Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement
Authors: Preston Roger JS , Harmon Shona , Ni Ainle Fionnuala B , Johnson Jennifer A , Cunningham Moya , Smith O. , White Barry , O’Donnell James S 
Published: April 2020

Abstract
Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (&lt;2-fold) anticoagulant activity compared to wild type APC. The anticoagulant activity of APC-L38D, however, was severely impaired. APC-L38D was unable to achieve half-maximal inhibition of endogenous thrombin potential (ETP) at APC concentrations as high as 150nM, compared to wild type APC, which achieved half-maximal inhibition at 7.2nM APC. To clarify the role of Leu-38 in facilitating APC anticoagulant function, we further studied the ability of APC-L38D to be stimulated in protein S-deficient plasma reconstituted with plasma-purified protein S. Co-incubation of wild type APC with increasing protein S concentration (12.5–200nM) caused a corresponding reduction in ETP (IC50 = 24nM protein S). In contrast, APC-L38D was unresponsive to protein S. In the presence of APC-L38D, ETP was reduced only 22% at 1.5μM protein S (10-fold higher than plasma free protein S). In a phospholipid-dependent FVa proteolysis time course assay, both wild type APC and APC-L38D rapidly reduced FVa cofactor activity, indicating that the observed impaired plasma anticoagulant activity of APC-L38D is not mediated by impaired interaction with anionic phospholipids or FVa. In a modified version of this assay, wild type APC-mediated FVa proteolysis was rapidly enhanced by added protein S, with half-maximal inhibition observed at 5nM protein S. In contrast, APC-L38D exhibited no protein S-enhanced FVa proteolysis. Cumulatively, these data confirm that Leu-38 mediates APC anticoagulant function in plasma by facilitating critical protein S cofactor enhancement of FVa proteolysis. Previous studies have shown that APC Gla domain mutations can influence EPCR binding, a pre-requisite for PAR-1 mediated cytoprotective signalling. Consequently, we assessed APC binding to sEPCR using surface plasmon resonance. Binding affinities of APC-L38D and wild type APC were very similar (KD 112±25nM versus 117±36nM). Furthermore, the ability of APC-L38D to protect EAhy926 cells from staurosporine-induced apoptosis was also investigated using RT-PCR quantification of pro- (bax) and anti- (bcl-2) apoptotic gene expression. Pre-incubation with APC-L38D significantly reduced the bax/bcl-2 ratio to the same extent as wild type APC. The EPCR-dependence of these anti-apoptotic activities was confirmed using RCR-252, (an inhibitory anti-EPCR antibody) which ablated the cytoprotective effect of both APC species. In conclusion, we demonstrate that a single amino acid substitution (L38D) can significantly impair APC anticoagulant activity due to elimination of protein S cofactor enhancement. However, despite the location of Leu-38 in the Gla domain, APC-L38D retains its ability to bind EPCR, and trigger PAR-1 mediated cytoprotective signalling 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 dissociate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.