Abstract
The antithrombotic effect of angiotensin(Ang)-(1-7) has been reported, but the mechanism of this effect is not known. We investigated the participation of platelets and receptor Mas-related mechanisms in this action. We used Western blotting to test for the presence of Mas protein in rat platelets and used fluorescent-labeled FAM-Ang-(1-7) to determine the specific binding for Ang-(1-7) and its displacement by the receptor Mas antagonist A-779 in rat platelets and in Mas(-/ -) and Mas(+/+) mice platelets. To test whether Ang-(1-7) induces NO release from platelets, we used the NO indicator DAF-FM. In addition we examined the role of Mas in the Ang-(1-7) antithrombotic effect on induced thrombi in the vena cava of male Mas(-/ -) and Mas(+/+) mice. The functional relevance of Mas in hemostasis was evaluated by determining bleeding time in Mas(+/+) and Mas(-/ -) mice. We observed the presence of Mas protein in platelets, as indicated by Western Blot, and displacement of the binding of fluorescent Ang-(1-7) to rat platelets by A-779. Furthermore, in Mas(+/+) mouse platelets we found specific binding for Ang-(1-7), which was absent in Mas(-/ -) mouse platelets. Ang-(1-7) released NO from rat and Mas(+/+) mouse platelets, and A-779 blocked this effect. The NO release stimulated by Ang-(1-7) was abolished in Mas(-/ -) mouse platelets. Ang-(1-7) inhibited thrombus formation in Mas(+/+) mice. Strikingly, this effect was abolished in Mas(-) (/) (-)mice. Moreover, Mas deficiency resulted in a significant decrease in bleeding time (8.50 +/- 1.47 vs. 4.28 +/- 0.66 min). This study is the first to show the presence of Mas protein and specific binding for Ang-(1-7) in rat and mouse platelets. Our data also suggest that the Ang-(1-7) antithrombotic effect involves Mas-mediated NO release from platelets. More importantly, we showed that the antithrombotic effect of Ang-(1-7) in vivo is Mas dependent and that Mas is functionally important in hemostasis.
Highlights
Platelets play a critical role in hemostasis and thrombosis, and drugs inhibiting platelet aggregation are valuable for preventing thrombotic events (1)
Mas Protein is Present in Rat Platelets Western-blotting revealed a single band corresponding to the Mas protein in protein extracts prepared from isolated rat platelets
Ang-(1-7) Binding to Platelets After Western blotting demonstrated the presence of Mas in platelets, we tested the specific binding of Ang-(1-7) in these cells
Summary
Platelets play a critical role in hemostasis and thrombosis, and drugs inhibiting platelet aggregation are valuable for preventing thrombotic events (1). Platelets, which are activated by a wide variety of conditions, adhere to the injured blood vessel wall and subsequently aggregate (2). Those processes may result in the formation of occlusive thrombi in the lumen of the injured vessel. These thrombi are the source of thromboembolic complications of atherosclerosis, such as heart attacks, stroke, and peripheral vascular disease. An important platelet-modulating agent is nitric oxide (NO), which exerts antiplatelet actions and plays a crucial role in thrombotic events (4). The NO generated by the endothelium or platelets regulates platelet activation, causing inhibition of adhesion and aggregation (4)
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