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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-06-1691.
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Blood, 15 March 2003, Vol. 101, No. 6, pp. 2277-2284
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Gla domain-mutated human protein C exhibiting enhanced
anticoagulant activity and increased phospholipid binding
Yong-Hui Sun,
Lei Shen, and
Björn Dahlbäck
From the Department of Laboratory Medicine, Division of
Clinical Chemistry, Lund University, University Hospital, Malmö,
Sweden.
Protein C is a member of the vitamin K- dependent protein
family. Proteins in this family have similar  -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 (WT APC), while QGN-APC and SEDY-APC did
not. Both normal activated factor V (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- 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 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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