Z-dependent Protease Inhibitor Research Articles

An inhibitory single-domain antibody against protein Z-dependent protease inhibitor promotes thrombin generation in hemophilia A and FXI deficiency.

Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia. To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity. We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay (TGA) in normal plasma, factor VIII (FVIII)- and FXI-deficient plasma. Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency. ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders.

Protein Z and Protein Z-dependent protease inhibitor in patients with acute ischemic stroke: A prospective mechanistic study

ObjectivesProtein Z (PZ) /Protein Z-dependent protease inhibitor (ZPI) (PZ/ZPI) system is a new anticoagulant system discovered in recent years, which plays an important role in many diseases. We aimed to compare the plasma PZ/ZPI levels of acute ischemic stroke (AIS) patients and non-stroke control participants and the role of PZ/ZPI in the development of stroke was preliminarily analyzed. Materials and methodsEnzyme linked immunosorbent assay (ELISA) was used to detect and compare plasma PZ levels of 86 patients with acute AIS and 85 non-stroke control patients. Multivariable Logistic regression was used to analyze whether PZ was an independent risk factor for AIS. ResultsIn the present study, plasma PZ is closely related to inflammatory response, coagulation process and platelet activation, and may participate in the development of AIS by inducing inflammatory responses and interfering with the coagulation process. ConclusionsOur results suggested that plasma PZ level is one of the independent risk factors of AIS, and plasma ZPI was closely related to coagulation and platelet parameter and may play a role in the coagulation process during AIS.

ZPI prevents ox‐LDL‐mediated endothelial injury leading to inhibition of EndMT, inflammation, apoptosis, and oxidative stress through activating Pi3k/Akt signal pathway

Oxidized low-density lipoprotein(ox-LDL)-mediated endothelial dysfunction exerts an essential role in the development of atherosclerosis. Protein Z-dependent protease inhibitor (ZPI), a member of the serine protease inhibitor superfamily, could inhibit the function of activated coagulation factor X (FXa) via interaction with protein Z (PZ). Studies have pointed out that ZPI was statistically related to atherosclerotic diseases, which may have a robust cardiovascular protective effect. However, the underlying mechanism of ZPI on ox-LDL-mediated endothelial injury requires further elucidation. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL (100 μg/ml) and ZPI (10 μg/ml). Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, oxidative stress, and endothelial-to-mesenchymal transition(EndMT) were analyzed by immunofluorescence(IF). Cell migration was measured using a wound-healing assay. Quantitativereal-time polymerase chain reactionand westernblot analysis were performed to determine messenger RNA and protein expression.Ox-LDL (100 μg/ml, 48 h) significantly reduced cell viability and migration, increased EndMT, inflammation, apoptosis, and oxidative stress. The related protein expression of phosphatidylinositol 3 kinase/protein kinase B (Pi3k/Akt) signal pathway in HUVECs was also simultaneously decreased. We also discovered that ZPI treatment could prevent ox-LDL-mediated endothelial injury through the improvement of cell viability and alleviation of apoptosis, oxidative stress, EndMT, and inflammation. Thus, the protective effect of ZPI on HUVECs may be mediated by activation of the Pi3k/Akt signal pathway.ZPI may exert an important protective role in HUVECs dysfunction triggered by ox-LDL via activation of the Pi3k/Akt signal pathway. Therefore, ZPI may possess potential therapeutic effects on atherosclerotic endothelial injury-related diseases.

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site

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.

Ozone exposure and prothrombosis: Mechanistic insights from a randomized controlled exposure trial

Epidemiological studies have associated ozone exposure with cardiovascular diseases, but the molecular mechanisms were not elucidated. We performed an untargeted serum proteomic analysis in a randomized, crossover, controlled exposure trial. We recruited 32 healthy young adults and asked them to receive filtered air and 200-ppb ozone exposures for 2 h in a random order before serum collection. Linear mixed-effect models were used to identify differentially expressed proteins (DEPs) between the two exposures and Gene Ontology enrichment and ingenuity pathway analysis were performed to determine their biological function. A total of 56 DEPs were identified. For example, acute ozone exposure increased coagulation factor X and factor VII-activating protease by 20.96% and 28.35%, respectively. Whereas, protein Z, protein Z-dependent protease inhibitor, and plasminogen decreased by 13.62%, 33.54%, and 10.47%, respectively. We also observed a 42.32% decrease in paraoxonase 3 and evident changes in four apolipoproteins. Additionally, we found 18.21% and 95.82% increases in L-selectin and β2-microglobulin, respectively, and significant changes in three complements. DEPs and enriched pathways suggest that short-term ozone exposure may promote coagulation, suppress fibrinolysis, disrupt lipoprotein metabolism, activate immune responses, and affect the complement system. These findings provide additional insights into the mechanisms linking acute ozone exposure to thrombosis.

