readings were taken. During the right experiment with SBP measurements, the same procedures were done. SBP measurements were performed when animals were calm (confirmed by a suitable pulse wave observed in a pulse channel). A few sample readings were taken, then at least 3 consecutive valid readings were performed, so each value of BP was the average of three readings.

Venous thrombosis model

Venous thrombosis was performed as previously described (18). Rats were anaesthetized with pentobarbital (40 mg/kg, intraperitoneally). The abdomen was opened, and the vena cava was ligated tightly with cotton thread below the left renal vein. Subsequently, the abdomen was closed with a double layer of sutures. Stasis-induced thrombi were allowed to age for 7 hours before injection of the drugs. After 8 hours, the abdomen was reopened, the vena cava was dissected, then thrombus was taken and weighed after 24 hours. Thrombus reduction was evaluated by the difference in thrombus weight between control and drug-treated rats.

Drug administration

EXP3174 (dissolved in phosphate buffered saline, PBS) was administered into the tail vein 7 hours after venous thrombosis induction at doses of 10 and 30 mg/kg. At the same time, control rats received PBS as vehiculum (VEH). For a positive control, heparin (HEP) was used at a dose of 50 IU/kg (19).

Bleeding time

Bleeding time (BT) was measured 8 hours after venous thrombosis induction. The standardized device was applied longitudinally on the tail. BT was measured from the moment the tail was surgically cut until the bleeding stopped completely (no rebleeding within 30 s) (4).

Platelet aggregation in whole blood

After BT measurement, at the end of in vivo experiment, blood (5 mL) was drawn from the right ventricle of the heart of the anesthetized rats and collected into tri-sodium citrate (3.15% w/v) in a ratio 9:1. Platelet aggregation was measured by the impedance method using an aggregometer (Chrono-Log, Whole blood lumi-aggregometer, USA) in whole blood (0.5 mL) (19). Platelet aggregation was evaluated by measuring the maximal extension of the aggregation curve at 6 min after the addition of collagen (5 µg/ml) and was expressed in ohms [Ω].

Overall plasma potentials

Two fibrin time curves, changing during fibrin generation and clot formation, were made by the registration of optical density (OD) via microplate reader (Dynex Tech., USA) as previously described (19). To determine overall hemostasis potential (OHP), CaCl2, thrombin, and t-PA in a Tris buffer were mixed with plasma samples (120 µL) that were obtained from blood centrifuged for 20 min at 2000 × g at 4ºC. To determine overall coagulation potential (OCP), a fibrin time curve was created without adding t-PA. The results are presented as values of OD, which were recorded every minute from time zero until 15 min. Based on the principle of integrals, the area under the curve, illustrating OHP and OCP, was expressed by summation of the OD values. Time to fibrin generation (TFG) was measured from the start of OD registration up to a noticeable increase and corresponded to the speed of the clotting initiation.

Euglobulin clot lysis time

Platelet poor plasma (PPP) was prepared by centrifugation (2000 × g, 10 min) at 4ºC from the blood containing tri-sodium citrate (3.15% w/v), immediately after blood samples collection. All the PPP was kept in –80ºC until use. The plasma euglobulin fraction was prepared by 20 times dilution of citrated plasma (0.5 mL) and acidification at pH 5.2. After being left for 1 hour at 4ºC, followed by centrifugation (2000 × g, 10 min), the precipitate was dissolved by 0.5 mL of 0.1 M tris HCl buffer pH 7.4. 50 µL of thrombin (100 IU/mL) was added to 300 µL of the plasma euglobulin fraction to induce clot formation. The fraction was incubated at 37ºC and the time measured from full clot to full lysis expressed the fibrinolytic activity of plasma (4).

Amidolytic activity of thrombin

To determine the thrombin activity, the chromogen substrate S-2238 with p-nitroaniline attached to the C-terminal amino acid was added in excess to the plasma samples with a Tris-HCL buffer. The enzyme activity was measured as the amount of p-nitroaniline released, which was determined spectrophotometrically at 405 nm.

Chemicals and drugs

EXP3174 (DuPont Merck Pharmaceutical, USA), pentobarbital (Biowet, Poland), calcium chloride, PBS, trisodium citrate (Polish Chemical Reagents, Poland), chromogene substrate S-2238 (Chromogenix-Instrumentation Laboratory SpA, Italy), collagen (Chronolog, USA), heparin (Polfa, Poland), recombinant tissue-type plasminogen activator (Boehringer Ingelheim, Poland), thrombin (Biomed, Poland), Tris buffer (Tris(hydroxymethyl)-aminomethane hydrochloride and Tris(hydroxymethyl)-aminomethane (Sigma, Poland) were used in the study.

