Modulation of activated protein C (APC) anticoagulant and cytoprotective function

Abstract

Activated protein C (APC) is an anticoagulant glycoprotein that attenuates thrombin generation. Moreover, it has recently been demonstrated that APC possesses anti-inflammatory, anti-apoptotic and endothelial cell barrier stabilizing properties. The first goal of this thesis was to study the role of N-linked carbohydrates (glycans) in modulating APC anticoagulant function and endothelial cytoprotective signalling via the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1). It was determined that removal of APC N-linked glycans using the enzyme PNGase resulted in a ~6-fold reduction in the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability. Furthermore, deglycosylation enhanced APC anti-apoptotic function on endothelial cells compared to untreated APC. Recombinant APC variants were generated in which each Asn-linked glycan attachment site was eliminated by amino acid substitution. The variant APC-N329Q displayed up to 5-fold enhanced protection of the endothelial barrier compared to wild type APC. Moreover, an APC variant (APC-L38D/N329Q) was generated which possessed minimal anticoagulant activity but 5-fold improved endothelial barrier protective function compared to wild type APC.