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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-06-1691.
Submitted June 10, 2002
Accepted November 1, 2002
Gla-domain mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding
Yong-Hui Sun, Lei Shen, and Bjorn Dahlback*
Department of Laboratory Medicine, Division of Clinical Chemistry, University Hospital, Lund University, Malmo, Sweden
* Corresponding author; email: bjorn.dahlback{at}klkemi.mas.lu.se.
Protein C is a member of the vitamin K-dependent protein family. Proteins in this family have similar Gla-domains but their affinities for negatively charged phospholipid membranes vary over 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, three 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 wt-APC, while QGN-APC and SEDY-APC did not. Both normal FVa and FVa Leiden (Arg506Gln) 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 3-7-fold enhanced binding as compared to 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 phospholipid 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.
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