Expression and diagnostic value of plasma protein Z and protein Z-dependent protease inhibitor in fetal growth restriction.

Investigating the expression levels of plasma protein Z (PZ) and protein Z-dependent protease inhibitor (ZPI) in fetal growth restriction (FGR) and to explore their diagnostic value in FGR. In this study, the number of pregnant women with FGR, healthy pregnant women (Healthy Control, HC), and childbearing-age women without pregnancy (Blank Control, BC) is 79, 79, and 60, respectively; their plasma PZ and ZPI levels in each group are determined by ELISAs. Then, the correlations between these indices and FGR were assessed using Spearman analysis. Moreover, these indices' diagnostic values for FGR are evaluated using the receiver operating characteristics (ROC) curves. The plasma levels of PZ and ZPI are significantly decreased in the HC and FGR groups compared against the BC group (P < 0.001), whilst the levels of PZ and ZPI in the FGR groups are lower than those in the HC group (P < 0.01) notably. PZ plasma concentration has positive relationship with ZPI concentrations in the HC and FGR groups. The combination of PZ and ZPI, with the Area under the Curve (AUC) 0.92 (95% CI = 0.88-0.96), the sensitivity 0.82, and the specificity 0.88, outperforms everyone. Plasma PZ and ZPI are significantly decreased in pregnant women with FGR, which can be used for pregnant women's FGR screening.

The Proteolytic Inactivation of Protein Z-Dependent Protease Inhibitor by Neutrophil Elastase Might Promote the Procoagulant Activity of Neutrophil Extracellular Traps in Sepsis.

Septic shock is the archetypal clinical setting in which extensive crosstalk between inflammation and coagulation dysregulates the latter. The main anticoagulant systems are systematically impaired, depleted, and/or downregulated. Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that not only targets coagulation factors Xa and XIa but also acts as an acute phase reactant whose plasma concentration rises in inflammatory settings. The objective of the present study was to assess the plasma ZPI antigen level in a cohort of patients suffering from septic shock with or without overt-disseminated intravascular coagulation (DIC). The plasma ZPI antigen level was approximately 2.5-fold higher in the patient group (n = 100; 38 with DIC and 62 without) than in healthy controls (n = 31). The elevation's magnitude did not appear to depend on the presence/absence of DIC. Furthermore, Western blots revealed the presence of cleaved ZPI in plasma from patients with severe sepsis, independently of the DIC status. In vitro, ZPI was proteolytically inactivated by purified neutrophil elastase (NE) and by NE on the surface of neutrophil extracellular traps (NETs). The electrophoretic pattern of ZPI after NE-catalyzed proteolysis was very similar to that resulting from the clotting process-suggesting that the cleaved ZPI observed in severe sepsis plasma is devoid of anticoagulant activity. Taken as a whole, our results (1) suggest that NE is involved in ZPI inactivation during sepsis, and (2) reveal a novel putative mechanism for the procoagulant activity of NETs in immunothrombosis.

Anti-inflammatory Activity of the Protein Z-Dependent Protease Inhibitor.

