Angiotensin Receptor 2 Over-Expression: A Novel Physiologic Mechanism for Thrombosis Protection.
Authors: Shariat-Madar Ziaeddin , Warnock Mark , Mahdi Fakhri , Srikanth Sujata , Krijanovski Yelena , Schmaier Alvin H. 
Published: October 2019

Abstract
Recent in vitro evidence from our laboratory and others suggests that the plasma kallikrein/kinin system (KKS) is a physiologic counterbalance to the renin angiotensin system (RAS). We sought animal models to examine this hypothesis. Bradykinin (BK) is a potent physiologic stimulator for tPA liberation and NO and prostacyclin formation. We hypothesized that the bradykinin B2 receptor knockout mouse (BKB2R−/−) would be prothrombotic. The Rose Bengal model for mouse carotid artery thrombosis was utilized to characterize the thrombosis risk of BKB2R−/− mice. Contrary to our hypothesis, BKB2R−/− mice had delayed time to thrombosis on this model [78±7 min versus 31±3 min Control (CN)]. When control mice were treated with increasing concentrations (0.9–9.1 mg/kg IP) of a BKB2R antagonist, HOE140, the thrombosis time in CN mice also delayed from 25±1 to 73±5 min. Investigations sought the mechanism(s) for thrombosis protection in BKB2R−/− mice. Plasma levels for coagulation factors II, VII, IX, X, XII, HK, fibrinogen, antithrombin, plasminogen, and tPA are not significantly different from controls. The BKB2R−/− mice have elevated plasma PAI-1 (10.7±1 ng/ml versus 7.6±1 ng/ml CN, p<0.04) and prekallikrein (0.29±0.03 U/ml versus 0.23±0.01 U/ml CN, p<0.02) levels, but reduced plasma FXI levels (0.23±0.03 U/ml versus 0.39±0.01 U/ml CN, p<0.02). Other mechanisms for thromboprotection were sought. Recent in vitro evidence suggests that the BKB2R cross talks with angiotensin II (AngII) receptors. We postulated that in the absence of the BKB2R, there might be increased AngII that could stimulate one of its receptors. Plasma AngII is elevated in BKB2R−/− mice (64±6 pg/ml versus 44±6 pg/ml CN, p<0.023). Accordingly, plasma NO (61±5 micromolar in BKB2R−/− mice versus 24±2 micromolar in CN mice, p<0.001) and prostacyclin (30±11 ng/ml in BKB2R−/− mice versus 8±2.6 ng/ml in CN mice, p<0.032) are elevated. The importance of NO for thrombosis protection of these animals is shown by further experiments that indicate that treatment of the BKB2R−/− mice with LNAME (2 mg/ml drinking water for 1 week) shortens the time to thrombosis in these animals from 78±7 min to 33±2 min, p<0.0001. Further, treatment of BKB2R−/− mice with nimesulide, a COX2 inhibitor at 50 microgram/ml in drinking water for 1 week, also reduces the time to thrombosis from 85±2.3 min in untreated mice to 49±2.9 min in treated mice (p<0.0003). NO and prostacyclin elevation in the BKB2R−/− mice can only occur if the angiotensin receptor 2 (ATR2) or the angiotensin1–7 receptor is over-expressed. On real time PCR, there is increased ATR2 mRNA in kidney, heart, and liver in the BKB2R−/− mice. Alternatively, mRNA of the bradykinin B1 receptor, angiotensin receptor 1, and the angiotensin1–7 receptor are not over-expressed. On quantitative immunoblot, more ATR2 antigen is present in kidney of BKB2R−/− mice than CN. Confirmation that the ATR2 is responsible for thromboprotection in the BKB2R−/− mice was obtained by treating these mice with an antagonist to this receptor, PD123319. After 1 week of osmotic pump infusion of PD123319 at 50 microgram/kg/day, the time to thrombosis in the BKB2R−/− mice changed from 76±3 min to 30±5 min, p<0.001. These data indicate that the BKB2R of the KKS and ATR2 of the RAS cross talk in vivo and that over-expression of the ATR2 receptor influences thrombosis risk. These data suggest that changes in the RAS influence thrombosis risk. This investigation links blood pressure control with thrombosis risk.