Endothelial protein C receptor-assisted transport of activated protein C across the mouse blood-brain barrier. Journal of Cerebral Blood Flow and Metabolism

Department of Neurosurgery, Center for Neurodegenerative and Vascular Brain Disorders, University of Rochester Medical Center, Rochester, New York 14642, USA.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism (Impact Factor: 5.34). 10/2008; 29(1):25-33. DOI: 10.1038/jcbfm.2008.117
Source: PubMed

ABSTRACT Activated protein C (APC), a serine-protease with anticoagulant, anti-inflammatory, and cytoprotective activities, is neuroprotective and holds potential to treat different neurologic disorders. It is unknown whether APC crosses the blood-brain barrier (BBB) to reach its therapeutic targets in the brain. By using a brain vascular perfusion technique, we show that (125)I-labeled plasma-derived mouse APC enters the brain from cerebrovascular circulation by a concentration-dependent mechanism. The permeability surface area product of (125)I-APC (0.1 nmol/L) in different forebrain regions ranged from 3.11 to 4.13 microL/min/g brain. This was approximately 80- to 110-fold greater than for (14)C-inulin, a simultaneously infused reference tracer. The K(m) value for APC BBB cortical transport was 1.6+/-0.2 nmol/L. Recombinant APC variants with reduced anticoagulant activity, 5A-APC and 3K3A-APC, but not protein C, exhibited high affinity for the APC BBB transport system. Blockade of APC-binding site on endothelial protein C receptor (EPCR), but not blockade of its protease-activated receptor-1 (PAR1) catalytic site, inhibited by >85% APC entry into the brain. APC brain uptake was reduced by 64% in severely deficient EPCR mice, but not in PAR1 null mice. These data suggest that APC and its variants with reduced anticoagulant activity cross the BBB via EPCR-mediated saturable transport.

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Available from: Francis J Castellino, Feb 13, 2014
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    • "In additional experiments, we applied another antibody against EPCR, RCR-252. This antibody was shown previously to effectively block APC binding to EPCR on mouse endothelium (Deane et al. 2009) and to inhibit the proliferative effect of APC in rat keratinocytes (our unpublished observation). RCR-252, similarly to P-20, completely blocked the action of APC on neurons at glutamate toxicity (Fig. 2). "
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    ABSTRACT: Activated protein C (APC) is an anticoagulant and anti-inflammatory factor that acts via endothelial protein C receptor (EPCR). Interestingly, APC also exhibits neuroprotective activities. In the present study, we demonstrate for the first time expression of EPCR, the receptor for APC, in rat cortical and hippocampal neurons. Moreover, exposing the neurons to glutamate excitotoxicity we studied the functional consequence of the expression of EPCR. By cytotoxicity assay we showed that EPCR was necessary for the APC-mediated protective effect in both neuronal cell types in culture. The effect of APC was abrogated in the presence of blocking EPCR antibodies. Analysis of neuronal death by cell labelling with dyes which allow distinguishing living and dead cells confirmed that the anti-apoptotic effect of APC was dependent on both EPCR and protease-activated receptor-1. Thus, we suggest that binding of APC to EPCR on neurons and subsequent activation of protease-activated receptor-1 by the complex of APC-EPCR promotes survival mechanisms after exposure of neurons to damaging factors.
    Journal of Neurochemistry 09/2009; 111(4):967-75. DOI:10.1111/j.1471-4159.2009.06380.x · 4.24 Impact Factor
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    • "Although the inflammatory reactions might be different after ischemia/reperfusion, based on previous work with wt-APC demonstrating its effectiveness in ischemia reperfusion models (Shibata et al., 2001; Cheng et al., 2003, 2006), one might expect that 3K3A-APC will also be effective after transient brain ischemia as it is after permanent ischemia. It has been recently reported that wt-APC and its analogs with reduced anticoagulant activity including 3K3A-APC cross the intact blood-brain barrier via EPCR-mediated transport (Deane et al., 2008). Therefore it is possible that the direct action of 3K3A-APC on neurons contributes to the observed neuroprotection in vivo. "
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    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.
    European Journal of Neuroscience 04/2009; 29(6):1119-30. DOI:10.1111/j.1460-9568.2009.06664.x · 3.67 Impact Factor
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    • "APC-mediated cleavage of protease activated receptor 1 (PAR1) results in anti-apoptotic and antiinflammatory signaling in endothelial cells including brain endothelium of the blood-brain barrier (BBB), monocytes and tissue macrophages (Mosnier et al., 2007a), and neurons (Guo et al., 2004). Recent studies demonstrated that APC and its variants with reduced anticoagulant activity cross the BBB via endothelial protein C receptor (EPCR)-assisted transport (Deane et al., 2008). APC is neuroprotective in different rodent models of stroke including transient ischemia and embolic stroke (Shibata et al., 2001, Cheng et al., 2003, Zlokovic et al., 2005), spinal cord injury (Taoka et al., 2000) and multiple sclerosis (Han et al., 2008). "
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    ABSTRACT: Activated protein C (APC) is a protease with anticoagulant and cytoprotective activities. APC is neuroprotective in rodent models of stroke. But, an APC variant with reduced anticoagulant activity, 3K3A-APC, compared to wild-type APC shows greater neuroprotection with no risk for bleeding in stroke models. To determine whether 3K3A-APC exhibits species-dependent neuroprotection similar to that as seen with wild-type APC, we studied murine and human recombinant 3K3A-APC mutants which show approximately 80% reduced anticoagulant activity. Murine 3K3A-APC (0.2 mg/kg i.v.) administered at 4 h after embolic stroke improved substantially functional outcome and reduced by 80% the infract volume 7 days after stroke. Human 3K3A-APC was neuroprotective after embolic stroke in mice, but at significantly higher concentrations (i.e. 2 mg/kg i.v.). Species-dependent neuroprotection, i.e. murine > human 3K3A-APC, was confirmed in a mouse model of permanent middle cerebral artery occlusion. Human 3K3A-APC had by fivefold greater cytoprotective activity than murine 3K3A-APC in oxygen-glucose deprivation model in human brain endothelial cells, whereas murine 3K3A-APC was by 2.5-fold more potent than human 3K3A-APC in a mouse model of NMDA-induced neuronal apoptosis. Thus, 3K3A-APC exhibits species-dependent neuroprotection which should be taken into account when designing human trials for ischemic stroke with APC mutants.
    Journal of Neurochemistry 04/2009; 109(1):116-24. DOI:10.1111/j.1471-4159.2009.05921.x · 4.24 Impact Factor
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