The protein Z (PZ)-dependent plasma protease inhibitor (ZPI) is a glycoprotein that inhibits factor XIa and, in the presence of PZ, FXa. Recently, ZPI has been shown to be an acute-phase protein (APP). As usually APPs downregulate the harmful effects of inflammation, we tested whether ZPI could modulate the increase of cytokines observed in inflammatory states. We observed that recombinant human ZPI (rhZPI) significantly decreases the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor- α (TNF-α) induced by lipopolysaccharide (LPS) in a whole blood model. This inhibitory effect was unaffected by the presence of PZ or heparin. A ZPI mutant within the reactive loop center ZPI (Y387A), lacking anticoagulant activity, still had an anti-inflammatory activity. Surprisingly, rhZPI did not inhibit the synthesis of IL-6 or TNF-α when purified monocytes were stimulated by LPS, whereas the inhibitory effect was evidenced when lymphocytes were added to monocytes. The requirement of lymphocytes could be due to the synthesis of CCL5 (RANTES), a chemokine mainly produced by activated lymphocytes which is induced by rhZPI, and which can reduce the production of proinflammatory cytokines in whole blood. Lastly, we observed that the intraperitoneal injection of rhZPI significantly decreased LPS-induced IL-6 and TNF-α production in mouse plasma.

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

Protein Z (PZ) dependent protease inhibitor (ZPI) is a natural anticoagulant inhibiting blood coagulation proteases fXa and fXIa. Despite being a member of the serpin superfamily, it possesses unique structural features such as activation by PZ, regulating its inhibitory function. In order to understand the Reactive Centre Loop (RCL) dynamics of ZPI, which is absolutely critical for its activity, we performed Molecular Dynamics (MD) simulation on ZPI and its E371 and S359 variants located at important conserved functional sites. Unexpectedly, the RCL of E371 variants, (E371K, E371R, and E371Q), were shown to be very stable due to compensatory interactions at the proximal end of RCL. Interestingly, RCL flexibility was shown to be enhanced in the double mutant K318E-E371K due to the repulsive effect of increased negative charge on top of the breach region. Principal component analysis (PCA) coupled with residue wise interaction network analysis(RIN) revealed correlated motion between the RCL and the PZ binding regions in the WT. However, a loss of regulation in correlated motion between RCL and PZ binding hotspot Tyr240 in the double mutant was also observed. Additionally, the S359F and S359I mutations resulted in increased RCL flexibility owing to the disruption of stabilizing hydrogen bonding interaction at the distal end of strand S5A. Thus, the current study proposes that the overall stabilizing interactions of S5A is a major regulator of proper loop movement of ZPI for its activity. The results would be beneficial to engineer activity compromised ZPI as a prophylactic agent for the treatment of hemophilia. Communicated by Ramaswamy H. Sarma

Plasma concentration of protein Z-dependent protease inhibitor and ZPI exon 3 mutations in preeclampsia

Protein Z-dependent protease inhibitor (ZPI) serves as a cofactor of inhibition of FXa and FXIa by protein Z. The levels of protein Z and polymorphisms have been shown in preeclampsia (PE) patients, but the plasma levels of ZPI and ZPI gene mutations were not reported yet. The principal aim of this study was to identify the concentration of ZPI and gene polymorphism in PE. ZPI levels were determined in 113 PE patients (age: 29.9 ± 3.9 years) and in 106 controls (normal pregnancy, age: 27.0 ± 2.8 years). ZPI was measured by enzyme-linked immunosorbent assay kit, and the gene polymorphism was determined by polymerase chain reaction and sequencing. The results showed ZPI antigen was found to be significantly lower in PE patients than in controls (ZPI, 1.24 ± 0.29 mg/L vs. 1.94 ± 0.35 mg/L, p < 0.05). The exon-3 missense mutations were distributed in patients and controls and there was no convincing correlation between these mutations and PE. It was of interest to observe a close relationship between the genotypes of the exon 3 polymorphisms 181 A > G and 481 A > T in the ZPI gene. Impact statement What is already known on this subject? The occurrence of PE is closely related to dysfunction of coagulation, and it is known that the decrease of PZ level can increase the occurrence probability of PE, while the polymorphism of PZ is not related to the occurrence of PE. As a cofactor of PZ, the content and polymorphism of ZPI which related to the occurrence of PE is worth further study. What the results of this study add? ZPI antigen was found to be significantly lower in PE patients than in controls, but there was no convincing correlation between exon-3 mutations and PE. What the implications are of these findings for clinical practice and/or further research? Our results support the view that ZPI plays a significant role in anticoagulant, and the genotype of the 181 gene polymorphism in exon-3 and 481 gene polymorphism in exon-3 are closely related. Other mutations like 435T > G(Phe145Leu), 972G > A(Trp324X), 1151A > G(Gln384Arg) are necessary to confirm the association between ZPI and prothrombotic state including PE.

