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.

Activated Protein C Analog With Reduced Anti-Coagulant Activity Improves Functional Recovery and Reduces Bleeding Risk Following Controlled Cortical Impact

“…Therefore, both neuronal-protective and vascular-protective effects are fully preserved in 3K3A-APC mutant. In fact, recent studies using a permanent distal middle cerebral occlusion model of stroke in mice have demonstrated that 3K3A-APC has superior neuroprotection and reduced risk for bleeding (Guo et al, 2009a;Wang et al, 2009). Although, it is difficult to determine the exact contributions of direct neuroprotection and vasculoprotection versus reduced risk for bleeding in the overall beneficial effects of 3K3A-APC therapy compared to wt-APC in stroke and TBI models in vivo, it has been reported that 3K3A-APC has superior cytoprotective effects compared to wt-APC in cultured neurons challenged by N-methy-Daspratate and brain endothelial cells subjected to oxygen/glucose deprivation (Guo et al, 2009a).…”

“…The mechanism(s) for the augmented cytoprotection of 3K3A-APC versus wt-APC remains to be elucidated. In addition to the altered amino acid composition in the loop 37, different post-translational modifications of the mutant protein can occur that could change contents of negatively charged sialic acid and/or the distribution and branching of the Nlinked glycan species causing an increased ability of mutant 3K3A-APC to interact with PAR-1 and/or other putative APC receptors (Wang et al, 2009;Guo et al, 2009a). Thus, enhanced neuroprotective and/or vasculoprotective direct activities may contribute to the observed protection in vivo after TBI in addition to elimination of the bleeding risk.…”

“…The recombinant activated PCs were prepared as described (Gale et al, 2002;Mosnier et al, 2004) with modifications (Guo et al, 2009a;Wang et al, 2009). The cells were maintained in suspension in CD OptiCHO medium (Invitrogen, Carlsbad, CA) containing 2 mM CaCl 2 , 10 μg/ml vitamin K and 2 mM GlutaMAX (Invitrogen).…”

“…This action is mediated by highly specific proteolytic degradation of factors Va and VIIIa, with contributions from various plasma cofactors (Mosnier et al, 2007). Independent of its anticoagulant pathway, APC exerts direct vasculoprotective and neuronal-protective activities that require proteolytic activation of protease-activated receptor-1 (PAR-1) and endothelial protein C receptor (EPCR) on endothelial cells (Cheng et al, 2003(Cheng et al, , 2006 and PAR-1 and PAR-3 on mouse cortical neurons (Guo et al, , 2009aZhong et al, 2009) as well as PAR-1 and EPCR on rat hippocampal neurons (Gorbacheva et al, 2009(Gorbacheva et al, , 2010. Cellular effects of APC include cytoprotective alterations in gene expression profiles, anti-apoptotic and antiinflammatory activities and stabilization of endothelial barriers (Joyce et al, 2001;Riewald et al, 2002;Cheng et al, 2003;Domotor et al, 2003;Mosnier and Griffin, 2003;Feistritzer and Riewald, 2005;Finigan et al, 2005;Cheng et al, 2006).…”

“…In contrast to wt-APC, late post-ischemic therapy with 3K3A-APC, an APC analog with greatly reduced anticoagulant activity (Gale et al, 2002), had superior beneficial effects and no risk for intracerebral bleeding after stroke in rodents (Guo et al, 2009a;Wang et al, 2009). 3K3A-APC contains 3 alanine substitutions for 3 protease domain residues which reduce factor Va binding and inactivation (Gale et al, 2002), but do not affect APC exosites recognizing PAR-1 and EPCR, resulting in normal cytoprotection (Mosnier et al, 2004).…”