Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C

Abstract

Activated protein C (APC) reduces mortality of severe sepsis patients but increases the risk of serious bleeding. APC exerts anticoagulant activity by proteolysis of factors Va/VIIIa. APC also exerts antiinflammatory and antiapoptotic effects and stabilizes endothelial barrier function by APC-initiated cell signaling that requires two receptors, endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR1). The relative importance of APC's various activities for efficacy in sepsis is unknown. We used protein engineering of mouse APC and genetically altered mice to clarify mechanisms for the efficacy of APC in mouse sepsis models. Mortality reduction in LPS-induced endotoxemia required the enzymatic active site of APC, EPCR, and PAR-1, highlighting a key role for APC's cytoprotective actions. A recombinant APC variant with normal signaling but <10% anticoagulant activity (5A-APC) was as effective as wild-type APC in reducing mortality after LPS challenge, and enhanced the survival of mice subjected to peritonitis induced by gram-positive or -negative bacteria or to polymicrobial peritoneal sepsis triggered by colon ascendens stent implantation. Thus, APC's efficacy in severe sepsis is predominantly based on EPCR- and PAR1-dependent cell signaling, and APC variants with normal cell signaling but reduced anticoagulant activities retain efficacy while reducing the risk of bleeding.

Figure 1.

APC reduces the mortality of LPS-induced endotoxemia. 7-d survival of mice (n = 20 per group) infused with 10 μg APC (○), 2 μg APC (Δ), PBS (•), or 10 μg S360A-APC (□; n = 10 per group) over a 20-min period before challenge with an LD₅₀ (A) or LD₉₀ (B) of LPS. The statistical significance of mortality and survival time was determined by the Kaplan-Meyer log-rank test.

Figure 2.

APC treatment reduces apoptosis and LPS-induced vascular permeability. Nuclear HOECHST stain (A–C) and TUNEL assay (D–F) on liver sections of control mice (no LPS; A and D), LPS-challenged mice (B and E), and LPS-challenged mice treated with APC (C and F). (G) Quantitative analysis of apoptosis in liver, lung, and spleen tissue. (H) APC treatment reduces caspase-3 activity. All analyses were performed 24 h after LPS challenge. *, P ≤ 0.05 using the Student's t test. (I) Vascular permeability was determined before (no LPS) or 8 h after LPS challenge by infusion of FITC-labeled high MW dextran and rhodamine-labeled low MW dextran. (J) Quantitation of rhodamine–dextran extravasation by morphometry. (K) Permeability barrier protection by APC occurs predominantly in smaller vessels. *, P ≤ 0.05 compared with LPS, as determined by the Student's t test. (L) MPO activity as a marker for neutrophil activity in lung tissue 24 h after LPS challenge. APC reduces lung inflammation. *, P ≤ 0.01 compared with PBS control lungs; and #, P ≤ 0.03 compared with LPS challenged lungs, as determined by the Student's t test. Data represent the mean ± SD of 10 randomly chosen fields per section, with five sections per mouse and five mice each in the treatment and control groups. Bars, 100 μm.

Figure 3.

APC effect on lymphocyte apoptosis. Representative example of apoptosis detection in lymphocyte subpopulations as detected by FITC-labeled caspase-3 antibody (A), or double staining with YO-PRO-1 and 7-AAD nuclear stains (B). Analysis was performed 24 h after administration of LPS, PBS, or LPS and APC. Equivalent experiments were used to generate the data summarized in Tables I and II. APC does not reduce lymphocyte apoptosis. Numbers indicate the percentage of positive cells.

Figure 4.

Endotoxemia mortality reduction by APC requires EPCR and PAR1. (A) Survival of PAR1^−/− mice (n = 17 per group) infused with 10 μg APC (○) or PBS (•), followed LPS-challenge. APC treatment of PAR1^−/− mice prolongs survival time (P < 0.05). (B) Survival of mice (n = 12 per group) with reduced EPCR function (EPCR^δ/δ) infused over 20 min with 10 μg APC (○) or PBS (•), followed by LPS challenge. The statistical significance of mortality and survival time was determined by the Kaplan-Meyer log-rank test.

Figure 5.

Anticoagulant and cytoprotective activities of mouse APC variants. (A) Anticoagulant activity of recombinant mouse WT APC (Δ) and APC variants (230/231-APC [◊], 3K3A-APC [▪], and 5A-APC [*]) determined in vitro in plasma. (B) Profibrinolytic activity of WT APC (Δ) and APC variants (230/231-APC [◊], 3K3A-APC [▪], 5A-APC [*], and S360A-APC [□]). (C) Antiapoptotic activity of WT APC (Δ) and APC variants (3K3A-APC [▪] and 5A-APC [*]) in assays of staurosporin-induced (0.25 μM, 24 h) mouse endothelial cell apoptosis. (D) Antiinflammatory activity of WT APC (Δ), WT protein C zymogen (□), and 5A-APC (*) induced in mouse Raw264.7 macrophage-like cells. Data represent the mean ± SD. (E and F) APC effect on in vivo thrombin generation in endotoxin-challenged mice. Endotoxin-challenged mice (n = 6 per group) were infused with PBS (▴), 10 (•) or 2 (○) μg WT APC, or APC variants, and thrombin generation was determined at 2 h (E) and at 30 min (F) after APC infusion by measuring plasma levels of TAT complex. Administration of 2 μg 5A-APC did not significantly alter in vivo thrombin generation.

Figure 6.

5A-APC reduces mortality of endotoxemia. (A) Survival of mice (n = 10 per group) that were infused with PBS (•, control), 10 μg 5A-APC (□), or 2 μg (▪) 5A-APC for 20 min, followed by a 40-mg/kg i.p. injection of LPS. (B) Survival of mice (n = 10 per group) that were given a 10-μg i.v. bolus of 5A-APC (○) or PBS (□) 3 h after receiving 40 mg/kg LPS via i.p. injection. 5A-APC reduces mortality. The statistical significance of mortality reduction by APC treatment was determined by the Kaplan-Meyer log-rank test.

Figure 7.

5A-APC reduces mortality in bacterial sepsis. (A) Survival of mice (n = 10 per group) given either a 10-μg (□) or 2-μg (▪) i.v. bolus of 5A-APC or PBS (•, control), followed immediately by i.p. infection with 10⁸ S. aureus. (B) Survival of mice (n = 20 per group) given a 2-μg i.v. bolus of either 5A-APC (▪) or PBS (•, control) 3 and 10 h after i.p. infection with 10⁸ S. aureus. (C) Survival of mice (n = 20 per group) given a 2-μg i.v. bolus of either 5A-APC (▪) or PBS (•, control) at 3 and 10 h after i.p. infection with 2.5 × 10⁸ E. coli (O55:B5). (D) Survival of mice (n = 22 per group) after CASP. Mice were given a 2-μg i.v. bolus of either 5A-APC (▪) or S360A-APC (•) at 3 and 10 h after CASP. The statistical significance of mortality reduction by APC treatment was determined by the Kaplan-Meyer log-rank test.