Serpins, New Therapeutic Targets for Hemophilia.

Hemostasis is a tightly regulated process characterized by a finely tuned balance between procoagulant and anticoagulant systems. Among inherited hemostatic conditions, hemophilia is one of the most well-known bleeding disorders. Hemophilia A (HA) and B (HB) are due to deficiencies in coagulation factor VIII (FVIII) or FIX, respectively, leading to unwanted bleeding. Until recently, hemophilia treatment has consisted of prophylactic replacement therapy using plasma-derived or recombinant FVIII in cases of HA or FIX in cases of HB. Because FVIII and FIX deficiencies lead to an imbalance between procoagulant and anticoagulant systems, a recent upcoming strategy implies blocking of endogenous anticoagulant proteins to compensate for the procoagulant factor deficit, thus restoring hemostatic equilibrium. Important physiological proteins of the anticoagulant pathways belong to the serpin (serine protease inhibitor) family and, recently, different experimental and clinical studies have demonstrated that targeting natural serpins could decrease bleeding in hemophilia. Here, we aim to review the different, recent studies demonstrating that blocking serpins such as antithrombin, protein Z-dependent protease inhibitor, and protease nexin-1 or modifying a serpin like α1-antitrypsin could rebalance coagulation in hemophilia. Furthermore, we underline the potential therapeutic use of serpins for the treatment of hemophilia.

Plasma Concentrations of Protein Z and Protein Z-Dependent Protease Inhibitor in Thalassemia Major Patients

Thalassemia is the most common congenital hemolytic disorder by a partial or complete deficiency in globin chain synthesis. Recent investigations have suggested a correlation between protein Z (PZ) and protein Z-dependent protease inhibitor (ZPI) deficiency and thrombosis. So, the aim of this study was to evaluate the PZ and ZPI levels in beta (β)-thalassemia patients. In this study, 40 patients with thalassemia major and 40 control subjects were selected. PZ and ZPI serum levels were measured by sandwich ELISA technique. PZ and ZPI were significantly higher in thalassemic patient group than control group. Also, PZ levels and PLT and Hb counts were significantly higher in splenectomized thalassemic patients than non-splenectomized ones. The exact mechanism of increased PZ and ZPI have an impact on hypercoagulable state in β-thalassemia major could not be found in our study. More studies are still needed to understand these challenges in β-thalassemia major patients.

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

BackgroundProtein Z‐dependent protease inhibitor (ZPI), is an important anticoagulant protein in plasma that functions in complex with its cofactor, protein Z (PZ) to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. Recent studies suggest that the ZPI‐PZ anticoagulant complex is a promising target for restoring hemostasis in hemophilia (Girard, et al, J Thromb Haemost, 2019, 17, 149‐156). ObjectiveEngineering a ZPI mutant as a novel antagonist of ZPI anticoagulant function. MethodsWe engineered two alanine mutations in human ZPI, one in the reactive loop P1 Y387 residue to inactivate the FXa/FXIa inhibitory function, and the second in the K239 binding interface residue to enhance the affinity of the inactive ZPI for PZ. The mutant was expressed, purified, and characterized by in vitro and plasma assays. ResultsThe mutant, Y387A/K239A (ZPI‐2A), bound PZ >20‐fold tighter than WT ZPI or a PZ antibody (PZAb). FXa inhibition assays showed that ZPI‐2A effectively neutralized ZPI/PZ anti‐FXa activity with a ~three‐fold molar excess over wild type ZPI (WT ZPI) whether FXa was bound to FVa in prothrombinase or unbound. Thrombin generation assays in a purified system or in normal/hemophilia plasmas showed that ZPI/PZ activity was reversed by ZPI‐2A in a dose‐dependent manner, with a three‐fold molar excess sufficient to fully reverse ZPI/PZ inhibition of thrombin generation. ConclusionsZPI‐2A is a potent antagonist of ZPI/PZ anticoagulant function, capable of fully blocking the anti‐FXa activity of plasma levels of ZPI/PZ at significantly lower doses than a PZAb and thus a promising prophylactic agent for treating hemophilia.

