Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.
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 preve...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4382112?pdf=render |
| _version_ | 1818555505405067264 |
|---|---|
| author | Anna P Andreou Maria Efthymiou Yao Yu Helena R Watts Faruq H Noormohamed Daqing Ma David A Lane James T B Crawley |
| author_facet | Anna P Andreou Maria Efthymiou Yao Yu Helena R Watts Faruq H Noormohamed Daqing Ma David A Lane James T B Crawley |
| author_sort | Anna P Andreou |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T09:54:20Z |
| format | Article |
| id | doaj.art-cf567f8bc92d4787a41eded2589b2bd7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-12T09:54:20Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-cf567f8bc92d4787a41eded2589b2bd72022-12-22T00:28:11ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01104e012241010.1371/journal.pone.0122410Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke.Anna P AndreouMaria EfthymiouYao YuHelena R WattsFaruq H NoormohamedDaqing MaDavid A LaneJames T B CrawleyIschaemic 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.http://europepmc.org/articles/PMC4382112?pdf=render |
| spellingShingle | Anna P Andreou Maria Efthymiou Yao Yu Helena R Watts Faruq H Noormohamed Daqing Ma David A Lane James T B Crawley Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke. PLoS ONE |
| title | Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke. |
| title_full | Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke. |
| title_fullStr | Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke. |
| title_full_unstemmed | Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke. |
| title_short | Protective effects of non-anticoagulant activated protein C variant (D36A/L38D/A39V) in a murine model of ischaemic stroke. |
| title_sort | protective effects of non anticoagulant activated protein c variant d36a l38d a39v in a murine model of ischaemic stroke |
| url | http://europepmc.org/articles/PMC4382112?pdf=render |
| work_keys_str_mv | AT annapandreou protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT mariaefthymiou protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT yaoyu protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT helenarwatts protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT faruqhnoormohamed protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT daqingma protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT davidalane protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke AT jamestbcrawley protectiveeffectsofnonanticoagulantactivatedproteincvariantd36al38da39vinamurinemodelofischaemicstroke |