Differential Neuroprotection and Risk for Bleeding From Activated Protein C With Varying Degrees of Anticoagulant Activity

Abstract

Background and Purpose-Activated protein C (APC), a protease with anticoagulant and cytoprotective activities, protects neurons and endothelium from ischemic injury. Drotrecogin-alfa activated, a hyperanticoagulant form of human recombinant APC, is currently being studied in patients with ischemic stroke. How changes in APC anticoagulant activity influence APC's neuroprotection and risk for bleeding is not clear. Methods-We used neuronal and brain endothelial cell injury models and middle cerebral artery occlusion in mice to compare efficacy and safety of drotrecogin-alfa activated and human 3K3A-APC, an APC nonanticoagulant mutant. Results-Drotrecogin-alfa activated and 3K3A-APC exhibited 148% and 10% of plasma-derived APC's anticoagulant activity and differ in the carbohydrate content. 3K3A-APC protected mouse neurons from N-methyl-D-aspartate-induced apoptosis and human brain endothelial cell from oxygen-glucose deprivation with 1.8-and 3.1-fold greater efficacy than drotrecogin-alfa activated. Given 5 minutes before transient middle cerebral artery occlusion, 3K3A-APC and drotrecogin-alfa activated (0.5 and 2 mg/kg intravenously) reduced comparably and dose-dependently the infarction lesion up to 85%. 3K3A-APC, but not drotrecogin-alfa activated, improved neurological score dose-dependently (PϽ0.05). 3K3A-APC did not cause bleeding. In contrast, drotrecogin-alfa activated dose-dependently increased hemoglobin content in postischemic brain. After permanent middle cerebral artery occlusion, 3K3A-APC multidose therapy (1 mg/kg intravenously at 12 hours and 1, 3, 5, and 7 days) improved functional recovery and reduced infarction by 60% with no risk for bleeding, whereas drotrecogin-alfa activated increased hemoglobin deposition in the postischemic brain and showed relatively modest neuroprotection. Conclusions-Nonanticoagulant

Activated Protein C Attenuates Systemic Lupus Erythematosus and Lupus Nephritis in MRL-Fas(lpr) Mice

“…In this study, we used Drotrecogin alfa activated (Xigris; Lilly Deutschland), a human recombinant activated protein C that was shown to be effective in mice. Xigris (Lilly Deutschland) had a neuroprotective effect in murine neuronal and brain endothelial cell injury models (25) and prevented septic-like reactions after injection of histones into mice (26). In sepsis, it was initially thought that activated protein C has antithrombotic and profibrinolytic functions, but recently, it has been postulated that cytoprotective effects are responsible for reduced mortality.…”

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.…”

“…Although our recent study demonstrated potential of APC as a therapy for TBI (Petraglia et al, 2010), the anticoagulant properties of wt-APC may carry a significant risk for intracerebral bleeding, as shown in patients with severe sepsis (Bernard et al, 2001) and in rodent models of stroke when APC was administered at later time points after ischemia (Wang et al, 2009;Guo et al, 2009a,b). 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).…”

“…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).…”