Statistical analysis

Statistical analysis was performed using the Mann-Whitney test. Multiple group comparisons were performed by Kruskal-Wallis nonparametric ANOVA, followed by Dunn’s multiple comparisons test. The P < 0.05 was considered significant. The OHP and OCP were calculated as an area under the curve with TP4.1 pharmacometric software (ThothProTM, Poland).

RESULTS

The effect of EXP3174 on a formed thrombus

There were no significant differences between a 7- and 8-hour old thrombus in the control, VEH-treated rats (5.15 ± 1.05 mg versus 7.04 ± 1.02 mg). Administration of EXP3174 resulted in a reduction of the thrombus mass. EXP3174 significantly reduced the weight of formed thrombi to 2.40 ± 0.78 mg and 1.59 ± 0.74 mg for doses 10 and 30 mg/kg, respectively, P < 0.01 (Fig. 1). HEP, administered as a positive control, significantly reduced the weight of formed thrombi to 0.38 ± 0.08 mg, P < 0.001 (Fig. 1).

Fig. 1. Thrombus weight in control- (VEH), EXP3174 10 mg- and EXP3174 30 mg-treated rats. Heparin (HEP) was used as a positive control (18). **P < 0.01, ***P < 0.001 versus VEH; n = 8 – 10.

The effect of EXP3174 on systolic blood pressure and hemostatic parameters

Both doses of EXP3174 produced significant decreases in SBP from 162 ± 7 mmHg to 143 ± 5 mmHg and 131 ± 3 mmHg for 10 and 30 mg/kg, respectively, P < 0.05. However, no changes in BT after administration of EXP3174 were observed. Collagen-induced aggregation decreased significantly after EXP3174 administration only at a dose of 30 mg/kg, P < 0.05. Both doses of EXP3174 caused a significant reduction in ECLT (P < 0.05). Only the higher dose of EXP3174 significantly decreased thrombin amidolytic activity (P < 0.01). Decreases in OCP and OHP were observed after both doses of EXP3174, while significant prolongation of TFG was observed only at a higher dose (P < 0.05) (Table 1).

Table 1. The influence of EXP3174 on systolic blood pressure and hemostatic parameters.

*P < 0.05; **P < 0.01 versus VEH; n = 8 – 10.
Abbreviations: BT, bleeding time; ECLT, euglobulin clot lysis time; OCP, overall coagulation potential; OHP, overall hemostatic potential; SBP, systolic blood pressure; TFG, time to fibrin generation.

DISCUSSION

Thrombus resolution as a multifaceted process depends on the availability of plasminogen activators, platelet-derived profibrinolytic and antifibrynolitic factors, as well as thrombin generation (20). Thus, we attempted to check which elements of hemostasis mediate the EXP3174-induced process of thrombus resolution. Using a therapeutic model of thrombosis, we found that EXP3174 dose-dependently decreased the weight of the formed venous thrombi. The changes in thrombus weight were associated with an enhancement of plasma fibrinolytic potential, a marked reduction of platelet aggregation, and inhibition of the coagulation process.

EXP3174 was used here at hypotensive dose of 10 mg/kg, which was shown to block completely AT1 receptors in rats within 1 hour and inhibit the pressor response to Ang II (8). Inhibition of the Ang II pressor response correlated with the log of the steady state plasma EXP3174 concentration in a sigmoidal fashion with an IC50 of about 200 ng/mL. The similar plasma EXP3174 concentration was observed clinically after single oral administration of 50 mg of losartan to human healthy subjects (21, 22). To evaluate the dose-response relationship in hemostasis we used also the higher dose of 30 mg/kg.

We observed a marked increase in plasma fibrinolytic activity after EXP3174 treatment, expressed as a shortening of ECLT, which is closely related to both t-PA and PAI-1 levels (23). Since most of plasma t-PA amounts are complexed with PAI-1 and do not express the activity, the amounts of free PAI-1 could also be a regulator for euglobulin clot lysis (23). We assume that the profibrinolytic effect of EXP3174 may result from increased t-PA release from endothelium as well as reduced PAI-1 release from platelets. Moreover, the involvement of endothelial NO in the effect of EXP3174 on fibrinolysis could be considered. It was shown that EXP3174 independently of AT1-mediated signaling stimulates eNOS phosphorylation and suppresses tumor necrosis factor alpha-induced apoptosis by activating VEGFR2/PI3K/Akt pathway in bovine aortic endothelial cells (16). It has been shown that elimination of Ang II action causes immediate thrombolysis (30 minute after intravenous administration of angiotensin converting enzyme inhibitor) in rats, which was blunted and delayed by pharmacological suppression of NO synthesis with L-NAME (24). What is more, it was demonstrated that during the AT1 blockade, the activation of AT2 in rat aorta occurred with increased level of cGMP leading to increase in NO production (25).

