Antiplatelet properties of a novel, non-NO-based soluble guanylate cyclase activator, BAY 41-2272
Introduction
Nitric oxide (NO) is an important regulator of numerous, diverse physiological processes in the cardiovascular, gastrointestinal, respiratory, nervous and immune systems Moncada et al., 1991, Hobbs et al., 1999. Many of the biological actions of NO are mediated via activation of the intracellular receptor, soluble guanylate cyclase (sGC; Hobbs, 1997, Hobbs and Ignarro, 1996), which catalyses the formation of the second messenger cyclic 3′,5′-guanosine monophosphate (cGMP). In the cardiovascular system, this NO–sGC–cGMP axis is fundamental to the regulation of blood pressure (Vallance et al., 1989) and exerts a critical antithrombotic/cytoprotective influence on the blood vessel wall. This is mediated primarily via inhibition of the adherence of both platelets Radomski et al., 1987a, Radomski et al., 1987b and leukocytes Kubes et al., 1991, Niu et al., 1996 to the vascular endothelium. Previous studies have demonstrated that activation of sGC within platelets potently inhibits aggregation in vitro Radomski et al., 1987a, Moro et al., 1996 and in vivo Bhardwaj et al., 1988, Radomski et al., 1992 and synergises in this regard with a second endothelial-derived factor, prostacyclin (PGI2; Radomski et al., 1987b). Moreover, NO/cGMP is important in the regulation of leukocyte rolling, adhesion and extravasation Kubes et al., 1991, Niu et al., 1996. Platelets have often been used as a simple, efficient cell-based assay for evaluating the potency and biological activity of drugs stimulating the sGC–cGMP transduction pathway. Such compounds may be useful therapeutics for the treatment of several cardiovascular disorders involving impaired endogenous NO production.
YC-1, a novel non-NO-based sGC activator, was identified recently and shown to relax vascular smooth muscle in vitro, cause a fall in systemic blood pressure in vivo and inhibit platelet aggregation Mulsch et al., 1997, Wu et al., 1995. Moreover, the actions of YC-1 were haem dependent and synergistic with NO Stone and Marletta, 1998, Friebe and Koesling, 1998. More recently, a significantly more potent family of non-NO-based sGC activators, devoid of inhibitory PDE activity, have been reported Stasch et al., 2001, Stasch et al., 2002a, Stasch et al., 2002b, Becker et al., 2001, Straub et al., 2001. These compounds are potent vasodilators in vitro and in vivo and exhibit antiplatelet activity. Such agents represent useful pharmacological tools (particularly to differentiate between cGMP-dependent and -independent effects of NO) and have significant therapeutic potential (e.g., in the treatment of angina and/or heart failure).
In the present study we have undertaken a thorough and systematic study of the in vitro and in vivo antiplatelet effects of BAY 41-2272 to evaluate the role of cGMP-dependent inhibition of platelet aggregation and the potential therapeutic benefits of this family of sGC activators.
Section snippets
Platelet-rich plasma and washed platelet suspensions
Human blood was collected from individuals who had not taken NSAIDs for the previous 14 days and PRP and prostacyclin (PGI2)-washed platelet suspensions (WP) were prepared as described previously (Radomski and Moncada, 1983).
Platelet aggregation was measured in a whole-blood ionised calcium lumi-aggregometer (Chronolog, Havertown, PA). Compounds (BAY 41-2272, 0.01–100 μM; GSNO, 0.01–10 μM; PGI2, 3 ng/ml) were incubated with PRP or WP (2.5×108 platelets/ml) for 1 min before addition of ADP (1–4
Platelet-rich plasma
BAY 41-2272 (10–100 μM) produced a concentration-dependent inhibition of ADP (1–4 μM)-induced aggregation Fig. 1, Fig. 2. PGI2 (3 ng/ml) and GSNO (10 μM) inhibited ADP (4 μM)-induced aggregation by approximately 25% (Fig. 3). However, in combination with BAY 41-2272 (10 μM), PGI2 and GSNO elicited a synergistic inhibition of platelet reactivity such that aggregation was inhibited by 70% and 85%, respectively (Fig. 3).
Washed platelets
BAY 41-2272 (10 nM–1 μM) produced a concentration-dependent inhibition of
Discussion
NO is an important regulator of the reactivity of circulating cells and endothelial-derived NO maintains a constant antithrombotic influence in the vasculature to prevent platelet (and leukocyte) activation and adherence Radomski et al., 1987a, Radomski et al., 1987b, Kubes et al., 1991. The effects of NO in modulating platelet reactivity are thought to be mediated via the activation of sGC, formation of cGMP and subsequent activation of a cGMP-dependent protein kinase (G-kinase). In turn, this
Acknowledgements
A.J.H. is the recipient of a Wellcome Trust Career Development Fellowship. The authors thank Dr. Eduardo Salas for his extensive contribution to the work described in this manuscript.
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