Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation.
Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 x 10(5) m(-1).s(-1), a dissociation rate constant of 7.61 x 10(-2) s(-1) and equilibrium binding constant (K(D)) of 147 nm. It was activated by thrombin over endothelial cells with a K(m) of 213 nm and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K(m) (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.
Preston
Roger J S
RJ
Department of Haematology, Division of Investigative Science, Hammersmith Campus, Imperial College London, UK.
Villegas-Mendez
Ana
A
Sun
Yong-Hui
YH
Hermida
José
J
Simioni
Paolo
P
Philippou
Helen
H
Dahlbäck
Björn
B
Lane
David A
DA
D015703:Antigens, CD; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D000074283:Endothelial Protein C Receptor; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry / Q000378:metabolism*; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; D006023:Glycoproteins; C478370:PROCR protein, human; D010743:Phospholipids; D011486:Protein C; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins