Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity.
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.
Guo
Huang
H
Department for Neurosurgery and Neurology, Center for Neurodegenerative and Vascular Brain Disorders, University of Rochester Medical Center, Rochester, NY 14642, USA.
Singh
Itender
I
Wang
Yaoming
Y
Deane
Rashid
R
Barrett
Theresa
T
Fernández
José A
JA
Chow
Nienwen
N
Griffin
John H
JH
Zlokovic
Berislav V
BV
D000704:Analysis of Variance; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000925:Anticoagulants / Q000378:metabolism / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001921:Brain / Q000166:cytology; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D042783:Endothelial Cells / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity; D005260:Female; D005947:Glucose / Q000172:deficiency; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000188:drug therapy; D020287:In Situ Nick-End Labeling / Q000379:methods; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation / Q000502:physiology*; D016202:N-Methylaspartate / Q000633:toxicity; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D018696:Neuroprotective Agents / Q000378:metabolism / Q000627:therapeutic use*; D011247:Pregnancy; D011486:Protein C / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use*; D044462:Receptors, Proteinase-Activated / Q000235:genetics; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000906:Antibodies; D000925:Anticoagulants; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D006454:Hemoglobins; D018696:Neuroprotective Agents; D011486:Protein C; D044462:Receptors, Proteinase-Activated; D016159:Tumor Suppressor Protein p53; D016202:N-Methylaspartate; D053148:Caspase 3; D053453:Caspase 9; D005947:Glucose