Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity

Abstract

The anticoagulant activated protein C (APC) protects neurons and endothelium via protease activated receptor (PAR)1, PAR3 and endothelial protein C receptor. APC is neuroprotective in stroke models. Bleeding complications may limit the pharmacologic utility of APC. Here, we compared the 3K3A-APC mutant with 80% reduced anticoagulant activity and wild-type (wt)-APC. Murine 3K3A-APC compared with wt-APC protected mouse cortical neurons from N-methyl-D-aspartate-induced apoptosis with twofold greater efficacy and more potently reduced N-methyl-D-aspartate excitotoxic lesions in vivo. Human 3K3A-APC protected human brain endothelial cells (BECs) from oxygen/glucose deprivation with 1.7-fold greater efficacy than wt-APC. 3K3A-APC neuronal protection required PAR1 and PAR3, as shown by using PAR-specific blocking antibodies and PAR1- and PAR3-deficient cells and mice. BEC protection required endothelial protein C receptor and PAR1. In neurons and BECs, 3K3A-APC blocked caspase-9 and -3 activation and induction of p53, and decreased the Bax/Bcl-2 pro-apoptotic ratio. After distal middle cerebral artery occlusion (dMCAO) in mice, murine 3K3A-APC compared with vehicle given 4:00 h after dMCAO improved the functional outcome and reduced the infarction volume by 50% within 3 days. 3K3A-APC compared with wt-APC multi-dosing therapy at 12:00 h, 1, 3, 5 and 7 days after dMCAO significantly improved functional recovery and reduced the infarction volume by 75% and 38%, respectively, within 7 days. The wt-APC, but not 3K3A-APC, significantly increased the risk of intracerebral bleeding as indicated by a 50% increase in hemoglobin levels in the ischemic hemisphere. Thus, 3K3A-APC offers a new approach for safer and more efficacious treatments of neurodegenerative disorders and stroke with APC.

FIG. 1

Murine recombinant 3K3A-APC blocks NMDA-induced apoptosis in mouse cortical neurons. (a) Immunostaining for terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) and Hoechst at 24:00 h after NMDA in the absence or presence of murine recombinant 3K3A-APC (5 nm). (b) Dose-dependent neuroprotective effects of murine 3K3A-APC (green) and wt-APC (yellow) at 24:00 h of NMDA (*P < 0.01 by two-way ANOVA). Cell survival was quantified with a WST-8 assay. NMDA label at zero concentration APC on the abscissa shows cell survival of neurons treated with NMDA only without 3K3A-APC or wt-APC. Vehicle denotes the basal cell survival rate of neurons in the culture medium in the absence of NMDA. S360A-APC indicates cell survival of neurons treated with NMDA in the presence of enzymatically inactive APC (negative control, brown). (c) IC⁵⁰ (inhibiting concentration) values for 3K3A-APC vs. wt-APC were calculated from experiments shown in (b). All values are mean ± SEM.

FIG. 2

Effects of murine recombinant 3K3A-APC on activation of caspases-9 and -3, p53 levels and Bax and Bcl-2 expression in NMDA-treated mouse neurons. Caspase-9 (a) and caspase-3 (b) activities in neurons treated with NMDA in the absence or presence of caspase-9 inhibitor (10 µm; z-LEDH-fmk) and/or caspase-3 inhibitor (50 µm; Ac-DEVD-CHO), murine 3K3A-APC, wt-APC and enzymatically inactive S360A-APC at 5 nm. (c) Western blots for p53 in nuclear extracts, Bax and Bcl-2 in whole-cell extracts from NMDA-treated cells in the absence or presence of murine 3K3A-APC (5 nm). Histone H1 was used as a control for loading of nuclear proteins and β-actin as a control for loading of the whole-cell lysate proteins. (d) Intensity of p53, Bax and Bcl-2 signals measured by scanning densitometry and effects of 3K3A-APC in experiments as in (c). Signal for nuclear p53 was normalized by histone H1 abundance, and signal for Bax and Bcl-2 with β-actin. All values are mean + SEM. NS, non-significant.

FIG. 3

The neuroprotection of 3K3A-APC in NMDA-treated neurons is mediated by PAR1 and PAR3. (a) Cortical neurons treated with NMDA and incubated with recombinant murine 3K3A-APC (5 nm and various cleavage-site-blocking antibodies (20 µg/mL) that specifically block the actions of PAR1 (H-111), PAR2 (SAM-11), PAR3 (H-103) and PAR4 (S-20). N-terminal-blocking PAR1 (S-19) and PAR2 (S-19), and C-terminal-blocking PAR3 (M-20) and PAR4 (M-20) antibodies (20 µg/mL) were used as negative controls. Cell survival was quantified with WST-8 assay as in Fig. 1b. (b) Cortical neurons from PAR1^−/− and PAR3^−/− mice were treated with NMDA and incubated with and without 3K3A-APC (5 nm). Cell survival was quantified with a WST-8 assay. All values are mean + SEM. NS, non-significant.

