Activated protein C analog promotes neurogenesis and improves neurological outcome after focal ischemic stroke in mice via protease activated receptor 1

Subcortical white matter (WM) stroke accounts for 25% of all strokes and is the second leading cause of dementia. Despite such clinical importance, we still do not have an effective treatment for ischemic WM stroke, and the mechanisms of WM postischemic neuroprotection remain elusive. 3K3A-activated protein C (APC) is a signaling-selective analogue of endogenous blood protease APC that is currently in development as a neuroprotectant for ischemic stroke patients. Here, we show that 3K3A-APC protects WM tracts and oligodendrocytes from ischemic injury in the corpus callosum in middle-aged mice by activating protease-activated receptor 1 (PAR1) and PAR3. We show that PAR1 and PAR3 were also required for 3K3A-APC’s suppression of post–WM stroke microglia and astrocyte responses and overall improvement in neuropathologic and functional outcomes. Our data provide new insights into the neuroprotective APC pathway in the WM and illustrate 3K3A-APC’s potential for treating WM stroke in humans, possibly including multiple WM strokes that result in vascular dementia.

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Activated protein C, protease activated receptor 1, and neuroprotection

Blood ◽

10.1182/blood-2018-02-769026 ◽

2018 ◽

Vol 132 (2) ◽

pp. 159-169 ◽

Cited By ~ 39

Author(s):

John H. Griffin ◽

Berislav V. Zlokovic ◽

Laurent O. Mosnier

Keyword(s):

Ischemic Stroke ◽

Cell Signaling ◽

Protein C ◽

Anticoagulant Activity ◽

Single Agent ◽

Active Form ◽

Activated Protein C ◽

Stroke Patients ◽

Protease Activated Receptor 1

Abstract Protein C is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts potent anticoagulant activity. In addition to its antithrombotic role as a plasma protease, pharmacologic APC is a pleiotropic protease that activates diverse homeostatic cell signaling pathways via multiple receptors on many cells. Engineering of APC by site-directed mutagenesis provided a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks >90% anticoagulant activity but retains normal cell signaling activities. This 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein–coupled receptor, protease activated receptor 1. Potent neuroprotection in murine ischemic stroke models is linked to 3K3A-APC–induced signaling that arises due to APC’s cleavage in protease activated receptor 1 at a noncanonical Arg46 site. This cleavage causes biased signaling that provides a major explanation for APC’s in vivo mechanism of action for neuroprotective activities. 3K3A-APC appeared to be safe in ischemic stroke patients and reduced bleeding in the brain after tissue plasminogen activator therapy in a recent phase 2 clinical trial. Hence, it merits further clinical testing for its efficacy in ischemic stroke patients. Recent studies using human fetal neural stem and progenitor cells show that 3K3A-APC promotes neurogenesis in vitro as well as in vivo in the murine middle cerebral artery occlusion stroke model. These recent advances should encourage translational research centered on signaling selective APC’s for both single-agent therapies and multiagent combination therapies for ischemic stroke and other neuropathologies.

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Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

Biochemical Journal ◽

10.1042/bj20030341 ◽

2003 ◽

Vol 373 (1) ◽

pp. 65-70 ◽

Cited By ~ 163

Author(s):

Laurent O. MOSNIER ◽

John H. GRIFFIN

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Active Site ◽

Protein C ◽

Activated Protein C ◽

Cell Protein ◽

Protease Activated Receptor 1 ◽

Induced Apoptosis ◽

Apoptotic Effects ◽

Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

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PAR1 biased signaling is required for activated protein C in vivo benefits in sepsis and stroke

Blood ◽

10.1182/blood-2017-10-810895 ◽

2018 ◽

Vol 131 (11) ◽

pp. 1163-1171 ◽

Cited By ~ 36

Author(s):

Ranjeet K. Sinha ◽

Yaoming Wang ◽

Zhen Zhao ◽

Xiao Xu ◽

Laurent Burnier ◽

...

Keyword(s):

Ischemic Stroke ◽

Mechanism Of Action ◽

Protein C ◽

Activated Protein C ◽

Point Mutations ◽

Key Points ◽

Biased Signaling ◽

Lethal Sepsis

Key Points R41Q and R46Q point mutations in PAR1 in mice enabled studies of APC’s in vivo mechanism of action in lethal sepsis and ischemic stroke. APC-biased, PAR1-dependent signaling due to cleavage at R46 in PAR1 is required for APC’s in vivo benefits in sepsis and ischemic stroke.

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Recombinant murine-activated protein C is neuroprotective in a murine ischemic stroke model

Blood Cells Molecules and Diseases ◽

10.1016/s1079-9796(03)00034-2 ◽

2003 ◽

Vol 30 (3) ◽

pp. 271-276 ◽

Cited By ~ 54

Author(s):

José A Fernández ◽

Xiao Xu ◽

Dong Liu ◽

Berislav V Zlokovic ◽

John H Griffin

Keyword(s):

Ischemic Stroke ◽

Protein C ◽

Activated Protein C ◽

Stroke Model

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1340 Activated protein C resistance in Chinese patients with ischemic stroke

Journal of the Neurological Sciences ◽

10.1016/s0022-510x(05)81704-6 ◽

2005 ◽

Vol 238 ◽

pp. S441-S442

Keyword(s):

Ischemic Stroke ◽

Protein C ◽

Activated Protein C ◽

Chinese Patients ◽

Activated Protein C Resistance

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Ischemic stroke and activated protein C resistance

JAMA ◽

10.1001/jama.274.16.1266b ◽

1995 ◽

Vol 274 (16) ◽

pp. 1266b-1266

Author(s):

C. Bachmeyer

Keyword(s):

Ischemic Stroke ◽

Protein C ◽

Activated Protein C ◽

Activated Protein C Resistance

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Endothelial Protein C Receptor (EPCR), Protease Activated Receptor-1 (PAR-1) and Their Interplay in Cancer Growth and Metastatic Dissemination

Cancers ◽

10.3390/cancers11010051 ◽

2019 ◽

Vol 11 (1) ◽

pp. 51 ◽

Cited By ~ 3

Author(s):

Marek Z. Wojtukiewicz ◽

Dominika Hempel ◽

Ewa Sierko ◽

Stephanie C. Tucker ◽

Kenneth V. Honn

Keyword(s):

Cell Surface ◽

Cancer Patients ◽

Cancer Progression ◽

Protein C ◽

Critical Role ◽

Activated Protein C ◽

Cancer Growth ◽

Endothelial Protein C Receptor ◽

Surface Receptors ◽

Protease Activated Receptor 1

Endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR-1) by themselves play important role in cancer growth and dissemination. Moreover, interactions between the two receptors are essential for tumor progression. EPCR is a cell surface transmembrane glycoprotein localized predominantly on endothelial cells (ECs). It is a vital component of the activated protein C (APC)—mediated anticoagulant and cytoprotective signaling cascade. PAR-1, which belongs to a family of G protein–coupled cell surface receptors, is also widely distributed on endothelial and blood cells, where it plays a critical role in hemostasis. Both EPCR and PAR-1, generally considered coagulation-related receptors, are implicated in carcinogenesis and dissemination of diverse tumor types, and their expression correlates with clinical outcome of cancer patients. Existing data explain some mechanisms by which EPCR/PAR-1 affects cancer growth and metastasis; however, the exact molecular basis of cancer invasion associated with the signaling is still obscure. Here, we discuss the role of EPCR and PAR-1 reciprocal interactions in cancer progression as well as potential therapeutic options targeted specifically to interact with EPCR/PAR-1-induced signaling in cancer patients.

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Abstract 102: Reduced Hemorrhagic Transformation and Neurological Worsening with Neuroprotection (3K3A-APC) During Thrombolysis and Thrombectomy

Stroke ◽

10.1161/str.51.suppl_1.102 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Kyle Ogami ◽

Shlee Song ◽

Patrick Lyden ◽

Ariana Anderson ◽

Keyword(s):

Protein C ◽

Activated Protein C ◽

Hemorrhagic Transformation ◽

Significant Variable ◽

Gradient Echo ◽

Stroke Patients ◽

Hemorrhagic Infarction ◽

The Central Nervous System ◽

Protease Activated Receptor 1

Background: Agonism of protease-activated receptor 1 (PAR1) potently protects neurons and vasculature in the central nervous system during stroke. We evaluated the effects of 3K3A-APC, a recombinant variant of activated protein C active at PAR1, in acute ischemic stroke patients during conventional recanalization with thrombolysis or thrombectomy or both. We hypothesized that 3K3A-APC would reduce post-treatment hemorrhage and symptomatic neurologic deterioration. Methods: Using the NeuroNEXT trial NN104 (RHAPSODY) database, susceptibility weighted and gradient echo images were graded for intracerebral hemorrhage size according to radiographic criteria described in ECASS (Hemorrhagic infarction type 1 and 2 and parenchymal hematoma type 1 and 2) at four time points (0, 7, 30, and 90 days after treatment). Of 110 participants enrolled, 101 had appropriate imaging for analysis. Images were evaluated and graded by two blinded, independent raters with an expert providing consensus reads. Utilizing NIHSS following drug treatment, neurological worsening was defined as an increase in NIHSS ≥ 4 points. We compared placebo versus drug administration using multi-variate regression. Results: Of 101 participants evaluated, those receiving placebo (n=41) were significantly more likely to have hemorrhage at any time compared with those receiving any amount of drug (n=60) (p = 0.04; CI: 1.080 - 7.544; OR = 2.73). Age was found to be the only significant variable leading independently to increased neurological worsening (p = 0.038; CI: 1.015-1.193; OR = 1.086). Conclusion: The neuroprotectant 3K3A-APC showed significant reduction in hemorrhagic transformation when co-administered with conventional recanalization therapy. Further studies are underway for determining the effect of 3K3A-APC on functional outcome.

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