Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S.
Protein S is a cofactor for tissue factor pathway inhibitor (TFPI) that critically reduces the inhibition constant for FXa to below the plasma concentration of TFPI. TFPI Kunitz domain 3 is required for this enhancement to occur. To delineate the molecular mechanism underlying enhancement of TFPI function, in the present study, we produced a panel of Kunitz domain 3 variants of TFPI encompassing all 12 surface-exposed charged residues. Thrombin-generation assays in TFPI-depleted plasma identified a novel variant, TFPI E226Q, which exhibited minimal enhancement by protein S. This was confirmed in purified FXa inhibition assays in which no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentration-dependent binding of protein S to wild-type TFPI, but almost no binding to TFPI E226Q. We conclude that the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S.
Centre for Haematology, Faculty of Medicine, Imperial College London, Du Cane Road, London, United Kingdom. j.ahnstrom@imperial.ac.uk
Josefin
J
Ahnström
Helena M
HM
Andersson
Verity
V
Hockey
Yiran
Y
Meng
Thomas A J
TA
McKinnon
Tsutomu
T
Hamuro
James T B
JT
Crawley
David A
DA
Lane
D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D017293:Protein S / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000378:metabolism
D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D017293:Protein S; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D013917:Thrombin; D015951:Factor Xa