Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor

Abstract

Protein S (PS) is a cofactor for activated protein C (APC), which inactivates coagulation factors (F) Va and VIIIa. Deficiency of protein C or PS is associated with risk of thrombosis. We found that PS also has APC-independent anticoagulant activity (PS-direct) and directly inhibits thrombin generated by FXa/FVa (prothrombinase complex). Here we report that PS contains Zn(2+) that is required for PS-direct and that is lost during certain purification procedures. Immunoaffinity-purified PS contained 1.4 +/- 0.6 Zn(2+)/mol, whereas MonoQ-purified and commercial PS contained 0.15 +/- 0.15 Zn(2+)/mol. This may explain the controversy regarding the validity of PS-direct. Zn(2+) content correlated positively with PS-direct in prothrombinase assays and clotting assays, but APC-cofactor activity of PS was independent of Zn(2+) content. PS-direct and Zn(2+) were restored to inactive PS under mildly denaturing conditions. Conversely, o-phenanthroline reversibly impaired the PS-direct of active PS. Zn(2+)-containing PS bound FXa more efficiently (K(d)(app)=9.3 nM) than Zn(2+)-deficient PS (K(d)(app)=110 nM). PS bound TFPI efficiently, independently of Zn(2+) content (K(d)(app)=21 nM). Antibodies that block PS-direct preferentially recognized Zn(2+)-containing PS, suggesting conformation differences at or near the interface of 2 laminin G-like domains near the PS C terminus. Thus, Zn(2+) is required for PS-direct and efficient FXa binding and may play a role in stabilizing PS conformation.

Figure 1.

PS-direct correlates with Zn²⁺ content. PS-direct of each PS is plotted as a function of its Zn²⁺ content. 1/IC₅₀, 1/[PS] required to inhibit the rate of thrombin generation by 50% in the standardized prothrombinase assay. Typical initial rate of thrombin generation in the control without PS was 7 nM/min.

Figure 2.

PS containing Zn²⁺ expresses PS-direct in plasma. A) Endogenous thrombin potential assays were performed as described in Materials and Methods. NHP (dashed curve) or PSdP or mixtures of the 2 plasmas were compared to PSdP supplemented with 2 doses each of immunoaffinity-purified PS (IAP PS) that contained Zn²⁺ and conventionally MonoQ-purified PS (MQ PS, dotted curve) that was Zn²⁺ deficient. For clarity, only 1 dose of each PS and 1 mixture of NHP and PSdP are shown. B) Linear response of lag time in the ETP assay for various mixtures of NHP/PSdP or NHP/TFPIdP. Standard curves were used to estimate the PS-direct of Zn²⁺-containing or Zn²⁺-deficient PS added to PSdP or TFPIdP. C) Similar experiment as in A, but using TFPIdP in place of PSdP. All results were confirmed on separate dates.

Figure 3.

PS-direct is enhanced after Zn²⁺ treatment under mildly denaturing conditions. A) MonoQ-purified PS with poor PS-direct was treated with 10 μM Zn²⁺ under the conditions indicated (see Materials and Methods). After dialysis, PS was tested for ability to inhibit generation of thrombin from prothrombin by FXa/FVa. IC₅₀(μM) was determined in each case. PS-direct was expressed as 1/IC₅₀; 1/IC₅₀ ≤ 0.5 was indicated as 0.5. B) Dose response of MonoQ-purified PS with poor PS-direct, with or without treatment with 10 μM Zn²⁺in the presence of 6 M urea or pH 2.7 buffer, followed by dialysis as in Materials and Methods. For comparison, PS-direct of typical IAP PS is also illustrated (dashed line). This experiment was repeated 4 times (Table 2).

Figure 4.

PS-direct is lost or regained with loss or regain of Zn²⁺. PS that contained Zn²⁺ and had good PS-direct (lighter bars) and Zn²⁺-deficient PS with weak PS-direct (darker bars) were each treated for 10 min with 10 mM of the Zn²⁺-chelator, o-phenanthroline. Half of each treated PS was then dialyzed against TBS (middle set of bars), and the other half of each was dialyzed against TBS-10 μM Zn²⁺ (last set of bars). Each was then dialyzed twice against TBS. PS antigen level was determined, and several concentrations were tested for ability to inhibit generation of thrombin from prothrombin by FXa/FVa. Reciprocal of IC₅₀ is indicated. Data from 2 experiments performed on different days were combined.

Figure 5.

Binding of FXa to PS. A) Binding of FXa to Zn²⁺-containing PS (squares) or to Zn²⁺-deficient commercial PS (circles) immobilized on a plate at 2 μg/ml. Results were confirmed in 2 separate experiments, and results for Zn²⁺-containing PS confirm our earlier report . B) SPR binding experiments. See Materials and Methods for details. Peak binding from 5–6 sensograms obtained using different concentrations of FXa binding to Zn²⁺-containing PS (squares) or to commercial Zn²⁺-deficient PS (circles). Weak binding of prothrombin (FII, triangle) to Zn²⁺-containing PS is used as a control; consistent results for several higher concentrations of FII are not shown. Global fits of sensograms corresponding to these data are shown in Table 3.

Figure 6.

Binding of TFPI to PS. A) Binding of TFPI to Zn²⁺-containing PS (squares) or to Zn²⁺-deficient commercial PS (circles) immobilized on a plate at 2 μg/ml. Results were confirmed in a separate experiment. B) SPR binding experiments. See Materials and Methods for details. Peak binding of 2 different concentrations of TFPI to Zn²⁺-containing PS and 3 different concentrations of TFPI to commercial Zn²⁺-deficient PS. Data for the 2 PS were indistinguishable and were combined in one graph. Global fit of sensograms corresponding to these data is shown in Table 3.

Figure 7.

Antibodies against PS residues 621–635 (PSP14) preferentially recognize PS that contains Zn²⁺. Several PS preparations were coated to the wells of a microtiter plate at the variable concentrations indicated on the x axis. Immunoaffinity-purified antibodies against PSP14 were incubated in the wells, and bound antibodies were detected with biotinylated secondary antibodies as described in Materials and Methods. ERL PS, untreated Zn²⁺-deficient commercial PS; urea/Zn²⁺ ERL PS, same PS after treatment with Zn²⁺ in urea as in Fig. 3; IAP PS, Zn²⁺-containing immunoaffinity-purified PS.

Figure 8.

Zn²⁺ content and PS-direct do not correlate with multimeric forms of PS. Native-PAGE was used to examine monomeric and multimeric forms of PS. Commercial PS that was >95% monomeric and Zn²⁺-deficient was untreated (lanes 2, 6); activated with Zn²⁺ in 6 M urea (lane 1) or with Zn²⁺ at pH 2.7 (lanes 4, 8); or taken through dialysis steps without Zn²⁺ treatment (lanes 3, 7, from 2 different experiments). PS partially purified by conventional chromatography steps not including MonoQ contained Zn²⁺ and had PS-direct (lane 5). Monomers (M), dimers (D), and trimers (T) indicated at right . Zn (+), Zn²⁺ content ≥ 0.7 atoms/mol PS; PS-direct (+), PS-direct ≥ 2.6 (IC₅₀≤0.39 μM). Two experiments from different days are shown.