Activated protein C β-glycoform promotes enhanced noncanonical PAR1 proteolysis and superior resistance to ischemic injury

Activated protein C (APC) plays multiple regulatory roles that cumulatively serve to limit thrombus development¹  and attenuate inflammation.²  APC engagement with the endothelial cell protein C receptor (EPCR) facilitates proteolysis-dependent activation of protease-activated receptor 1 (PAR1),³  which protects the endothelial cell barrier from disruptive agents,⁴  inhibits apoptosis,^5,6  and limits cytokine expression from cells exposed to proinflammatory stimuli.^6,7 

Recombinant APC signaling attenuates neurovascular sequelae associated with experimental murine ischemic stroke^8-10  and limits neurotoxicity caused by subsequent thrombolytic tissue plasminogen activator (t-PA) administration.^11,12  Recombinant APC therefore represents a promising neuroprotective drug candidate to treat ischemic stroke, and a recombinant nonanticoagulant APC variant is currently being evaluated as an adjunctive therapy to minimize the adverse effects of t-PA administration.^13,14 

A recombinant APC variant (APC^N329Q) that mimics the glycosylation pattern of the endogenous plasma APC-β glycoform¹⁵  exhibits significantly enhanced PAR1-dependent cytoprotective activity on endothelial cells compared with wild-type APC.¹⁶  In this study, we sought to determine the molecular basis for superior APC-β cytoprotective signaling and establish whether ablation of the APC-β glycosylation sequon to accelerate cytoprotective signaling represents a novel means by which to increase the therapeutic efficacy of APC in an ischemic stroke model.

A complete description of the reagents used in this study is included in the supplemental Material, found on the Blood Web site.

Recombinant human and murine wild-type protein C and variants were generated, expressed, and purified largely as previously described.^16,17  A detailed description is provided in the supplemental Material.

PAR1 reporter–transfected 293T cells were incubated with APC in serum-free Dulbecco’s modified Eagle medium supplemented with 3 mM CaCl₂ and 0.6 mM MgCl₂ for 3 hours, after which the supernatant was removed and alkaline phosphatase (AP) activity measured using an AP substrate (QUANTI-Blue, Invivogen) at 650 nm.

Analysis of APC inhibition of cytokine secretion from RAW264.7 cells was performed as previously described.¹⁸  Tumor necrosis factor-alpha (TNFα) and IL-6 were measured by enyme-linked immunosorbent assay (ELISA) (R&D Systems).

Ischemic stroke in mice was induced as previously described¹⁹  and is further detailed in the supplemental Material. Briefly, 200 µL of 10 mg/kg t-PA (10% bolus, 90% perfusion during 20 minutes) with or without 0.2 mg/kg mAPC^PS or mAPC^PS/N329Q (50% bolus, 50% perfusion during 20 minutes) were given intravenously 3 hours after vessel occlusion. After 24 hours, mice were killed and their brains removed and frozen in isopentane. 20 μm cryostat-cut coronal brain sections were stained with thionine and analyzed (ImageJ 1.48V). For volume analysis, one section of every 10 was stained covering the entire lesion, and lesion (unstained) areas were measured.

We hypothesized that enhanced cytoprotective PAR1 signaling by APC-β was a consequence of accelerated EPCR-dependent PAR1 proteolysis. To test this, 293T cells cotransfected with EPCR and PAR1 linked to an N-terminal alkaline phosphatase (AP) cleavage reporter²⁰  were treated with recombinant APC-β (APC^N329Q) or N-glycosidase PNGase F–treated APC (APC^PNG), in which all N-linked glycans were excised.¹⁶  Interestingly, both APC^PNG and APC^N329Q demonstrated up to a fourfold increased PAR1 proteolysis when compared with wild-type APC (Figure 1A) or other APC glycoform variants (data not shown).

Generation of unique tethered ligands by APC or thrombin, caused by distinct cleavage events at Arg-46 or Arg-41, respectively, on PAR1, has been proposed as a potential mechanism to explain the disparate outcomes of PAR1 signaling initiated by each protease.^21,22  To determine whether the enhanced cytoprotective activity exhibited by APC^N329Q was a consequence of enhanced PAR1 proteolysis at the noncanonical Arg-46 cleavage site, 293T cells were cotransfected with EPCR and either PAR1^R41Q-AP or PAR1^R46Q-AP reporter variants (Figure 1B-C). Notably, cleavage at the Arg-46 site on PAR1 was especially sensitive to the loss of N-linked glycosylation after PNGase F treatment, and particularly loss of glycosylation at the APC-β sequon (Figure 1B). In contrast, Arg-41 proteolysis was limited in the presence of APC and largely insensitive to glycosylation status (Figure 1C). The increased rate of Arg-46 proteolysis on PAR1 by APC^PNG and APC^N329Q provides a novel molecular explanation for the enhanced cytoprotective activity of APC^N329Q on endothelial cells and supports the emerging concept of biased PAR1 agonism to achieve downstream cytoprotective signaling.

