Over-Expression of the Mas Receptor Decreases Arterial Thrombosis Risk in B2R KO Mice by Elevating NO and Prostacyclin and Reducing GPVI Activation

Bradykinin B2 receptor KO mice (B2R KO) were recognized to have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and prostacyclin (Blood 108:192, 2006). It was proposed that the combined presence of elevated AngII and AT2R are essential for the thrombosis protection phenotype. However, after losartan treatment, an angiotensin receptor 1 antagonist, thrombosis remains delayed in B2R KO despite reduction in AngII levels from 258±64 to 40±15 pg/ml. An additional pathway for thrombosis protection in B2R KO mice was sought.

Losartan treatment lowers ACE mRNA in mice along with lowering AngII levels. However, even in the presence of low AngII levels, plasma Ang1-7 levels, the breakdown product of AngII, in losartan-treated B2R KO are increased [21±2.2 pg/ml vs 14.4±0.7 pg/ml in wild type mice (WT)]. Upon examination, B2R KO also have elevated Mas mRNA, the receptor for angiotensin1-7, with over-expressed renal Mas protein. Treatment of B2R KO with A-779, a Mas antagonist, shortens carotid artery thrombosis time (58±4 to 38±4 min) and tail bleeding time (170±13 to 88±8 sec) and lowers plasma concentrations of nitrate (from 21.5±3.6 micromolar in untreated B2R KO to 15±5 micromolar in A-779-treated mice) and 6-keto-PGF1alpha (259±103 pg/ml in untreated B2R KO to 132±58 pg/ml). ADP- or thrombin-induced platelet (plt) activation are not abnormal in B2R KO, either by aggregation studies or by flow cytometric studies examining for fibrinogen binding or JON/A integrin epitope binding and P-Selectin expression, respectively. Although static and flow adherence to collagen was normal, B2R KO plts have defective spreading on collagen (20±0.6 microns in B2R KO versus 27±0.4 microns in WT, p<0.0001) under static conditions. Resting B2R KO plts constitutively also have increased DAF-FM fluorescence (649±41 AFU versus 405±36 AFU, p=0.0012), suggesting increased plt NO without stimulation. GSNO, an NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibits murine plt spreading on collagen in a concentration-dependent manner. B2R KO plts have reduced GPVI activation using CRP or convulxin even though GPVI expression on B2R KO and WT plts are similar. Further, B2R KO plts have reduced Syk phosphorylation after convulxin stimulation and reduced phosphorylation of Src family kinases when spreading on collagen. Treatment of B2R KO plts in vivo with A-779 corrects the spreading and Src family kinase phosphorylation defects upon collagen exposure, suggesting an in vivo role of the Mas receptor in the plt function defect and thrombosis delay.

B2R KO are protected from thrombosis by over-expression of two receptors (AT2R and Mas) from the renin-angiotensin system resulting in increased NO and prostacyclin and an acquired platelet function defect. The receptors AT2R and MAS compensate for the absence of the B2R. These studies show novel mechanism(s) whereby receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo.

No relevant conflicts of interest to declare.