Bradykinin B2 Receptor KO Mice Are Protected From Thrombosis by A Platelet Spreading Defect

Bradykinin B2 receptor KO mice (B2R KO) have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO mice, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and PGI2 (Blood 108:192, 2006). The combined presence of elevated AngII and AT2R was essential to have the thrombosis protection phenotype. However, thrombosis remained delayed in B2R KO mice despite reduction in AngII levels from 258+64 to 40+15 pg/ml using losartan, an angiotension 1 receptor antagonist. These data suggested an additional pathway for thrombosis protection in B2R KO mice.

B2R KO mice have elevated bradykinin and bradykinin 1–5 levels with normal thrombin generation times. Losartan treatment lowered ACE mRNA in mice. B2R KO mice also had elevated Mas mRNA, the receptor for angiotensin1-7, with overexpressed renal AT2R and Mas antigens. Treatment of B2R KO with A-779, a Mas antagonist, shortened thrombosis time (58+4 to 38+4 min) and tail bleeding time (170+13 to 88+8 sec) and lowered plasma concentrations of nitrate (21.5+3.6 to 15+5 micromolar) and 6-keto-PGF1alpha (259+103 to 132+58 pg/ml). Immunofluorescent staining and immunoblot demonstrate AT2R and Mas antigen on murine platelets. “Threshold” ADP- and thrombin-induced platelet aggregation was identical for B2R KO and wild type platelets. Platelet activation by arachidonic acid, ionomycin, thrombin, or collagen as measured by flow cytometry (the activated α_2bβ₃ integrin complex and P-selectin) was similar between B2R KO and wild type platelets. Although static and flow adherence to collagen was normal, B2R KO platelets had defects in spreading on collagen (20+0.6 versus 27+0.4 microns WT, p<0.0001) under both static and flow conditions. B2R KO platelets had increased DAF-FM fluorescence (649+41 versus 405+36 AFU, p=0.0012), suggesting increased platelet NO. Increasing concentrations of GSNO, a NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibited mouse platelet spreading in a concentration-dependent manner. B2R KO and wild type platelets did not show a difference in cGMP [631+143 (Mean+SD) vs 594+80 fmol/108 plts] or cAMP [505+227 vs 568+151 fmol/108 plts] levels. However, B2R KO platelets had increased constitutive and GSNO-induced total protein nitrosylation on biotin-switch assay. N-acetylcysteine, a protein nitrosylation inhibitor and antioxidant, corrected the spreading defect in B2R KO platelets.

B2R KO mice are protected from thrombosis by over-expression of two receptors from the renin-angiotensin system that results in increased NO and prostacyclin and an acquired platelet spreading defect. These data suggest that a platelet spreading defect alone protects from thrombosis and indicate that the receptors AT2R and MAS compensate the absence of the B2R. Last, these studies show a novel mechanism(s) by which receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo.

No relevant conflicts of interest to declare.