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.
Highlights
Ischaemic stroke is a leading cause of mortality and morbidity worldwide
Consistent with a previous report [8], an estimated 5–20% of the human and murine protein C(Ca-ins) variant was secreted as disulphide-linked dimers as evidenced by detection of an additional ~120kDa band by Western blotting, and that disappeared upon reduction
These results suggest that a proportion of this protein C(Ca-ins) variant does not contain the intended engineered disulphide bond, which leaves surface exposed free cysteines available that can lead to dimerisation
Summary
Ischaemic stroke is a leading cause of mortality and morbidity worldwide. Tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy to promote clot dissolution and reduce further ischemia if administered within 4.5 hours of stroke onset.[1]. The anticoagulant activities of the human APC variants were determined based on their ability to inhibit tissue factor (TF)-induced thrombin generation, using calibrated automated thrombography assays, as previously described.[9,29,30] Both normal pooled human plasma and protein C-deficient plasma (Affinity Biologicals) were used.
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