Cytoprotective-selective activated protein C therapy for ischaemic stroke

Abstract

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotective-selective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

Keywords: EPCR; PAR1; PAR3; Stroke; activated protein C.

Figure 1. The protein C system

Depicted are protein C activation (middle) and the anticoagulant (left) and cytoprotective (right) protein C pathways. Efficient activation of protein C zymogen (yellow) (middle of scheme) by thrombin (IIa) (green) requires thrombomodulin (TM) (red) and the endothelial protein C receptor (EPCR) (blue). Retention of APC bound to EPCR allows APC to express multiple cytoprotective activities (right side of scheme) that involve PAR-1. These cytoprotective activities include APC-mediated anti-inflammatory and anti-apoptotic activities, alterations of gene expression profiles, and protection of endothelial barrier functions. Dissociation of APC from EPCR allows binding of APC to negatively charged cell membrane surfaces and expression of APC’s anticoagulant activity (left side of scheme) that involves limited proteolysis of the activated cofactors Va (fVa) and VIIIa (fVIIIa) to yield the inactivated cofactors, fVi and fVIIIi.

Figure 2. APC neuroprotective effects and tPA-induced hemorrhage during murine ischemic stroke

in vivo neuroprotective effects of recombinant mouse wt-APC in C57Bl6 mice during focal ischemic stroke. APC or vehicle was given 10 min after MCAO. Motor neurological score (A), total brain injury volume (B) and post-ischemic cerebral blood flow (C). Brain hemorrhage mediated by tPA after transient ischemia in mice was determined 24 h after transient MCAO for mice receiving the indicated treatments (D), where the bottom two images show brains for mice receiving either 0.2 mg/kg APC with tPA or 2.0 mg/kg APC at 3h after tPA. Bleeding in the ischemic brain was quantified by hemoglobin after treatment with or without rh-tPA (0.2 mg/kg) and with or without simultaneous infusion or infusion 3 h post-tPA of mouse wt-APC (0–2 mg/kg) (E). Panels A-C are from Cheng et al. Nat Med 2003 (34), panels D–E are from Cheng et al. Nat Med 2006 (38).

Figure 3. APC space-filling model showing amino acid residues of activity-selective APC mutants

The model of APC depicts the N-terminal Gla domain at the bottom which binds EPCR and phospholipid membranes, the EGF-1 and EGF-2 domains in the middle and the protease domain at the top with the “active site” residues noted in red. On the top of the model, blue highlights five basic residues (KKK191-193 and RR229/230) which form a large positively charged exosite that recognizes factor Va; mutations of these residues reduce anticoagulant activity but not cytoprotective activity. Purple highlights two residues (R222 and D237) which when mutated to Cys can form a disulfide bond, causing loss of most anticoagulant activity but retention of cytoprotective activity. Light green highlights the L38D mutation that reduces anticoagulant activity due to reduced protein S enhancement. Anticoagulant-selective APC mutants include mutations of E330 and E333 to Ala (orange) that selectively reduce PAR1 signaling, the E149A mutation (yellow) in the C terminus of the light chain, and mutation of L8 in the GLA-domain (dark green) that selectively disrupts APC binding to EPCR but not to negatively charged phospholipids. The model is based on the x-ray crystallographic structure of APC (1AUT) (90).

Figure 4. Functional selectivity and biased agonism due to canonical and non-canonical PAR1 activation

Thrombin and APC display functional selectivity for PAR1 activation that results in either proinflammatory effects or cytoprotective effects, respectively. Thrombin cleaves PAR1 at Arg41 (“canonical cleavage”) and APC cleaves PAR1 at Arg46 (non-canonical cleavage”). The N-terminal tethered-ligands with the new N-terminal sequences as represented by the TRAP peptide that exists after cleavage at Arg41 or the TR47 peptide that exists after cleavage at Arg46 can accordingly cause activation of different signaling pathways, and is termed ‘biased agonism.’ The conformer subsets of PAR1 that are stabilized by TRAP or TR47 peptides preferentially promote signaling mediated by G-proteins or by β-arrestin. The agonist bias is thus directly related to the differing PAR1 cleavage sites. This figure was originally published in Blood (64). © the American Society of Hematology.

Figure 5. Multiple neuroprotective effects of APC on the neurovascular unit following ischemic injury

APC neuroprotective effects and its ability to prevent tPA-induced neurotoxicity in the neurovascular unit include (i) protection of the BBB integrity, (ii) anti-inflammatory and anti-apoptotic effects on brain endothelial cells, and (iii) anti-apoptotic effects on neurons and other brain cells. This figure was modified from Zlokovic and Griffin, Trends Neuroscience 2011 (30).

Figure 6. Dosing of cytoprotective-selective 3K3A-APC in healthy adult volunteers

The surface contours for the APC protease domain showing positive (blue) and negative (red) side-chains for wt-APC (A) and 3K3A-APC (B). The yellow rectangle highlights the comparison of the protease domain for 3K3A-APC (containing 3 Ala substitutions at Lys191, Lys192, and Lys193) with wt-APC to indicate the loss of three positively charged surface residues from an exosite that is required for normal recognition of factor Va. Loss of this factor Va-binding site results in reduced anticoagulant activity (~5% of wt-APC) but not any loss of cytoprotective actions, hence the designation “cytoprotective-selective” for the 3K3A-APC variant. (C) As the result of its reduced anticoagulant activity, 3K3A-APC can reach high plasma concentrations without adverse effects in healthy human subjects after a brief i.v. infusion of a single dose (data in panel C) or after five repeated high doses every 12 h (data not shown). Panel C is from Lyden et al. Curr Pharm Design 2013 (89).