Ewa Chabielska

Angiotensin II AT1 Receptor Antagonists Inhibit Platelet Adhesion and Aggregation by Nitric Oxide Release

Abstract—This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist–stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 &mgr;mol/L, which was attenuated by NO synthase inhibitor NG-nitro-l-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with NG-nitro-l-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist–stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

Angiotensin II enhances thrombosis development in renovascular hypertensive rats

There is an increased number of in vitro evidence that angiotensin II (Ang II) may promote thrombosis. However there are no in vivo experiments exploring the effect of Ang II on thrombus formation. In the present study we have investigated the influence of Ang II on venous thrombosis in renovascular hypertensive rats. Furthermore, we examined the role of AT(1) receptor and Ang II metabolites: angiotensin III (Ang III) and angiotensin IV (Ang IV) in the mechanisms of Ang II action. The contribution of coagulation and fibrinolytic systems in the mode of Ang II action was also determined. Venous thrombosis was induced by ligation of vena cava. Ang II infused into rats developing venous thrombosis caused dose-dependent increase in thrombus weight, which was partially reversed by losartan, selective AT(1) antagonist. Ang III did not influence the thrombus formation in hypertensive rats, while Ang IV caused a marked increase in thrombus weight only in one of the used doses. Our study shows that Ang II via AT(1) receptor enhances thrombosis development. The prothrombotic effect of Ang II may partially depend on enhanced leukocytes adhesion to endothelial cells accompanied by accelerated fibrin formation and increased plasma level of PAI-1. Moreover, Ang II action is partially mediated by one of its metabolites - Ang IV.

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant - An in-vivo study

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

The antithrombotic effect of angiotensin-(1-7) closely resembles that of losartan.

Angiotensin-(1-7) [Ang-(1-7)] is the bioactive peptide which may be responsible for some of the pharmacological effects of losartan. Our previous study has demonstrated the antithrombotic action of losartan in a model of experimental thrombosis. In the present study, we compared the antithrombotic action of losartan and Ang-(1-7). Acute (10 mg/kg, p.o.) and chronic (10 mg/kg, p.o., three weeks) losartan administration to spontaneously hypertensive rats (SHR) induced a decrease in thrombus weight (1.6±0.6 mg and 1.2±0.3 mg respectively vs. control 2.9±0.8 mg; p<0.05, p<0.05). A similar reduction was observed in two-kidney, one-clip hypertensive rats (2K-1C) receiving acute losartan administration (1.39±0.29 mg vs. 3.25±0.62 mg; p<0.01). Infusion of Ang-(1-7) to 2K-1C rats also reduced the thrombus weight (1.01±0.34 mg, 1.23±0.38 mg and 2.17±0.36 mg for 1, 10, 100 pmol/kg/min, respectively vs. 3.58±0.6 mg control; p<0.01, p<0.01, p<0.05). Losartan produced a decrease in systolic blood pressure (BP) in SHR as well as in 2K-1C rats, while Ang-(1-7) infusion had no effect on BP. Acute losartan dosing to 2K-1C rats decreased platelet adhesion to fibrillar collagen (24.9±1.0% vs. control 31.5±1.1%, p<0.001). The incubation of platelet samples with Ang-(1-7) (10-6 and 10 —5 M) also reduced adhesion to fibrillar collagen (38.4±0.1% and 33.8±0.8% respectively vs. control 40.0±0.6%; p<0.05, p<0.001). There were no apparent changes in prothrombin time, activated partial thromboplastin time and euglobulin clot lysis time in losartan and Ang-(1-7)-treated groups. We conclude that, like losartan, Ang-(1-7) is able to act as an antithrombotic agent.

Proteasome inhibitor prevents experimental arterial thrombosis in renovascular hypertensive rats

Recent studies indicate that highly selective proteasome inhibitors can be useful in prevention of some cardiovascular events. Here we demonstrate that proteasome inhibitor, Z-Ile-Glu (Ot-Bu) Ala-Leucinal (PSI), is active in the prevention of platelet-dependent arterial thrombosis induced in renovascular hypertensive rats (two-kidney, one clip Goldblatt model, and 2K1C, n=5). The administration of PSI intravenously at a single dose of 0.3 mg/kg before induction of arterial thrombosis markedly increased carotid final flow rate, as compared to control (vehicle) group (10.36 +/- 1.8 ml/min and 1.2 +/- 1.2 ml/min, respectively), significantly decreased the wet (1.23 +/- 0.23 mg and 4.1 +/- 0.94 mg, respectively), and dry (0.46 +/- 0.145 mg and 1.46 +/- 0.39, respectively) thrombus weight, and completely prevented arterial occlusion. Moreover, platelets from PSI - treated thrombotic 2K1C rats, showed in response to collagen a significant inhibition of aggregation in the whole blood (10.26 +/- 0.6 ohms vs. 15.51 +/- 0.91 ohms in the control group). In contrast, collagen-induced platelet aggregation was not inhibited in vitro, after pre-treatment of the blood with PSI at the concentration of 10 microM that effectively inhibited the 20S proteasome activity in platelets, indicating that ex vivo anti-aggregatory effect of PSI proceeds through an indirect mechanism not associated with suppression of 20S proteasome activity in platelets. In conclusion, our in vivo findings demonstrate that proteasome inhibitor, Z-Ile-Glu(Ot-Bu)Ala-Leucinal, prevents the development of arterial thrombosis in renovascular hypertensive rats and effectively suppresses platelet aggregation by an indirect mechanism. Thus the data provide a new insight into the potential role for the proteasome-dependent pathway in cardiovascular events.

