Effect of variations in the conserved residues E371 and S359 on the structural dynamics of protein Z dependent protease inhibitor (ZPI): a molecular dynamic simulation study

Characterization of the protein Z–dependent protease inhibitor

Dear Sirs, Protein Z (PZ) is a vitamin K-dependent, plasma protein that serves as a cofactor for the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI) in the presence of calcium ions and phospholipids. ZPI is a member of the serpin superfamily of proteinase inhibitors and not only inhibits factor Xa in a PZ-dependent fashion, but also inhibits factor XIa in the absence of cofactors. Both PZ and ZPI are thought to be produced predominantly by the liver (1). To assess other potential sources of PZ besides the liver, tissue northern analysis was performed and showed substantial PZ mRNA expression (~1.4 kb) in the kidney (▶Fig. 1A). Subsequent immunohistochemistry studies using two monoclonal anti-PZ antibodies (Mab 2048, Mab 2306.BF12.2) (2) demonstrated PZ staining in the renal distal and collecting tubules based on tubular morphology and location in paraffin and frozen sections of the renal cortex (not shown) and the renal medulla (Fig. 1C). ZPI was also detected in kidney tubules by immunohistochemical analysis using two monoclonal antibodies (Mab 4242.2, Mab 4336.1E5) (3) in the same location as PZ (Fig. 1D). As Northern analysis has shown little if any ZPI expression in the kidney, compared to that in the liver (4), it is conceivable that the ZPI detected by immunohistochemistry was produced elsewhere and then bound to tubular PZ (2). Notably, PZ and ZPI immune-reactivity was not seen in glomeruli or other renal vascular structures. PZ and ZPI expression was also detected in the liver by immunohistochemistry, but not in lung, heart, spleen, or vasculature (not shown). Figure 1 PZ and ZPI in human kidney tubules Madin-Darby canine kidney cells (MDCK, CCL34, ATCC, Manassas, VA, USA) are derived from canine distal tubule cells, have retained a polarised phenotype, and have been used extensively to examine the sorting of proteins and other molecules. Studies of MDCK cells stably expressing recombinant human PZ, ZPI and both PZ and ZPI cultured on Transwell filter units (0.4 μm, Fisher Scientific, Pittsburgh, PA, USA) (5) demonstrated that these proteins were secreted apically, i.e. toward the tubular lumen (not shown). Random urine testing of six laboratory personnel showed the presence of PZ (61.3 ± 9.3 ng/ml; 2.4 ± 0.4% of the plasma level), but no ZPI by monoclonal sandwich immunoassays (2, 3). Western blotting using rabbit polyclonal antibodies of concentrated (YM 10, Millipore, Billerica, MA, USA) urine specimens demonstrated apparent full-length PZ (62,000 molecular weight [MW]), with little evidence of proteolytic degradation and a trace of ZPI reactivity at 72,000 and 50,000 MW. These results suggest that at least some of the PZ expressed by tubular cells is secreted into the urine, whereas the bulk of ZPI is either not secreted into the urine or is degraded to forms of low molecular weight. Glomerular filtration appears less likely as a source of urinary PZ, since PZ and ZPI circulate as a relatively high-molecular-weight complex in plasma (2). Prothrombin, factor XI and prekallikrein are also produced by kidney tubules (6–10) and the F1 fragment of prothrombin (PTF1) and PZ (11, 12) have been detected in kidney stones. Northern analysis of mouse tissues demonstrates kidney expression of PZ (not shown) and mouse urine (n=5) contains PZ at 1.9 ± 0.6% the PZ level of mouse plasma. Therefore the mouse will likely provide an appropriate model to investigate the physiologic relevance of urinary PZ.

Structural basis for catalytic activation of protein Z–dependent protease inhibitor (ZPI) by protein Z

Characterization of the protein Z–dependent protease inhibitor

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Protein Z Dependent Protease Inhibitor Inhibition of Factor XIa Is Accelerated by Unfractionated Heparin in the Presence and Absence of Protein Z: Further Evidence of the Potential Significance of XIa Inhibitory Activity.

