Biased agonism of protease-activated receptor-1 regulates thromboinflammation in murine sickle cell disease

Abstract

Sickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein-coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.

Graphical abstract

Figure 1.

Inhibition of endogenous APC by SPC-54 exacerbates the thromboinflammation at steady state in HbSS mice. (A) Four-month-old HbAA (gray) and HbSS (blue) mice were treated with 10 mg/kg (i.p.) IgG (solid bars), or SPC54 (white hashed bars), and samples were collected 24 hours later. Plasma levels of TAT (B), IL-6 (C), IL-18 (D), sVCAM (E), sICAM (F), VWF (G), sP-sel (H), and PF4 (I). Data are represented by mean ± standard error of the mean (SEM) of 5 to 6 mice per group and analyzed by 2-way analysis of variance (ANOVA) and Tukey post hoc test. Asterisks directly above the bars indicate statistical significance of SS mice to AA mice in the same treatment group. Asterisks over brackets indicate difference from IgG-treated mice. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. AA, controls; i.p., intraperitoneal; SS, sickle mice.

Figure 2.

APC inhibition exacerbates hepatic congestion and lung neutrophil accumulation. (A) Representative images of liver sections from HbSS mice treated with IgG or SPC-54 stained with H&E (top) and Prussian Blue (bottom). Black arrow indicates sinusoidal congestion, and green arrow indicates iron-laden macrophages. Quantification of liver congestion (B) and liver necrosis (C) by 3 blinded observers. (D) Plasma levels of alanine aminotransferase (ALT). (E) Quantification of Prussian blue stain per total area. (F) Representative images of lung sections stained for neutrophils (brown). Scale bar, 50 μm; red asterisk (∗) denotes neutrophils. (G) Quantification of neutrophils (PMN) averaged over 10 high powered (40×) fields. Data are represented by mean ± SEM of 5 to 6 mice per group and analyzed by 2-way ANOVA and Tukey post hoc test. Asterisks directly above the bars indicate the statistical significance of SS mice to AA mice in the same treatment group. Asterisks over brackets indicate difference from IgG-treated mice. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. AA, controls; PMN, polymorphonuclear lymphocytes; SS, sickle mice.

Figure 3.

Inhibition of endogenous APC by SPC54 exacerbates thromboinflammation in HbSS mice after TNF-α challenge. (A) Male and female HbSS mice were treated with IgG or SPC-54 (10 mg/kg, IP) 19 hours before SAL or TNF-α (2 μg/kg, IP) and plasma was collected 5 hours later. Plasma levels of TAT (B), IL-6 (C), IL-18 (D), sVCAM1 (E), sICAM (F), VWF (G), and sP-sel (H). Data represent mean ± SEM mean of 5 to 10 mice per group. Asterisks above brackets indicate statistical significance by 1-way ANOVA with Kruskal-Wallis posttest. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. Sal, saline.

Figure 4.

Effect of PAR1 biased agonism on biomarkers of coagulation and inflammation. SS bone marrow was transplanted into lethally irradiated WT (gray), R41Q (red), and R46Q (blue) mice. Four months later, mice were treated with SAL (steady state, solid bars) or TNF-α (2 mg/kg, IP) (white hashed bars), and plasma was collected after 5 hours (A). Plasma levels of TAT (B), IL-6 (C), HMGB1 (D), IL-18 (E), sVCAM-1 (F), sICAM (G), VWF (H), sP-sel (I), and PF4 (J). Data represent mean ± SEM for 6 to 8 mice (SAL, steady state) and 15 to 17 mice per group (TNF-α challenge). Asterisks above bar represent statistical significance vs SAL-treated mice of same genotype by 2-way ANOVA and Tukey post hoc test. Asterisks above brackets indicate comparison. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. SAL, saline.

Figure 5.

Hepatic congestion is enhanced in SS^BM/R46Q mice. (A) Livers were collected and stained with H&E; representative images of livers from SS^BM/WT, SS^BM/R41Q, and SS^BM/R46Q mice during steady state (SAL) and TNF-α challenge; scale bar represents 50 μm. Three blinded observers scored congestion (B) and necrosis (C). Data represent mean ± SEM. Black arrow denotes sinusoidal congestion, and green arrow denotes iron-laden macrophages. There were 6 to 8 mice (SAL, steady state) and 15 to 17 mice (TNF-α challenge) per group. Asterisks above bar represent statistical significance vs SAL-treated mice of same genotype by 2-way ANOVA and Tukey post hoc test. Asterisks above brackets indicate comparison. ∗P < .05. SAL, saline.