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.