Endothelial protein C receptor-assisted transport of activated protein C across the mouse blood-brain barrier - PubMed (original) (raw)

Endothelial protein C receptor-assisted transport of activated protein C across the mouse blood-brain barrier

Rashid Deane et al. J Cereb Blood Flow Metab. 2009 Jan.

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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Conflict of interest statement

Conflict of Interest Disclosures: Dr. Zlokovic is a scientific founder of Socratech LLC a start-up biotechnology company with a mission to develop new therapies for the diseases of the aging brain including Alzheimer’s and stroke.

Figures

Figure 1

Figure 1. Time-dependent transport of 125I-labeled plasma-derived mouse APC across the mouse blood-brain barrier studied by the vascular brain perfusion method

(A) Uptake into the parietal cortex from the cerebral circulation of 125I-APC (0.1 nM; TCA-precipitable radioactivity) and simultaneously infused 14C-inulin (extracellular space reference marker) was expressed as a distribution space, Vd, corrected for the residual vascular radioactivity with 99mTc-albumin (Eqs. 1 and 2). Vd values of both tracers were plotted against the perfusion time. (B) TCA-precipitable 125I-APC (0.1 nM) radioactivity uptake into the whole brain homogenate, capillary-depleted brain and isolated brain microvessels after 10 min of cerebrovascular perfusion was corrected for the residual vascular radioactivity (99mTc-albumin) and expressed as a distribution space Vd normalized for each fraction per gram of the cerebral cortex. Values are mean ± SEM, n = 3–5 mice per group.

Figure 2

Figure 2. Concentration-dependent transport of 125I-labeled plasma-derived APC into the brain

(A) Permeability surface area (PS) product of TCA-precipitable 125I-APC radioactivity after 10 min of cerebrovascular arterial perfusion with 125I-APC (0.1 nM) and various concentrations of unlabeled APC. PS values were computed using eq. 3. (B) APC influx into the parietal cortex at various concentrations was calculated using eq 5. Values are mean ± SEM, n=3–6 mice per group.

Figure 3

Figure 3. APC variants with reduced anticoagulant activity cross-inhibit 125I-labeled plasma-derived APC transport into the brain

TCA- precipitable 125I-APC (0.1 nM) uptake into the parietal cortex corrected for the vascular space distribution (99mTc-albumin) after 10 min of cerebrovascular arterial perfusion in the absence and presence of 10 nM of unlabeled APC, 5A-APC, 3K3A-APC and mouse plasma-derived protein C (PC). Values are mean ± SEM, n=3–6 mice per group.

Figure 4

Figure 4. APC transport across the BBB from the cerebrvascular circulation into the brain requires EPCR

(A) 125I-APC (0.1 nM; TCA-precipitable radioactivity) uptake into the parietal cortex corrected for the residual vascular radioactivity (99mTc-albumin; eq. 1) after 10 min of the carotid arterial perfusion in the absence and presence of antibodies that specifically block (RCR-252) or do not block (RCR-92) APC binding site on EPCR and PAR1 catalytic site (H111). All antibodies were studied at 20 μg/ml. (B) 125I-APC (0.1 nM; TCA-precipitable radioactivity) uptake into the parietal cortex corrected for 99mTc-albumin residual vascular radioactivity after 10 min of the carotid arterial perfusion in severely-deficient EPCR mice and in PAR1 null mice. (C) Western blot analysis of EPCR in brain microvessels from control (EPCR+/+) and severely-depleted EPCR (EPCRδ/δ) mice (right), and quantification of EPCR relative abundance in brain capillaries from EPCR+/+ and EPCRδ/δ mice (left). β-actin was used to standardize protein levels. Values are mean ± SEM, n=3–4 mice per group.

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