Abstract-This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT 1 )-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT 1 -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 AT 1 -receptor antagonists on platelet adhesion to collagen and thromboxane A 2 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 mol/L, which was attenuated by NO synthase inhibitor N G -nitro-L-arginine methyl ester. The angiotensin II AT 1 -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 AT 1 -receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N G -nitro-L-arginine methyl ester. Neither the AT 2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A 2 /prostaglandin H 2 receptor antagonist SQ29,548 had any effect on angiotensin II AT 1 -receptor antagonist-stimulated NO release in platelets and endothelial cells. Key Words: platelets Ⅲ nitric oxide Ⅲ endothelium Ⅲ angiotensin II Ⅲ angiotensin antagonist P latelets play an important role in arterial thrombosis and the onset of acute myocardial infarction after atherosclerotic plaque rupture. Inhibition of platelet aggregation has become a critical step in preventing thrombotic events that are associated with stroke, heart attack, and peripheral arterial thrombosis. 1 Thrombosis is a multicellular event in which other cells, such as endothelial cells, are involved in the regulation of platelet reactivity. During the past several years, clear evidence has emerged that a concerted action of nitric oxide (NO) generated by either endothelial or platelet NO synthases regulates platelet activation, causing inhibition of adhesion and aggregation. 2,3 Recently developed nonpeptide angiotensin II (Ang II) AT 1 -receptor antagonists (AT 1 -As) make up a new generation of antihypertensive agents that also modulate hemostasis, 4,5 and apparently this effect is not solely a result of Ang II-receptor blockage. Ang II induces an early phase of platelet activation 6 and increases secretion of plasminogen activator inhibitor type I from vascular endothelial cells, 7 whereas AT 1 -As inhibit the vasoconstrictor and platelet aggregation effects induced by tromboxane A 2 (T...

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.

To cite this article: Szemraj J, Walkowiak B, Kawecka I, Janiszewska G, Buczko W, Bartkowiak J, Chabielska E. A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity -a staphylokinase variant. I. In vitro study. J Thromb Haemost 2005; 3: 2156-65.Summary. We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent -hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity.The plasminogen activation rate (determined by K M and K cat ) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125 I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.Keywords: antiplatelet activity, hirudin and K2 domain of t-PA, recombinant protein, staphylokinase, thrombolysis, thrombolytic and antithrombotic agents.

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 (H 2 O 2 ) 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 H 2 O 2 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.