Gla domain-mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Abstract

Protein C is a member of the vitamin K-dependent protein family. Proteins in this family have similar γ-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12, (QGN), S23E32D33Y44 (SEDY), and Q10G11N12S23E32D33Y44(QGNSEDY), were created. In plasma-based coagulation assays, the activated form of QGNSEDY (QGNSEDY-APC) demonstrated approximately 20-fold higher anticoagulant activity than wild-type activated protein C (WTAPC), while QGN-APC and SEDY-APC did not. Both normal activated factor V (FVa) and FVa Leiden (Arg506GIn) were degraded much more efficiently by QGNSEDY-APC than by WT APC in the presence as well as in the absence of protein S. Binding of protein C variants to negatively charged phospholipid membranes was investigated using light scattering and the BIAcore technique. QGNSEDY demonstrated 3- to 7-fold enhanced binding as compared with WT protein C, suggesting the membrane affinity to be influenced by several residues located at different parts of the Gla domain. The anticoagulant activity as well as phospho-lipid binding ability was only enhanced when multiple regions of the Gla domain were modified. The results provide insights into the molecular mechanisms that are involved in determining the binding affinity of the interaction between Gla domains and phospholipid membranes. The unique properties of QGNSEDY-APC suggest this APC variant possibly to have greater therapeutic potential than WT APC. © 2003 by The American Society of Hematology.