Protein S enhances the tissue factor pathway inhibitor inhibition of factor Xa but not its inhibition of factor VIIa-tissue factor.

Abstract

The tissue factor (TF) pathway of blood coagulation is initiated when factor VIIa binds to TF and proteolytically activates FIX and FX. The key regulator of this pathway is TF pathway inhibitor (TFPI), a multivalent Kunitz-type proteinase inhibitor that directly inhibits FXa and produces FXa-dependent feedback inhibition of the FVIIa–TF complex. In the final quaternary inhibitory complex containing FXa, TFPI, FVIIa and TF, the Kunitz-2 domain of TFPI interacts with FXa and the Kunitz-1 domain interacts with FVIIa. The formation of this complex is frequently described as a two-step process in which TFPI binds to FXa, and the FXa–TFPI complex subsequently binds to FVIIa–TF. Kinetic studies, however, strongly suggest that TFPI interacts with the tertiary FVIIa–TF–FXa complex before FXa is released from the FVIIa–TF complex after the cleavage of FX [1]. Protein S is a vitamin K-dependent protein that not only serves as a cofactor for the inactivation of FVa and FVIIIa by activated protein C (APC), but also appears to possess APC-independent anticoagulant activity [2, 3]. Recently, Hackeng et al. made the important observation that protein S enhances the interaction of TFPI with FXa in the presence of Ca2+ and phospholipids, and suggested that protein S-mediated enhancement of the FXa–TFPI interaction may also accelerate the inhibition of FVIIa–TF by TFPI [4]. Studies in which the inhibition of FVIIa–TF by TFPI is assessed by the generation of FXa, however, must be interpreted with caution, due to the inhibition of FXa by TFPI. Therefore, we have re-examined the effect of protein S on FVIIa–TF inhibition by TFPI using methods designed to minimize the confounding inhibitory effect of TFPI on FXa activity. First, the effect of protein S on TFPI inhibition of FXa was confirmed by repeating the experiments of Hackeng et al. [4], in which FXa was incubated with substrate S-2222 in the presence of TFPI, Ca2+ and phospholipids with or without protein S, and S-2222 hydrolysis was followed by measuring the p-nitroaniline released (ΔA405) at various concentrations of protein S. Protein S significantly enhanced the TFPI inhibition of FXa, with an apparent IC50 (concentration achieving 50% potentiation of the TFPI inhibition of FXa) of 10 nm (Fig. 1A). Maximum stimulation was observed at ∼ 160 nm protein S (Fig. 1A, inset). Protein S did not inhibit FXa in the absence of TFPI (data not shown). Similar results were obtained with protein S purchased from two independent vendors, Haematologic Technologies Inc. (Essex Junction, VT, USA) and Enzyme Research Laboratories (Southbend, IN, USA). These findings are consistent with those reported by Hackeng et al. [4], and confirm that protein S stimulates the TFPI inhibition of FXa in the presence of Ca2+ and phospholipids. Effect of protein S on tissue factor (TF) pathway inhibitor (TFPI) inhibition of FXa and FVIIa–TF. Reactions in Tris-buffered saline (50 mm Tris, 150 mm NaCl, pH 7.4) contained 5 mm CaCl2 and 25 μm phospholipid vesicles [60% phosphatidylcholine, 20% phosphatidylserine and 20% phosphatidylethanolamine (PC/PS/PE)], prepared as previously described [6]. TF was relipidated in PC/PS/PE as previously described [6]. (A) Effect of protein S on TFPI inhibition of FXa. Various concentrations of protein S (0–160 nm) were premixed with TFPI (1.5 nm) and S-2222 (500 μm) in reaction buffer. FXa (0.2 nm) was added to start the reaction. FXa activity was followed at 25 °C by measuring the rate of p-nitroaniline release (A405). Final velocities (vst) of S-2222 hydrolysis at each protein S concentration were calculated by non-linear regression as described by Morrison and Walsh [7], using the program Grafit from Erithacus software (Sigma, St Louis, MO, USA). IC50 (concentration of protein S yielding 50% potentiation of the TFPI inhibition of FXa) was obtained by fitting the vst values to the IC50 four-parameter logistic equation of Halfman [8]. Inset: Typical progress curves for S-2222 hydrolysis by FXa incubated with TFPI and various concentrations of protein S. (B) Effect of protein S on TFPI inhibition of the FVIIa–TF activation of FX as described by Hackeng et al. [4]. FX (160 nm) was mixed with TFPI (1 nm) with or without protein S (100 nm) before addition of FVIIa–TF (1 pm) to start the reaction. At different times, aliquots were withdrawn and quenched with 20 mm EDTA. The FXa activity generated was then determined by amidolytic assay using S-2222. (C) Measurement of the FVIIa–TF