Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Fionnuala Ni Ainle,Jennifer A. Johnson,Owen P. Smith,Moya S. Cunningham,Shona Harmon,Barry White,Roger J.S. Preston,James S. O'Donnell

doi:10.1074/jbc.m802338200

Abstract

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Highlights

Recent studies have shown that Activated protein C (APC) bound to endothelial cell protein C receptor (EPCR) via its Gla domain can activate protease-activated receptor-1 (PAR-1) on endothelial cells, triggering complex intracellular signaling [7]
A single amino acid substitution (L38D) is sufficient to entirely ablate the ability of protein S to function as a cofactor for this APC variant yet does not alter APC-mediated proteolysis of factor Va (FVa) in the absence of protein S
Despite the fact that this key amino acid is located in the APC Gla domain, we show that this APC variant interacts normally with EPCR and that it retains its ability to initiate PAR-1 protective signaling in an identical fashion to that of wild type APC

Summary

Introduction

Recent studies have shown that APC bound to EPCR via its Gla domain can activate protease-activated receptor-1 (PAR-1) on endothelial cells, triggering complex intracellular signaling [7]. In both protein S-dependent FVa proteolysis assays using purified coagulation proteins and in the plasma milieu, this APC Gla domain variant exhibited minimal residual anticoagulant activity compared with wild type APC.

Results

Conclusion