Antithrombin (AT) and protein Z‐dependent protease inhibitor (ZPI) are two serpin inhibitors in plasma that contribute to regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has a narrower specificity, thus being capable of inhibiting only factors Xa (fXa) and XIa. To investigate the contribution of residues of the reactive center loop (RCL) to restricting the specificity of ZPI, we engineered two AT mutants in which the P1‐Arg of the RCL was replaced with P1‐Tyr of ZPI (AT‐R393Y) in one construct and the P12‐P3’ residues were replaced with the corresponding residues of ZPI in the other. The mutants were characterized with respect to their ability to react with coagulation proteases, chymotrypsin and a fXa mutant containing a Ser substitution for Asp‐189 at its primary specificity pocket (D189S). The reactivity of AT‐R393Y with all coagulation proteases including fXa was dramatically impaired, however, the serpin mutant inhibited chymotrypsin with >four orders of magnitude higher specificity. On the other hand, the P12‐P3’ mutant of AT did not exhibit improved reactivity with chymotrypsin, but reacted with a normal reactivity with fXa, though the reaction was not enhanced by heparin cofactors. The fXa‐D189S mutant exibited similar poor reactivity with both AT and AT‐R393Y, however, the protease mutant exhibited normal or improved reactivity with ZPI and AT‐P12‐P3’. These results suggest that P1‐Tyr as well as other unique structural features within the ZPI RCL are responsible for restricting the protease specificity of the serpin.

Expression of Protein Z (PZ) and Protein Z-Dependent Protease Inhibitor (ZPI) In Situ in Human Malignant Tissues.

Engineering a protein Z‐dependent protease inhibitor (ZPI) mutant as a novel antagonist of ZPI anticoagulant function for hemophilia treatment

Serpins, characterized by a conserved structural fold, serve diverse biological roles. Protein Z-dependent protease inhibitor (ZPI), a serpin superfamily member, acts as an endogenous anticoagulant by inhibiting clotting factors Xa (fXa) and XIa (fXIa). Beyond anticoagulation, ZPI has roles in inflammation, cancer, and immune regulation. However, its exact pathophysiological role is yet to be fully characterized. To elucidate ZPI's evolutionary trajectory and non-haemostatic roles, we conducted a comprehensive phylogenetic analysis integrating sequence, gene structure, and synteny data. We identified a lamprey-specific serpin, ZPIL_AGTL_PMA, containing both an inhibitory reactive center loop (RCL) and an angiotensin II (Ang II) motif. This finding suggests that ZPIL_AGTL_PMA represents an ancestral bifunctional serpin from which ZPI and angiotensinogen (AGT), a non-inhibitory serpin involved in blood pressure regulation, evolved by sub-functionalization in jawed vertebrates. This bifunctionality within a single gene in lamprey likely reflects an ancestral vertebrate trait. Gene cluster analyses showed serpinA10 (ZPI) as possibly the earliest member, with other Clade A serpins arising via subsequent duplication. The chromosomal location of this gene cluster is conserved in most vertebrates, except Carnivores and Suidea. Sequence analysis indicated potential non-inhibitory ZPI variants in certain species with atypical non-serine residues at the P1' position within its RCL, a critical determinant of inhibitory serpin activity. The close evolutionary relationship between ZPI and AGT further suggests mechanistic interplay between coagulation and blood pressure regulation, highlighting shared regulatory pathways involving these serpins. Together, these findings expand the functional landscape of ZPI and underscore the dynamic evolution of serpin-mediated physiological processes.

Antithrombin (AT) and protein Z-dependent protease inhibitor (ZPI) are among two physiological serpin inhibitors in plasma that are involved in the regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has narrower protease specificity, inhibiting only factors Xa (fXa) and XIa. Unlike an Arg at the P1 site of the AT reactive center loop (RCL), this residue is a Tyr in ZPI. To investigate the contribution of P1 Tyr in restricting the specificity of ZPI, we engineered an AT mutant in which the P1 Arg of the RCL was replaced with the P1 Tyr of ZPI (AT-R393Y). The reactivity of AT-R393Y with fXa and thrombin was decreased 155- and 970-fold, respectively. However, the serpin mutant inhibited chymotrypsin with an efficiency higher by >4 orders of magnitude. By contrast, chymotrypsin did not exhibit any reactivity with ZPI. The substitution of Asp-189 of fXa with the corresponding residue of chymotrypsin (Ser) did not improve the reactivity of the protease mutant with AT-R393Y; however, the fXa mutant reacted normally with ZPI. These results suggest that the contribution of P1 Tyr to restricting the protease specificity of ZPI is RCL context-dependent and that in addition to P1 Tyr, other structural features within and/or outside the ZPI RCL are involved in determining the protease specificity of the serpin. The results further suggest that thrombin is less tolerant than fXa in accommodating the nonoptimal P1 Tyr of the AT mutant in its active-site pocket.