activation of FX using a modified enzyme-linked immunosorbent assay (ELISA) method. The reaction was carried out exactly as described in (B). To measure the FXa generated, quenched mixtures were diluted in excess TFPI to fully complex FXa with TFPI. TFPI–FXa complexes were then assayed with a TFPI–FXa-specific ELISA kit (American Diagnostica Inc., Stamford, CT, USA). (D) Effect of protein S on TFPI inhibition of the FVIIa–TF activation of FIX in the presence of FX. [3H]FIX (100 nm) was mixed with FX (50 nm) and TFPI (3 nm) with or without protein S (200 nm). FVIIa–TF (500 pm, prepared by mixing a 1 : 1 molar ratio of FVIIa with relipidated TF) was then added to start the reaction. At different times, aliquots were withdrawn and quenched in 20 mm EDTA. The FIX activation was followed by the release of the trichloroacetic acid-soluble activation peptide [5]. For (B), (C) and (D), the data were analyzed on the basis of the slow, tight binding mechanism as previously described [7] using the Enzyme kinetics program from Erithacus software. FVIIa–TF only, open circles; FVIIa–TF + protein S, closed circles; FVIIa–TF + TFPI, open triangles; FVIIa–TF + TFPI + protein S, closed triangles. Data for each panel represent averages of at least two independent measurements, which did not differ by more than 10%. The effect of protein S on TFPI inhibition of FVIIa–TF was similarly reassessed by following the TFPI inhibition of the FVIIa–TF-catalyzed FX activation in the presence and absence of protein S. For these experiments, FX and TFPI were incubated with FVIIa–TF with or without protein S in reaction buffer containing Ca2+ and phospholipids. At various time intervals, aliquots of the reaction were withdrawn and quenched with EDTA. Hackeng et al. [4] subsequently measured the FXa generated by amidolytic assay. Our replication of this assay is presented in Fig. 1B. As is evident from these data, protein S alone does not inhibit the FVIIa–TF activation of FX. Nevertheless, when compared to TFPI alone, the combination of TFPI with protein S appears to dramatically reduce FVIIa–TF activity. However, given the relatively higher molar concentration of TFPI than the quantity of FXa generated, the result could represent the potentiation of the TFPI inhibition of FXa by protein S. We therefore reassessed FXa generated in this system using a different approach. In this method, quenched reaction aliquots are diluted in buffer containing excess TFPI to completely complex the FXa generated to TFPI. TFPI–FXa complexes are then measured with the Imubind TFPI/Xa enzyme-linked immunosorbent assay kit from American Diagnostica Inc. (Stamford, CT, USA). In contrast to data obtained using the amidolytic assay (Fig. 1B), the inhibition of FVIIa–TF activation of FX by TFPI is not affected by the presence of protein S (Fig. 1C). To further confirm that the inhibition of FVIIa–TF activity by TFPI is not affected by protein S, FVIIa–TF-catalyzed activation of [3H]FIX was carried out in the presence of Ca2+, phospholipids, FX and TFPI with and without protein S. Here, FIXa generation was followed by measuring release of the trichloroacetic acid-soluble activation peptide from [3H]FIX as described by Bajaj and Birktoft [5]. Similar to the observations with FX activation, protein S did not enhance the TFPI inhibition of FVIIa–TF-catalyzed FIX activation in the presence of FX (Fig. 1D). In additional studies, FXa–TFPI complexes (1 nm) formed in the presence of Ca2+ and phospholipids with and without protein S (100 nm) were used to inhibit FVIIa–TF (100 pm)-catalyzed FX (160 nm) activation. Relative to the control with no inhibitor complexes, FXa–TFPI produced 90 ± 4% inhibition of FVIIa–TF activity at 2 min, as compared to 60 ± 6% inhibition produced by FXa–TFPI–protein S. Together, these data show that protein S enhances TFPI inhibition of FXa but not TFPI inhibition of FVIIa–TF. This is consistent with the notion that the predominant pathway for TFPI inhibition of FVIIa–TF involves its interaction with the tertiary complex FVIIa–TF–FXa produced during the activation of FX. The putative two-step pathway in which TFPI first binds FXa and then FXa–TFPI binds to FVIIa–TF does not appear to play a major role, although it can be demonstrated in vitro. The enhancing effect of protein S on FXa inhibition by TFPI is probably most important for the inhibition of FXa that escapes FVIIa–TF–FXa inhibition by TFPI. We thank S. P. Bajaj and S. Agah of UCLA/Orthopaedic Surgery for providing [3H]FIX. This work was supported by NIH grant HL 34462 to G. Broze. The authors state that they have no conflict of interest.