Abstract 33: Activated Protein C Light Chain Provides an Extended Binding Surface for Anticoagulant Cofactor Protein S
Plasma protein S whose deficiency is linked to increased risk for thrombosis provides anticoagulant cofactor activity for activated protein C (APC) by enhancing rates of inactivation of factors Va and VIIIa. Previous APC mutagenesis studies showed that residues 35-39 in the gamma-carboxyglutamic acid domain are required for normal interactions with protein S, indicating the APC Gla domain binds protein S. Here we used mutagenesis of APC to interrogate the surface of APC’s light chain to identify the extended binding surface for protein S. We characterized the ability of protein S to enhance the anticoagulant activity of multiple recombinant APC variants using factor Xa-1-stage clotting assays using normal pooled plasma and protein S-depleted plasma. Mutations of residues L38, K43, I73, F95, and W115 in APC significantly reduced protein S’s cofactor activity. An APC variant carrying all of these five mutations lost all of protein S cofactor activity. On the crystallographic structure of APC, these five residues delineate an extended surface on only one side of the APC light chain that identifies the putative protein S binding site which is found on a face that is opposite APC’s catalytic triad site. Each of the APC variants with single or multiple L38, K43, I73, F95, and W115 mutations showed a normal ability to cleave SEAP-labeled PAR1 at Arg 41 and Arg 46, implying that the protein S-binding surface does not bind EPCR or PAR1. In summary, mutagenesis studies identify an extended surface on a single face of APC’s light chain for binding protein S. This knowledge will enable design and interpretation of new APC biologics with enhanced translational value.
Author Disclosures: J.A. Fernandez: None. X. Xu: None. R.K. Sinha: None. L.O. Mosnier: None. J.H. Griffin: None.
- © 2017 by American Heart Association, Inc.