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nM KD and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2-7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action.

The pathological consequences of decreased protein Z (PZ) and/or Z-dependent protease inhibitor (ZPI) levels remain as yet unclear, despite a growing body of evidence which supports their involvement in an increased thrombotic risk. The purpose of the present study was 2-fold: to evaluate plasma concentrations of protein Z and ZPI in patients with essential thrombocythemia (ET) and to determine their significance in thrombotic complications. The median (range) plasma concentrations of PZ in our patients with ET were lower, but not significantly, than in healthy individuals: PZ (1.42 µg/mL, 0.36-3.14 µg/mL vs 1.6 µg/mL, 0.75-2.56 µg/mL, P = .08). On the other hand, the median (range) plasma concentrations of ZPI in the said patients with ET were meaningfully lower than in the reference group: ZPI (3.22 µg/mL, 0.85-6.97 µg/mL vs 4.41 µg/mL, 1.63-7.83 µg/mL, P = .0004). More importantly, the study revealed a statistically significant lower concentration of PZ and ZPI in patients with the presence of the JAK2V617F mutation relative to patients without the mutation, for PZ: 1.38 µg/mL, 0.36-2.6 µg/mL versus 1.63 µg/mL, 0.88-3.14 µg/mL, P = .03, and ZPI 2.89 µg/mL, 0.85-5.91 µg/mL versus 3.61 µg/mL, 1.53-6.97 µg/mL, P = .002. Additionally, significant differences between the concentrations of PZ and ZPI were found in patients with venous thrombotic episodes compared to healthy individuals, for PZ: 1.23 µg/mL, 0.82-1.99 µg/mL versus 1.6 µg/mL, 0.75-2.56 µg/mL, P = .043, and ZPI: 2.42 µg/mL, 0.85-4.21 µg/mL versus 4.41 µg/mL, 1.63-7.83 µg/mL, P < .0001. To recapitulate, our results suggest that the deficiency of PZ may increase tendency to thrombosis in patients with ET.

The protein ZPI (Z-dependent protease inhibitor) binds to PZ (protein Z), which enables ZPI to inhibit membrane-bound FXa (activated factor X). ZPI also inhibits FXIa (activated factor XI) independently of PZ. To study the PZ-independent ZPI function, we tested the in vitro and in vivo effect of disrupting the ZPI-PZ interaction by mutating ZPI Asp 293 to Ala (D293A). D293A mutation reduced PZ-dependent FXa inhibition without affecting FXIa inhibition. D293A also diminished FXIIa (activated FXII)-induced thrombin generation but reduced TF (tissue factor)-induced thrombin generation only at low TF concentrations. This suggests that D293A selectively inhibits the intrinsic pathway and the thrombin-FXI (factor XI) feedback loop that enhances low-dose TF-initiated coagulation. Wild-type and D293A ZPI both showed selectivity in inhibiting activated partial thromboplastin time but not prothrombin time. Increasing PZ in plasma enhances activated partial thromboplastin time inhibition and enables prothrombin time inhibition by wild-type but not D293A ZPI, further indicating that D293A ZPI selectively inhibits the intrinsic pathway independently of PZ. In mouse models, D293A inhibited FeCl3-induced occlusive carotid artery thrombosis and venous thrombosis in the inferior vena cava. Thus, PZ-independent ZPI function plays a major role in ZPI inhibition of occlusive thrombosis, and D293A ZPI is an effective antithrombotic. Importantly, administering D293A ZPI did not affect tail bleeding time and showed improved hemostasis in a saphenous vein hemostasis model as compared with wild-type ZPI. The PZ-binding defective variant of ZPI, D293A, selectively inhibits the intrinsic coagulation pathway and is a new anticoagulant with reduced bleeding risk.

Plasma concentration of protein Z and protein Z-dependent protease inhibitor in patients with haemophilia A