Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z

Abstract

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.

FIGURE 1.

SDS-PAGE analysis of PZ derivatives under non-reducing conditions. Lane 1, plasma-derived PZ (pPZ); lane 2, recombinant PZ (rPZ); lane 3, PZ-fX/LC; lane 4, GD-PZ; lane 6, molecular mass standards in kDa.

FIGURE 2.

Dependence of k_obs values on the concentration of PZ derivatives in ZPI inhibition of fXa and the competitive effect of GD-PZ on fXa inhibition by the PZ-ZPI complex. A, the k_obs values for the ZPI (5 nm) inhibition of fXa (0.5 nm) in the presence of increasing concentrations of pPZ (○), rPZ (•), and PZ-fX/LC chimera (□) were determined on PC/PS vesicles (20 μm) in TBS/Ca²⁺ at room temperature by an amidolytic activity assay described under “Experimental Procedures.” The non-linear regression analysis of data yielded K_d(app) values of 1.1 ± 0.1 nm for pPZ, 1.5 ± 0.2 nm for rPZ, and 8.7 ± 0.9 nm for PZ-fX/LC. B, the competitive effect of GD-PZ on inhibition of fXa (0.5 nm) was monitored in the presence of ZPI (5 nm) and pPZ (10 nm) on PC/PS vesicles (20 μm) in TBS/Ca²⁺ at room temperature by an amidolytic activity assay. The non-linear regression analysis of data yielded K_d(app) values of 29 ± 5 nm for the GD-PZ inhibition of the PZ cofactor activity. Data are derived from averages of 2–3 independent measurements from single batches of protein preparations.

FIGURE 3.

The binding of PZ derivatives to ZPI. An ELISA-based binding assay using a polyclonal anti-ZPI (2 μg/ml) as the capture antibody was used to evaluate the affinity of PZ derivatives for interaction with the serpin as described under “Experimental Procedures.” The symbols are: pPZ (○), rPZ (•), PZ-fX/LC chimera (□), and GD-PZ (▪). The non-linear regression analysis of data according to a hyperbolic equation yielded K_D values of 7.5 ± 0.5 nm for pPZ, 7.1 ± 0.1 nm for rPZ, 8.4 ± 0.1 nm for PZ-fX/LC, and 7.1 ± 0.2 nm for GD-PZ. Data are derived from averages of at least 2–3 independent measurements.

FIGURE 4.

Concentration dependence of the ZPI inhibition of fXa in the presence of PZ derivatives on PC/PS vesicles. The k_obs values for the ZPI inhibition of fXa (0.5 nm) in the presence of PZ derivatives: pPZ (○), rPZ (•), and PZ-fX/LC chimera (□) (100 nm each) were determined on PC/PS vesicles (20 μm) in TBS/Ca²⁺ at room temperature by an amidolytic activity assay described under “Experimental Procedures.” The k₂ values were calculated from the slopes of the straight lines and presented in Table 1. Data are derived from averages of at least 2–3 independent measurements.