Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S

Abstract

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 TFPIdepleted 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 concentrationdependent binding of protein S to wildtype 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. (Blood. 2012;120(25):5059-5062) IntroductionTissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor consisting of an acidic aminoterminal polypeptide, followed by 3 tandem Kunitz-type domains (Kunitz domains 1, 2, and 3) and a basic carboxyterminal tail. 1,2 TFPI exerts its anticoagulant function by inhibiting tissue factor (TF)-induced coagulation in the blood. [3][4][5] Purified assays have shown that FXa inhibition by TFPI occurs in a 2-step process that can be described by the inhibition constants K i and K i *, 6 respectively, in the following equation:In 2006, Hackeng et al identified protein S as a cofactor for TFPI that is capable of reducing the K i for FXa inhibition by TFPI by 10-fold. 7 More recently, Ndonwi et al showed that protein S enhancement of TFPI is dependent on the TFPI Kunitz domain 3. 8 A TFPI R199L variant showed partial loss of protein S cofactor function compared with wild-type (WT) TFPI. 8 The present study is an investigation of the role of all surface-exposed charged residues of TFPI Kunitz domain 3. Methods Generation, expression, and purification of TFPI variantsTen composite and individual point mutations were generated: D194Q/ R195Q/R199Q, E202Q/R204Q/K218Q, K213Q/R215Q/K232Q, E226Q/ E234Q/R237Q, D194Q, R195Q, R199Q, E226Q, E234Q, and R237Q (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Details of vector construction, protein expression, purification, and quantification can be found in the supplemental Methods. Thrombin-generation assay determined by CATCalibrated automated thrombogram (CAT) was performed in normal or TFPI-depleted plasma (supplemental Methods), as described previously. 9,10 Purified or WT TFPI and TFPI variants in concentrated conditioned medium (0-1.5nM) were added to the plasma. Purification status did not influence inhibitory function. FXa inhibition assayFXa (0.5nM) activity was monitored by the cleavage of the chromogenic substrate S-2765 (Chromogenix) in the presence of absence of TFPI (0-4nM) and protein S (0-320nM) essentially as descr...

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

“…17 Briefly, the TFPI expression vector was transiently transfected into HEK293T cells. After a 3-day expression, full-length TFPI was purified in 2 steps; heparin Sepharose FF chromatography followed by affinity purification using an anti-TFPI Kunitz domain 1 antibody (Sanquin).…”

“…15 The protein S enhancement of TFPI is dependent on the TFPI Kunitz domain 3, particularly TFPI Kunitz domain 3 residues Glu226 and Arg199. 16,17 The complementary interaction site on protein S for TFPI has to date not been the subject of any report, and the mechanism behind this enhancement has yet to be fully defined.Protein S is a 73-kDa, vitamin K-dependent, multidomain protein. 9 It comprises an N-terminal g-carboxylated glutamic acid (Gla) domain, a thrombin-sensitive region (TSR), 4 epidermal growth factor-like (EGF) domains and a C-terminal sex hormone-binding globulin (SHBG)-like domain.…”

“…on April 11, 2019. by guest www.bloodjournal.org From effect on the thrombin generation, whereas it efficiently enhanced TFPI-mediated inhibition of thrombin generation, as reported previously. 15,17,34 Inhibitory antibodies against TFPI and protein S were used to verify that inhibitory effects were a direct consequence of TFPI and TFPI cofactor function of protein S ( Figure 1B).The functional interaction site of protein S for TFPI was investigated using the library of 44 protein S variants. The screening of WT protein S and the protein S variants for their TFPI cofactor function was performed at 1 nM TFPI and 50 nM protein S ( Figure 1C).…”

“…The flow cells were perfused with protein S (0-1000 nM) and measurements were carried out by surface plasmon resonance (SPR) as described previously. 16,17 Results Screening of protein S variants for the TFPI cofactor function in plasmaThrombin generation was measured in protein S-depleted plasma. A dose-dependent decrease in thrombin generation was observed after the addition of increasing concentrations of TFPI (0-2 nM) in the absence of protein S. As expected, dose-dependent effects were detected by reduction of the peak and endogenous thrombin potential values, as well as an increase in lag time ( Figure 1A).…”

“…15,16 The EC 50 was determined by a one-phase exponential decay nonlinear curve fit. 16,17 Binding of protein S to TFPI investigated by surface plasmon resonancePurified recombinant TFPI was immobilized on a CM5 chip (GE Healthcare) using amine coupling according to the manufacturer's instructions. The flow cells were perfused with protein S (0-1000 nM) and measurements were carried out by surface plasmon resonance (SPR) as described previously.…”