Study of the mechanisms of aldosterone prothrombotic effect in rats.

Introduction: We investigated the role of primary haemostasis, fibrinolysis, nitric oxide (NO) and oxidative stress as well as mineralocorticoid receptors (MR) in acute aldosterone prothrombotic action. Materials and methods: Venous thrombosis was induced by stasis in Wistar rats. Aldosterone (ALDO; 10, 30, 100 µg/kg/h) was infused for 1 h. Eplerenone (EPL; 100 mg/kg, p.o.), a selective MR antagonist, was administered before ALDO infusion. Bleeding time (BT) and platelet adhesion to collagen were evaluated. The expression of nitric oxide synthase (NOS), NADPH oxidase, superoxide dismutase (SOD) and plasminogen activator inhibitor (PAI-1) was measured. NO, malonyl dialdehyde (MDA) and hydrogen peroxide (H2O2) plasma levels were assayed. Results: Significant enhancement of venous thrombosis was observed after ALDO infusion. ALDO shortened BT and increased platelet adhesion. Marked increases were observed in PAI-1, NADPH oxidase and SOD mRNA levels. MDA and H2O2 levels were augmented in ALDO-treated groups, and NOS expression and NO level were decreased. EPL reduced ALDO effects on thrombus formation, primary haemostasis, PAI-1 expression and MDA level. Conclusion: Short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, fibrinolysis, NO and oxidative stress-dependent pathways. The MR antagonist only partially diminished the ALDO effects, suggesting the involvement of additional mechanisms.

A potent 5-hydroxytryptamine receptor (5-HT2A) antagonist, DV-7028, delays arterial thrombosis development in rats

In our study, we demonstrated that DV-7028: (3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6, 7,8,9-tetrahydro-2H-pyrido [1,2,-a]-1,3,5-triazine-2, 4(3H)-dione maleate)--a selective 5-HT2A receptor antagonist, inhibited thrombus formation in the arterial thrombosis model and was completely ineffective in the prevention of venous thrombosis in the rat. In washed platelets prelabelled with 3H-serotonin, DV-7028 inhibited, in a dose-dependent manner, the collagen-induced secretion of serotonin. However, the uptake of serotonin into platelets was not affected by this substance. Administration of DV-7028 also inhibited platelet aggregation in the whole blood and platelet-rich plasma (PRP) induced by collagen, and diminished serotonin-induced aggregation of rat platelets in the presence of a sensitizing but nonaggregating amount of ADP, whereas it did not modify aggregation in PRP when induced by ADP. DV-7028 caused a concentration-dependent, almost parallel shift to the right of the concentration-response to serotonin for its pressor effect in the rat perfused tail artery. The present data demonstrate that DV-7028 exhibits 5-HT2A receptor antagonistic properties in the rat cardiovascular system, exhibits antithrombotic effect in the model of arterial but not venous thrombosis in rats. These results constitute further evidence of the possible importance of serotonin as a mediator of platelet thrombosis in arteries. Moreover, they can provide a useful tool for the prevention of various thrombotic diseases.

Thiol repletion prevents venous thrombosis in rats by nitric oxide/prostacyclin-dependent mechanism: relation to the antithrombotic action of captopril.

