title: Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.
abstract: Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.
authors.affiliation: Centre for Haematology, Imperial College London, London, United Kingdom; Section of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, London, United Kingdom.
authors.forename: Anna P
authors.initials: AP
authors.lastname: Andreou
authors.affiliation: Centre for Haematology, Imperial College London, London, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Efthymiou
authors.affiliation: Centre for Haematology, Imperial College London, London, United Kingdom.
authors.forename: Yao
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Section of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, London, United Kingdom.
authors.forename: Helena R
authors.initials: HR
authors.lastname: Watts
authors.affiliation: Section of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, London, United Kingdom.
authors.forename: Faruq H
authors.initials: FH
authors.lastname: Noormohamed
authors.affiliation: Section of Anaesthetics, Pain Medicine & Intensive Care, Imperial College London, London, United Kingdom.
authors.forename: Daqing
authors.initials: D
authors.lastname: Ma
authors.affiliation: Centre for Haematology, Imperial College London, London, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.affiliation: Centre for Haematology, Imperial College London, London, United Kingdom.
authors.forename: James T B
authors.initials: JT
authors.lastname: Crawley
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology / Q000627:therapeutic use*; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000097:blood / Q000188:drug therapy*; D007700:Kinetics; D008297:Male; D008810:Mice, Inbred C57BL; D020125:Mutation, Missense; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use*; D011486:Protein C / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D018046:Protein C Inhibitor / Q000737:chemistry / Q000494:pharmacology; D059748:Proteolysis; D015427:Reperfusion Injury / Q000097:blood / Q000517:prevention & control; D013917:Thrombin / Q000378:metabolism; D013918:Thrombin Time
chemical_list: D000925:Anticoagulants; D018696:Neuroprotective Agents; D011486:Protein C; D018046:Protein C Inhibitor; D013917:Thrombin
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