Activated protein C β-glycoform promotes enhanced noncanonical PAR1 proteolysis and superior resistance to ischemic injury

Abstract

Activated protein C (APC) is an anticoagulant protease that initiates cell signaling via protease-activated receptor 1 (PAR1) to regulate vascular integrity and inflammatory response. In this study, a recombinant APC variant (APCN329Q) mimicking the naturally occurring APC-β plasma glycoform was found to exhibit superior PAR1 proteolysis at a cleavage site that selectively mediates cytoprotective signaling. APCN329Q also enhanced integrin αMβ2-dependent PAR1 proteolysis to exert significantly improved antiinflammatory activity on macrophages compared with wild-type APC. Recent therapeutic applications of recombinant APC in ischemic stroke models have used APC variants with limited anticoagulant activity to negate potential bleeding side effects. Using a mouse model of ischemic stroke and late t-PA intervention, the neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPCPS) was compared with an identical APC variant except for the absence of glycosylation at the APC-β sequon (mAPCPS/N329Q). Remarkably, mAPCPS/N329Q limited cerebral ischemic injury and reduced brain lesion volume significantly more effectively than mAPCPS. Collectively, this study reveals the importance of APC glycosylation in controlling the efficacy of PAR1 proteolysis by APC and demonstrates the potential of novel APC variants with superior cytoprotective signaling function as enhanced therapeutic agents for the treatment of ischemic stroke.