Protein Z-dependent protease inhibitor (ZPI) is a physiologically significant inhibitor of prothrombinase function

Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.

Co-localization of Coagulation Factor X and its Inhibitory System, PZ/ZPI, in Human Endometrial Cancer Tissue.

Hemostatic system components contribute to cancer progression independently from their roles in hemostasis. It has been shown that protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) inhibit coagulation factor X (FX). The aim of the study was to analyze the expression of PZ/ZPI in relation to the main coagulation factor - FX in human endometrial cancer tissue. Immunohistochemical analysis was performed on 21 endometrial cancer specimens employing antibodies against ZPI, PZ and FX. Endometrial cancer cells showed a strong expression of ZPI and PZ and medium expression of FX. Normal endometrial tissue showed no expression of ZPI, PZ or FX. Strong expression of PZ and ZPI in endometrial cancer cells suggests a role of these proteins in endometrial cancer.

22 - Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI)

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membranebound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both Phosphatidylserine and polyunsaturated fatty acids were required to promoteinactivation of theZPI-PZcomplexor free ZPI, indicating that binding of thePZ-complexedor freeZPIto peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

High frequency of anti-protein Z antibodies in patients with anticardiolipin antibodies

Protein Z (PZ) is a vitamin K-dependent protein involved in the down-regulation of coagulation by forming a complex with the protein Z-dependent protease inhibitor. The complex inhibits the activated factor X on phospholipid surface. Presence of anti-PZ (aPZ) antibodies was first described in women with pathological pregnancies but the significance of aPZ antibodies in other pathological situations was poorly studied. In this work we analyzed the frequency of aPZ antibodies in a series of 86 consecutive patients with anticardiolipin (aCL) antibodies and studied the association of aPZ with other antiphospholipid (aPL) antibodies [lupus anticoagulant (LAC) and anti-ß2GP-1 antibodies] and the clinical signification of these aPZ antibodies in term of thrombosis or fetal loss. Anti-PZ antibodies (IgG and IgM) were detected using commercially available ELISA assays. The frequency of aPZ antibodies was 40.7% in the patient group versus 6.8% in a group of 59 healthy volunteers (p < 0.0001). The frequency of aPZ antibodies significantly increases (p < 0.05) in patients with a double or triple positivity of aPL antibodies and a higher frequency of aPZ antibodies was observed in patients with LAC (57.7%) than in patients without LAC (25.6%, p = 0.02). There were no significant differences in aPZ antibodies frequency between patients with and without thrombotic events. Interestingly, among the 8 women with recurrent foetal losses, aPZ antibodies were observed in 7 cases, in agreement with previous observations suggesting that aPZ antibodies may be associated with obstetrical complications.

Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI)

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membrane-bound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both phosphatidylserine and polyunsaturated fatty acids were required to promote inactivation of the ZPI-PZ complex or free ZPI, indicating that binding of the PZ-complexed or free ZPI to peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

Assessment of the Procoagulant Activity of Microparticles and the Protein Z System in Patients Undergoing Off-Pump Coronary Artery Bypass Surgery.

To understand the coagulation changes after off-pump coronary artery bypass (OPCAB) surgery, we evaluated the procoagulant activity of microparticles (MPs) and microparticles exposing tissue factor (MPs-TF), together with the levels of total tissue factor (TF), protein Z (PZ), protein Z-dependent protease inhibitor (ZPI), and factor X (FX) before (first day) and 1 week after surgery (seventh day) in plasma samples from 30 patients. Twenty healthy controls were also included. Compared to the controls, patients scheduled for surgery had significantly higher MPs-TF procoagulant activity and lower TF levels ( P = .0006, P = .02, respectively). In the whole cohort, median procoagulant activity of MPs-TF and median levels of TF and ZPI were significantly lower ( P = .02, P = .0003, and P = .004, respectively), while median levels of PZ and FX were significantly higher ( P = .02 and P = .002, respectively) on the seventh day compared to the first day. Our results suggest that OPCAB surgery has a significant effect on the procoagulant activity of MPs-TF and the PZ system.

Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity.

Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 μM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process.