We think that anticoagulant action may also be involved in the thrombolytic effect of EXP3174. We found both decreased OHP and OCP as well as a prolonged time to fibrin generation. We have previously shown that Ang II infusion increased fibrin generation and reduced TFG in 2K1C rats (19, 26). It was shown that Ang II activates thrombin generation in the mouse model of thrombosis (27), while LOS reduced expression of tissue factor in the endothelial cells of rat aorta via AT1 activation (2). Thus, the mechanism of anticoagulant action of EXP3174 may be related to the tissue factor/thrombin-depended pathway, since a reduction of thrombin activity was observed. Moreover, observed here EXP3174-prolonged TFG may be a result of reduced thrombin generation as well as may indicate on increased thrombus susceptibility to lysis. It was shown that thrombin reduction increased sensitivity of clot for lysis by mechanism dependent of TAFI inhibition (28).

Activated platelets provide a surface for the subsequent steps of the coagulation cascade leading to fibrin formation but also to secretion of PAI-1 which binds to the fibrin clot and inhibits fibrin-specific t-PA activity resulting in the prolongation of the clot lysis time (20). We observed here that EXP3174 administration resulted in reduction of platelet aggregation. It has already been reported that EXP3174 impairs collagen-induced platelet aggregation and adhesion, which was attributed to NO release within 30 min of EXP3174 administration (3, 29). EXP3174 had more than 70% greater potency in NO release in platelets than in endothelial cells, thus platelet-released NO may be a key mediator in antiplatelet action of EXP3174 (3). Interestingly, EXP3174 interacts also with the thromboxane A2/prostaglandin H2 receptor (9). Therefore, the effect of EXP3174 seems to be compound and the involvement of AT1-independent pathways should be taken into consideration. Although, in our study we did not evaluate gender interaction with the anti-aggregatory response to EXP3174, it is well known that one important factor affecting response to treatment with anti-aggregation drugs is gender (30).

We assume that the thrombolytic and anticoagulative effect of EXP3174 could be related also to its antiplatelet activity, which in turn reduces procoagulant and antifibrinolytic activity. Although, we did not evaluate the effect of EXP3174 on thrombus structure and its lysis, the effect of EXP3174 on platelets and fibrinolysis, may suggest the increased thrombus susceptibility to lysis. It is well known that the altered thrombus structure with more dense and more crosslinked clots is more stable mechanically and more resistant to fibrinolysis (31, 32). It was proposed that platelets can delay fibrinolysis and affect lysability of venous and arterial thrombi. The mechanism was related to the inhibition of t-PA by platelet-secreted PAI-1, that was shown to be a major factor responsible for the lytic resistance of a platelet-rich thrombus (33, 34). Thus, we assume that the reduced thrombus weight observed after EXP3174 administration may result also from reduced thrombus resistance to lysis.

Despite the marked inhibition of aggregation, EXP3174 did not influence the bleeding time. This parameter, although commonly used to assess the platelet/vessel wall interaction, can be influenced by numerous factors, including blood pressure. We assume that vasorelaxation and blood pressure lowering after EXP3174 balanced the decreased platelet aggregation without affecting bleeding time. However, the thrombolytic effect of EXP3174 could be related to the improvement of endothelial cell function. It is well known that drugs affecting platelet function improve endothelial vasodilatory function, increase eNOS level, reduce endothelial apoptosis as well as retain the endothelial cells’ viability (35).

Our previous study showed that Ang II infusion augmented the venous and arterial thrombus growth in a 2K1C hypertensive rats, confirming the pivotal role of AT1 in the thrombosis process (19, 26). The increased expression of AT1 receptors in the renal vasculature was observed in a 2K1C rat model of renovascular hypertension as well as in spontaneously hypertensive rats (36, 37). This upregulation of AT1 may mediate the hyperactivity to Ang II observed in hypertension. It was shown that LOS exerted stronger and structure-dependent suppression of AT1 receptor expression in hypertensive compared to normotensive rats (38, 39). Bearing in mind the above, we assume that antithrombotic effect of EXP3174 observed in our study may be related to a hypersensitivity state with upregulation of AT1 in renovascular hypertensive rats.

In summary, EXP3174, an active metabolite of LOS, administered in hypotensive doses, reduces thrombus weight in 2K1C rats. The thrombus weight reduction was a fibrinolytic, antiplatelet, and anticoagulative phenomenon.

Our present study documents novel and important properties of EXP3174, extending its action to those thrombolytic mechanisms which could be involved in the reduction of cardiovascular events during LOS treatment. Moreover, the unexpected changes in LOS metabolism related to cytochrome P450 enzyme polymorphisms as well as interactions with other drugs or herbs may significantly change the clinical effect of this drug, therefore treatment with its active metabolite - EXP3174 seems to be more reasonable, since the clinical effects of EXP3174 are not affected by metabolism-related disorders.

Acknowledgments: This work was supported by the project N/ST/ZB/18/003/2226 of the Medical University of Bialystok.

Conflict of interests: None declared.

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