α_Mβ₂ can replace EPCR as a coreceptor for PAR1-dependent APC antiinflammatory signaling on macrophages.²³  To determine whether APC glycosylation also negatively regulates α_Mβ₂-dependent PAR1 signaling, we tested the ability of APC^PNG and APC^N329Q to attenuate proinflammatory cytokine release from lipopolysaccharide (LPS)-stimulated macrophages. APC^PNG and APC^N329Q reduced TNFα and IL-6 release from LPS-treated macrophages significantly more effectively than wild-type APC (Figure 1D-G). 293T cells coexpressing recombinant human α_Mβ₂ and PAR1-AP were used to determine whether enhanced antiinflammatory activity was mediated by increased α_Mβ₂-dependent PAR1 proteolysis by APC^N329Q. Interestingly, the α_M subunit mediated all coreceptor activity for α_Mβ₂-PAR1 proteolysis by APC, and enabled APC cleavage of PAR1 at both canonical (Arg-41) and noncanonical (Arg-46) sites (supplemental Figure 1). Both APC^PNG and APC^N329Q significantly enhanced α_Mβ₂-dependent PAR1 proteolysis (Figure 1H), indicating that N-linked glycans at Asn-329 regulate cytoprotective signaling by APC by impairing PAR1 proteolysis, irrespective of the APC coreceptor used.

Thrombolysis with t-PA is the only Food and Drug Administration–approved thrombolytic treatment of ischemic stroke, but can only be safely administered within 3 hours of symptom onset because of significant neurotoxicity.²⁴  Recombinant APC variants with severely attenuated anticoagulant activity reduce t-PA–associated toxicity in murine models of ischemic stroke in a PAR1-dependent manner.^11,12  We hypothesized that a recombinant nonanticoagulant APC that consists solely in the β-glycoform would be more efficacious in reducing t-PA neurotoxicity than existing APC variants in a murine model of ischemic stroke with late t-PA intervention. Similar to its human equivalent, in vitro analysis indicated that loss of N-linked glycan attachment at Asn-329 in murine APC (mAPC^N329Q) enhanced PAR1 proteolytic and antiinflammatory activity (Figure 2A-C). To determine whether the therapeutic activity of an APC variant with reduced anticoagulant activity could be enhanced by selective de-glycosylation, a signaling-competent mAPC variant (mAPC-D36A/L38D/A39V; mAPC^PS) with severely impaired anticoagulant activity in murine plasma²⁵  was prepared. The therapeutic efficacy of this variant was compared with an identical mAPC variant prepared on an APC-β background (mAPC^PS/N329Q) (Figure 2D-E). As anticipated, sham-operated mice did not develop cerebral injury (supplemental Figure 2). Like other mAPC variants with reduced anticoagulant activity,¹²  mAPC^PS retained the ability to reduce t-PA–induced cerebral injury 24 hours after stroke and t-PA administration, albeit modestly in this model (Figure 2F-G). mAPC^PS/N329Q, however, significantly reduced brain lesion size compared with when t-PA was administered alone after the ischemic event (P = .006; Figure 2F-G). Remarkably, mAPC^PS/N329Q was also significantly superior in preventing brain lesions compared with mAPC^PS (P = .004; Figure 2F-G), despite their structures only differing by the presence or absence of a specific N-linked glycan.

This study shows that effective cytoprotective PAR1 signaling by APC is determined by specific N-linked glycans absent in certain naturally occurring APC glycoforms, revealing new mechanistic insights as to how PAR1 proteolysis by APC is regulated. Moreover, APC-β sequon disruption boosts recombinant nonanticoagulant APC amelioration of cerebral ischemic injury after late t-PA treatment in ischemic stroke. Although further studies are required to investigate which of the multiple PAR1-dependent neuroprotective functions mediated by APC in vivo¹³  are particularly enhanced by APC^PS/N329Q in this setting, recombinant APC variants capable of hyperactive cytoprotective signaling represent a novel approach to boost the efficacy of APC-based therapeutic strategies without increasing dosage or compromising safety.

The authors acknowledge Eva Molina and Maider Esparza for their excellent technical assistance.

This study was funded by a Science Foundation Ireland Starting Investigator Research Grant (09/SIRG/B1590) (R.J.S.P.), the National Children’s Research Centre (R.J.S.P.), a European Research Area Network (ERANET)-Neuron grant (PRI-PIMNEU-2011-1334), and the Instituto de Salud Carlos III (PI10/01432, PI13/00072, PI11/1458, RECAVA RD06/0014/0008).

Contribution: E.M.G., M.G.D., A.S., L.M.Q., C.D., and S.E.R., performed experiments and analyzed data; P.T.W., J.O., J.H., O.P.S., and J.S.O’D. analyzed data and planned experiments; R.M. and R.J.S.P. analyzed data, planned experiments, and conceived the study; and all authors contributed to the preparation of the manuscript.

Conflict-of-interest disclosure: R.J.S.P. has received honoraria from Octapharma and research funding from Bayer Healthcare Pharmaceuticals.

Correspondence: Roger J. S. Preston, School of Medicine, Trinity College Dublin, United Kingdom; e-mail: prestonr@tcd.ie; and Ramón Montes, Center for Applied Medical Research, University of Navarra, Pamplona, Spain; e-mail: montesdiazpamplona@outlook.com.