Protein Z-dependent Protease Inhibitor Research Articles

Polymorphisms of the protein Z-dependent protease inhibitor (ZPI) gene and the risk of venous thromboembolism

Letters to the Editor: Polymorphisms of the protein Z-dependent protease inhibitor (ZPI) gene and the risk of venous thromboembolism -

Human endothelial cells synthesize protein Z, but not the protein Z dependent inhibitor

Protein Z (PZ) is a vitamin K-dependent protein isolated from human plasma, and acts as a cofactor for a serpin, called protein Z-dependent protease inhibitor (ZPI). A prothrombotic phenotype has been reported in PZ deficient mice, and PZ deficiencies have been observed in patients with arterial thrombotic events. PZ was immunologically detected in the endothelium of atherosclerotic arteries, suggesting that endothelial cells could be involved in the production of PZ. In this study we analyzed the synthesis and release of PZ and ZPI by human umbilical vein endothelial cells (HUVEC), representative of the macrovasculature, and by HMEC-1, a microvascular endothelial cell line. PZ was quantified by a specific ELISA in the supernatant and in the lysates of both cellular types. Western blotting of the supernatants showed the presence of a band of 62 kDa, identical to PZ synthesized by the hepatoma cell line HepG2. mRNA of PZ was also detected in each cellular type. PZ biosynthesis was unaffected by inflammatory cytokines in HUVEC, whereas a slight decrease of mRNA and PZ antigen (53.5 +/- 14.5% of protein synthesis as compared to the control, p < 0.01) and a modest increase (126 +/- 8.5% as compared to the control, p < 0.05) were induced respectively byTumor Necrosis Factor (TNF)-alpha (25 ng/ml) and oncostatin M (5 ng/ml) in HMEC-1. Immunological studies showed the presence of PZ near the nucleus and a possible expression of PZ at the membrane. In addition, PZ was present in the endothelial cells of both normal arterial and venous vessel sections. In contrast, neither ZPI nor its mRNA was detected in endothelial cells.

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

The activity of the hemostatic system components facilitates tumor growth and dissemination. There exists a mechanism of direct inhibition of factor Xa that involves protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) system. ZPI also attenuates the activity of factor XIa. Data on the presence of PZ and ZPI in malignant tissue in situ are lacking.The aim of this study was to assess the localization of PZ and ZPI in situ in colon, breast, gastric, laryngeal, pancreatic, renal, endometrial cancer, non-small cell lung cancer (NSCLC), malignant melanoma and glial neoplasms. Studies were performed on tumor tissues obtained at surgical treatment of previously untreated patients. Immunohistochemical procedures according to the ABC method and Envision Double Staining System (Dako) employed a polyclonal antibody against PZ and a monoclonal antibody against ZPI. Protein Z was localized in cancer cell bodies in all types of malignant tumors examined. Staining intensity was more pronounced in less differentiated cancer cells of anaplastic gliomas. In contrast, more differentiated cancer cells of gastric cancer revealed stronger staining than less malignant ones. Cancer cell bodies of NSCLC and renal cancer were characterized by weak intensity of staining for PZ. In pancreatic and renal cancer, as well as in malignant melanoma, the intensity of staining for PZ in cancer cells was irregular: both high and low PZ expression was observed independently of the degree of malignancy. Protein Z was also revealed in association with macrophages in colon cancer, NSCLC and malignant melanoma. Expression of ZPI was observed in cancer cell bodies in all examined specimens. The staining intensity for ZPI was irregular: both strong and weak expression of ZPI was observed. Various percentages of ZPI-positive cancer cells were observed in different specimens of the tissues examined. In colon cancer, ZPI expression was detected in tumor infiltrating macrophages. Double staining studies revealed co-expression of both proteins in cancer cell bodies in all examined tumor types, although there were areas of tumor foci where only one protein (either ZP or ZPI) was localized. Our results suggest that PZ and ZPI, apart from being inhibitors of blood coagulation, may play an additional regulatory role in tumor progression.