Clinical and experimental data have recently accumulated for antithrombotic action of angiotensin-converting enzyme inhibitors (ACE-1s). We have shown previously that captopril (which contains a thiol group in the moiety) exerts more pronounced antithrombotic activity than does an equipotent dose of enalapril (the drug devoid of the thiol group). To clarify the relative importance of the presence of the thiol group in the molecule versus angiotensin-converting enzyme (ACE) inhibitory properties in the antithrombotic action of captopril, rats were treated with captopril (5 mg/kg twice daily; CAP), epicaptopril (stereoisomer of captopril devoid of ACE-inhibitory properties; 5 mg/kg twice daily; EPI), N-acetylcysteine (3.75 mg/kg twice daily; ACC), enalapril (3 mg/kg once daily; ENA), or distilled water (VEH) for 10 days, per os. After ligation of the vena cava, the incidence of the venous thrombosis and/or the thrombus weight decreased significantly in all but the ENA-treated groups when compared with control rats. The effect of CAP, EPI, and ACC was accompanied by a marked reduction of euglobulin clot lysis time and, with the exception of ACC, by an increase in prothrombin time in the blood collected from the site of the thrombus formation. Antithrombotic activity of EPI was completely abolished by nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or indomethacin, with the parallel reversal of fibrinolytic and coagulation parameters toward normal. Activated partial thromboplastin time, mean blood pressure, and bleeding time were not altered by either of the administered drugs. Thus, we demonstrated that thiol compounds exert antithrombotic activity by increasing fibrinolysis and/or suppression of the extrinsic pathway of the coagulation cascade in a nitric oxide/prostacyclin-dependent manner.

Losartan Inhibits Experimental Venous Thrombosis in Spontaneously Hypertensive Rats

The potential antithrombotic action of losartan, the AT1 receptor antagonist, in an experimental model of venous thrombosis in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) was tested. The involvement of nitric oxide and prostacyclin in this effect was also studied. Venous stasis was induced by ligation of the vena cava. Losartan, after administration of a single, hypotensive dose (10 mg/kg, p.o.), significantly reduced the thrombus weight in SHR but not in WKY. The antithrombotic activity of losartan in SHR was abolished by NG-nitro L-arginine methyl ester (L-NAME) (30 mg/kg s.c.) but not by indomethacin (2.5 mg/kg s.c.). No changes in primary hemostasis, platelet aggregation, coagulation parameters such as activated partial thromboplastin time, prothrombin time, euglobulin clot lysis time, and fibrinogen level, either in SHR or in WKY rats, were found. Our results indicate the NO-dependent mechanism in the antithrombotic effect of losartan on venous thrombosis in SHR.

Acute aldosterone infusion enhances thrombosis development in normotensive rats.

Thromb Haemost 2007; 98: 697–699 Dear Sir, It is commonly believed that the morning surge in blood pressure, along with haemostatic changes that promote thrombosis, contributes to cardiovascular events and death at this time of the day. Some studies have shown that aldosterone has circadian rhythmicity and that the major peak of aldosterone occurs during early morning hours (1). It has also been shown that acute systemic infusion of aldosterone in normotensive volunteers results in endothelial dysfunction (2). This observation suggests that even a short-lasting rise in aldosterone level may potentially promote the thrombotic process, by reducing the antithrombotic properties of the endothelium. Therefore, in the present study we wanted to elucidate whether acute infusion of aldosterone could affect experimental thrombosis in rats.The contribution of coagulation and fibrinolytic systems in the mode of aldosterone action was also determined. Furthermore, we investigated the role of the mineralocorticoid receptor in the mechanisms of aldosterone action. Male Wistar rats (300–350 g) were used in this study. All investigations were carried out at the same time of the day (09:00 a.m.) to minimize any effect of diurnal variation in the haemostatic system and vascular function. Aldosterone (ALDO) (Sigma-Aldrich, Poland; 3, 10 and 30 μg/kg/h) or VEH (0.4% ethanol; 2 ml/kg/h) was infused into the femoral vein 5 minutes (min) before the induction of venous thrombosis and was continued for 1 hour (h) and 5 min (Constant-Rate Infusion Pump, Kwapisz, Poland). Eplerenone (EP), a selective mineralocorticoid receptor antagonist (Pfizer Co., Poland), was administered (p.o., 5% aqueous gummi arabici solution) at a dose of 100 mg/ kg 30 min beforeALDO (30 μg/kg/h) infusion. Venous thrombosis was performed by ligation of the vena cava (3), following pentobarbital anesthesia (Vetbutal, Biowet, Poland, 45 mg/kg i.p.). After 1 h, blood samples were drawn from the right ventricle of the heart prior to thrombus removal. The blood was mixed with 3.13% sodium citrate (Sigma-Aldrich, Poland) in a volume ratio of 9:1 and centrifuged for 20 min at 3,500 x g, at 4oC, and then plasma was deep-frozen (-70oC). The venous thrombus was carefully collected from the vein and weighed. Serum aldosterone concentration was measured by the RIA procedure (DPC, Poland). Plasminogen activator inhibitor (PAI-I), tissue plasminogen activator (t-PA), thrombin activatable fibrinolysis inhibitor (TAFI) and tissue factor (TF) plasma levels Letters to the Editor