Elevated angiotensin II induces platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner during sepsis

Abstract

Thrombocytopenia is independently related with increased mortality in severe septic patients. Renin-angiotensin system (RAS) is elevated in septic subjects; accumulating studies show that angiotensin II (Ang II) stimulate the intrinsic apoptosis pathway by promoting reactive oxygen species (ROS) production. However, the mechanisms underlying the relationship of platelet apoptosis and RAS system in sepsis have not been fully elucidated. The present study aimed to elucidate whether the RAS was involved in the pathogenesis of sepsis-associated thrombocytopenia and explore the underlying mechanisms. We found that elevated plasma Ang II was associated with decreased platelet count in both patients with sepsis and experimental animals exposed to lipopolysaccharide (LPS). Besides, Ang II treatment induced platelet apoptosis in a concentration-dependent manner in primary isolated platelets, which was blocked by angiotensin II type 1 receptor (AT1R) antagonist losartan, but not by angiotensin II type 2 receptor (AT2R) antagonist PD123319. Moreover, inhibiting AT1R by losartan attenuated LPS-induced platelet apoptosis and alleviated sepsis-associated thrombocytopenia. Furthermore, Ang II treatment induced oxidative stress level in a concentration-dependent manner in primary isolated platelets, which was partially reversed by the AT1R antagonist losartan. The present study demonstrated that elevated Ang II directly stimulated platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner in sepsis-associated thrombocytopenia. The results would helpful for understanding the role of RAS system in sepsis-associated thrombocytopenia.

Keywords: angiotensin II (Ang II); angiotensin II receptor blocker (ARB); apoptosis; platelet; reactive oxygen species (ROS); thrombocytopenia.

FIGURE 1

Plasma Ang II correlates inversely with platelet count in patients with sepsis. Platelet count (A), plasma renin activity (B) and plasma Ang II (C) levels in septic patients (n = 42) and healthy volunteers (n = 11). (D) Correlation analysis between plasma Ang II levels and platelet count. *P <0.05, **P <0.01

FIGURE 2

Elevated plasma Ang II is associated with platelet apoptosis and thrombocytopenia in LPS‐induced endotoxemia mice. Plasma renin activity (A), plasma Ang II levels (B), platelet caspase‐3 activity (C), expressions of Bak, Bax, Bcl‐2 and Bcl‐XL in platelets (D) and platelet count (E) (n = 7). *P < 0.05, **P < 0.01. kDa, kiloDalton

FIGURE 3

Ang II treatment leads to platelet apoptosis in a concentration‐dependent manner in primary isolated platelets. Primary isolated mouse platelets were incubated with Ang II for 24 h. Caspase‐3 activity (A), expressions of Bak, Bax, Bcl‐2 and Bcl‐XL (B‐D) were determined in platelets (n = 4). *P < 0.05, **P <0.01. kDa, kiloDalton

FIGURE 4

Ang II treatment induces oxidative stress level in a concentration‐dependent manner in primary isolated platelets. Primary isolated mouse platelets were incubated with Ang II for 24 h. ROS production (A&B), levels of MDA, H₂O₂ and GPx activity (C) were determined in platelets (n = 4). Scale bars = 50 μm. **P < 0.01. kDa, kiloDalton

FIGURE 5

The effect of ROS scavenger NAC on platelet apoptosis and thrombocytopenia in LPS‐induced endotoxemia mice. Twenty‐four hours after NAC (100 mg/kg) treatment, caspase‐3 activity (A), expressions of Bak, Bax, Bcl‐2 and Bcl‐XL (B) in platelets, and platelet count (C) were determined (n = 7). **P < 0.01. kDa, kiloDalton

FIGURE 6

The effect of ROS scavenger NAC on Ang II‐induced oxidative stress and platelet apoptosis in primary isolated platelets. Primary isolated mouse platelets were incubated with 100 nmol l⁻¹ Ang II and/or 5 mmol l⁻¹ NAC for 24 h. ROS production (A&B), levels of MDA, H₂O₂ and GPx activity (C), caspase‐3 activity (D), and expressions of Bak, Bax, Bcl‐2 and Bcl‐XL (E) were determined in platelets (n = 4). Scale bars = 50 μm. **P < 0.01. kDa, kiloDalton

FIGURE 7

Ang II treatment leads to platelet apoptosis through promoting oxidative stress in primary isolated platelets in an AT1R‐dependent manner. Protein levels of AT1R and AT2R (A) in platelet obtained from control and LPS‐treated mice were determined. Primary isolated mouse platelets were incubated with 100 nmol l⁻¹ Ang II, 10 μmol l⁻¹ losartan, 10 μmol l⁻¹ PD123319 for 24 h. Caspase‐3 activity (B), expressions of Bak, Bax, Bcl‐2 and Bcl‐XL (C), ROS production (D&E), levels of MDA, H₂O₂ and GPx activity (F) were determined in platelets (n = 4). Scale bars = 50 μm. **P < 0.01. kDa, kiloDalton

FIGURE 8

The effect of AT1R antagonist losartan on platelet oxidative stress, apoptosis and thrombocytopenia in LPS‐induced endotoxemia mice. Levels of MDA, H₂O₂ and GPx activity were determined in platelets (A) at the indicated time‐points (n = 7). AT1R antagonist losartan was administered at the indicated doses. 24 h later, ROS production (B), levels of MDA, H₂O₂ and GPx activity (C), caspase‐3 activity (D), expressions of Bak, Bax, Bcl‐2 and Bcl‐XL (E) in platelets, and platelet count (F) were determined (n = 7). Scale bars = 50 μm. *P < 0.05, **P < 0.01. kDa, kiloDalton

FIGURE 9

Losartan treatment significantly improves survival in LPS‐induced endotoxemia mice in concentration‐dependent manner. Survival rates of mice treated with saline (LPS group, n = 26) versus mice receiving losartan (10, 20 and 30 mg/kg, respectively) before LPS (LPS + Losartan group, n = 26) were compared. *P < 0.05, **P < 0.01