FIG. 4

Murine 3K3A-APC protects against NMDA-induced lesions in vivo via PAR1 and PAR3. (a) Coronal sections of mouse brains at 48:00 h after infusions with NMDA with and without murine 3K3A-APC (0.2 µg). (b) Effects of murine 3K3A-APC (0.2 µg) and wt-APC (0.2 µg) on NMDA lesion volumes at 48:00 h. The white and black bars show the lesion volumes in control C57BL6 mice anesthetized with ketamine and xylazine or isoflurane, respectively. Values are mean + SEM, n = 5 mice per group. (c) NMDA-induced lesions in PAR1^−/−, PAR2^−/− and PAR3^−/− mice on C57BL6 background infused with vehicle or murine 3K3A-APC (0.2 µg) and in C57BL6 control mice infused with cleavage-site-blocking PAR2 antibody (SAM-11), PAR4 antibody (S-20) and vehicle or 3K3A-APC (0.2 µg). All values are mean + SEM. NS, non-significant.

FIG. 5

Human recombinant 3K3A-APC exerts antiapoptotic activity in hypoxic human BECs. (a) Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) and Hoechst staining of BECs subjected to OGD for 8:00 h in the absence or presence of human 3K3A-APC (10 nm). (b) Dose-dependent cytoprotective effects of human 3K3A-APC and wt-APC on OGD BECs were quantified with an LDH release assay after 8:00 h of exposure to OGD (*P < 0.01 by two-way ANOVA). The basal rate of BEC death under normoxic conditions is also shown. (c) IC₅₀ values of 3K3A-APC and wt-APC were calculated from Fig. 1b. All values are mean ± SEM.

FIG. 6

Effects of human 3K3A-APC on activation of caspases-9 and -3, p53 levels and Bax and Bcl-2 expression and requirements for EPCR and PAR1 in hypoxic human BECs. (a) Western blots for p53 in nuclear extracts, and Bax and Bcl-2 in whole-cell extracts from BECs treated with OGD for 3:00 h and incubated with and without human 3K3A-APC (10 nm). Histone H1 was used as a control for loading of nuclear p53 protein and β-actin as a control for loading of the whole-cell lysate proteins. (b) Intensity of p53, Bax and Bcl-2 signals measured by scanning densitometry and effects of human 3K3A-APC in experiments as in (a). Signal for nuclear p53 was normalized by histone H1 abundance, and signal for Bax and Bcl-2 with β-actin. Caspase-9 (c) and caspase-3 (d) activities in OGD BECs with and without caspase-9 inhibitor (10 µm; z-LEDH-fmk) and/or caspase-3 inhibitor (50 µm; Ac-DEVD-CHO) and human 3K3A-APC (10 nm). (e) BECs were treated with OGD for 8:00 h and incubated with human 3K3A-APC (10 nm) and various cleavage-site-blocking PAR1 (H-111), PAR2 (SAM-11), PAR3 (H-103) and PAR4 (S-20) antibodies (20 µg/mL) and antibodies against EPCR that either block (RCR252) or do not block (RCR92) APC binding. N-terminal-blocking PAR1 (S-19) and PAR2 (S-19) antibodies and C-terminal-blocking PAR3 (M-20) and PAR4 (M-20) antibodies (20 µg/mL) were used as negative controls. BEC injury was quantified with LDH assay as in Fig. 5b. All values are mean + SEM.

FIG. 7

Effects of murine recombinant 3K3A-APC on functional recovery and infarct volume after permanent dMCAO. Vehicle or murine 3K3A-APC (0.2 mg/kg) was administered via the tail vein at 4:00 h after permanent dMCAO. Functional tests and infarct volume were determined within 3 days (D) of dMCAO. (a) Foot-fault test. (b) Forelimb asymmetry. (c) Infarct volume. All values are mean + SEM. ^aP < 0.01, for 3K3A-APC compared with vehicle by repeated-measures ANOVA.

FIG. 8

Effects of murine recombinant 3K3A-APC and wt-APC on functional recovery and infarct volume after permanent dMCAO. Vehicle, murine wt-APC (0.2 mg/kg) or murine 3K3A-APC (0.2 mg/kg) was administered via the tail vein at multiple times at 12:00 h, 1, 3, 5 and 7 days after permanent dMCAO. Functional tests and infarct volume were determined within 7 days (D) of dMCAO. (a) Foot-fault test. (b) Forelimb asymmetry test. (c) Cresyl violet staining of brain coronal sections of ischemic mice treated with vehicle or 3K3A-APC for 7 days after dMCAO. (d) Infarct volume. (e) Hemoglobin levels in the ischemic hemisphere of mice treated with vehicle, wt-APC or 3K3A-APC for 7 days after dMCAO. All values are mean + SEM. ^aP < 0.01, for 3K3A-APC vs. wt-APC; ^bP < 0.01, for 3K3A-APC vs. vehicle by repeated-measures ANOVA.