Figure 4
Figure 4. The influence of Mas receptor on thrombosis risk in Klkb1−/− mice. (A) The contact activation system (CAS), kallikrein/kinin system (KKS), and renin-angiotensin system (RAS) interact with each other. ACE, angiotensin-converting enzyme; Ang-(1-7), angiotensin-(1-7); AngI, angiotensin I; AngII, angiotensin II; AT1R,angiotensin receptor 1; AT2R,angiotensin receptor 2; B2R, bradykinin B2 receptor; BK, bradykinin; BK-(1-5), the ACE breakdown product of BK (peptide RPPGF); FXII, factor XII; FXIIa, activated XII; HK, high-molecular-weight kininogen; Mas, the receptor Mas; PK, prekallikrein. The CAS is initiated by XII autoactivation on a surface to convert PK to plasma kallikrein. These 2 proteases reciprocally activate each other and amplify their activation. Plasma kallikrein cleaves HK to liberate BK in the KKS. BK binds to its receptor, B2R, to activate cells or be internalized and degraded. It is also degraded to BK-(1-5) by ACE. ACE interacts with the RAS because it converts AngI to AngII to bind its receptors, AT1R or AT2R. AngII also is degraded to Ang-(1-7) to bind to its receptor Mas. (B) Plasma bradykinin (BK) levels in Klkb1+/+ and Klkb1−/− mice (n = 6 in each genotype). (C) Renal mRNA of B2R in Klkb1+/+ (n = 6) and Klkb1−/− mice (n = 5). (D) Renal mRNA of Mas in Klkb1+/+ (n = 6) and Klkb1−/− mice (n = 5). (E) Immunoblots for renal Mas showing a representative gel from multiple individual studies of kidney lysates from 8 Klkb1+/+ and 7 Klkb1−/− mice. β-tubulin served as the loading control. (F) Ratio of Mas to β-tubulin as quantified by densitometry using ImageJ software. (G) Plasma angiotensin-(1-7) levels in Klkb1+/+ and Klkb1−/− mice (n = 9 in each group). (H) The influence of Mas antagonist A-779 on thrombosis time in Klkb1−/− mice. Klkb1+/+ and Klkb1−/− were treated with the Mas antagonist A-779 (n = 5 and n = 6, respectively) or phosphate-buffered saline (n = 9 and n = 5, respectively) using osmotic pumps as previously reported.18 In additional experiments, WT mice (n = 7) were treated with the Mas agonist AVE0991 added to the drinking water at 0.1 μM. Carotid artery occlusion times were determined using the rose bengal thrombosis assay after 7 days of treatment. Data are presented as mean ± SEM for all experiments. *Significant difference (P < .05) between the 2 groups on Student t test or 1-way ANOVA test when all groups were compared.

The influence of Mas receptor on thrombosis risk in Klkb1−/− mice. (A) The contact activation system (CAS), kallikrein/kinin system (KKS), and renin-angiotensin system (RAS) interact with each other. ACE, angiotensin-converting enzyme; Ang-(1-7), angiotensin-(1-7); AngI, angiotensin I; AngII, angiotensin II; AT1R,angiotensin receptor 1; AT2R,angiotensin receptor 2; B2R, bradykinin B2 receptor; BK, bradykinin; BK-(1-5), the ACE breakdown product of BK (peptide RPPGF); FXII, factor XII; FXIIa, activated XII; HK, high-molecular-weight kininogen; Mas, the receptor Mas; PK, prekallikrein. The CAS is initiated by XII autoactivation on a surface to convert PK to plasma kallikrein. These 2 proteases reciprocally activate each other and amplify their activation. Plasma kallikrein cleaves HK to liberate BK in the KKS. BK binds to its receptor, B2R, to activate cells or be internalized and degraded. It is also degraded to BK-(1-5) by ACE. ACE interacts with the RAS because it converts AngI to AngII to bind its receptors, AT1R or AT2R. AngII also is degraded to Ang-(1-7) to bind to its receptor Mas. (B) Plasma bradykinin (BK) levels in Klkb1+/+ and Klkb1−/− mice (n = 6 in each genotype). (C) Renal mRNA of B2R in Klkb1+/+ (n = 6) and Klkb1−/− mice (n = 5). (D) Renal mRNA of Mas in Klkb1+/+ (n = 6) and Klkb1−/− mice (n = 5). (E) Immunoblots for renal Mas showing a representative gel from multiple individual studies of kidney lysates from 8 Klkb1+/+ and 7 Klkb1−/− mice. β-tubulin served as the loading control. (F) Ratio of Mas to β-tubulin as quantified by densitometry using ImageJ software. (G) Plasma angiotensin-(1-7) levels in Klkb1+/+ and Klkb1−/− mice (n = 9 in each group). (H) The influence of Mas antagonist A-779 on thrombosis time in Klkb1−/− mice. Klkb1+/+ and Klkb1−/− were treated with the Mas antagonist A-779 (n = 5 and n = 6, respectively) or phosphate-buffered saline (n = 9 and n = 5, respectively) using osmotic pumps as previously reported.18  In additional experiments, WT mice (n = 7) were treated with the Mas agonist AVE0991 added to the drinking water at 0.1 μM. Carotid artery occlusion times were determined using the rose bengal thrombosis assay after 7 days of treatment. Data are presented as mean ± SEM for all experiments. *Significant difference (P < .05) between the 2 groups on Student t test or 1-way ANOVA test when all groups were compared.

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