Down-regulation of Factor IXa in the Factor Xase Complex by Protein Z-dependent Protease Inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a serpin inhibitor of coagulation factor (F) Xa dependent on protein Z, Ca2+, and phospholipids. In new studies, ZPI inhibited FIXa in the FXase complex. Since this observation could merely represent inhibition of the FXa product whose activity was measured, inhibition of FIXa was investigated five ways. 1) FXase incubation mixtures with/without ZPI/protein Z were diluted in EDTA; FXa activity was measured after reversal of its inhibition. 2) FXase incubation mixtures were immunoblotted for FXa product. 3) FX activation peptide region was 3H-labeled; release of 3H was used to measure FXase activity. 4) Activity was monitored in a FIXa-based clotting assay. 5) FIXa amidolytic activity was measured. In all cases, FIXa was inhibited by subphysiologic levels of ZPI. Unlike inhibition of FXa, inhibition of FIXa did not strictly require protein Z. Low concentrations of FVIIIa increased the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure. Unusual time courses were observed for inhibition of both FIXa in the FXase complex and FXa in the prothrombinase complex. Activity loss stabilized in <100 s at a level dependent on ZPI concentration, suggesting equilibrium interactions rather than typical covalent serpin-protease interactions. Surface plasmon resonance binding experiments revealed binding and dissociation of ZPI/FIXa with Kd (app) of 9-12 nm, similar to the concentration of ZPI needed for 50% inhibition. ZPI may be an unusual physiologic regulator of both the intrinsic FXase and the prothrombinase complexes.

Identification of Factor Xa Residues Critical for Interaction with Protein Z-dependent Protease Inhibitor: BOTH ACTIVE SITE AND EXOSITE INTERACTIONS ARE REQUIRED FOR INHIBITION

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.

Protein Z‐dependent protease inhibitor W303X mutation in venous thrombosis: response to Gonzalez‐Conejero et al

In reply to the study by Gonzalez-Conjero et al, we found their results on 218 Spanish White patients very interesting. As suggested in their article, there does indeed appear to be an ethnic bias and possible founder affect with regard to the W303X mutation in thrombosis patients. The frequency of both the Factor V Leiden mutation and the prothrombin 20210A variant also varies within White populations. The allelic frequencies for the factor V Leiden mutation in White subpopulations ranges from 90% were of northern European origin (McKinnon et al, 1997) with a strong influence from UK immigration. In our study we also identified another stop codon mutation (R67X) in three of our thrombosis patient cohort (n ¼ 250). It is unfortunate that Gonzalez-Conjero et al. did not extend their study to include the R67X mutation within the ZPI gene as well. Further studies of ZPI mutations in thrombosis patient populations are required, especially those in patients of northern European background, to evaluate the role of protein Z-dependent protease inhibitor in haemostasis and thrombosis.

Protein Z and protein Z-dependent protease inhibitor

To assess the potential roles of protein Z (PZ) and protein Z-dependent protease inhibitor (ZPI) in venous thrombosis, their plasma levels were measured in 426 individuals with venous thrombosis and 471 control individuals participating in the Leiden Thrombophilia Study. A relationship between the level of PZ or ZPI and venous thrombosis was not detected in the overall case-control study. PZ and ZPI circulate as a complex and their plasma levels are interdependent. Both PZ and ZPI are increased with oral contraceptive use and reduced with oral anticoagulant therapy.

Expression of Protein Z-Dependent Protease Inhibitor (ZPI) In Situ in Different Malignant Tumors.

Cancer progression is often associated with thromboembolic complications. Coagulation factors and inhibitors influence various processes involved in malignant tumor growth and metastatic dissemination. Recently a new coagulation inhibitor - protein Z-dependent protease inhibitor (ZPI) has been characterized. ZPI is a plasma proteinase inhibitor in the serpin superfamily. ZPI inhibits factor Xa activity in the presence of calcium and phospholipids. Protein Z augments its activity by more than 1000-fold. ZPI also attenuates the activity of factor XIa in the reaction which does not require the presence of calcium and phospholipids. ZPI inhibits the coagulation response prior to the formation of the prothrombinase complex. Up to now the data on the presence of ZPI in malignant tumors are obscure. The purpose of the study was to elucidate the solid phase interactions between various types of maligant tissues and ZPI that may contribute to tumor progression. The tissues from colon, breast, gastric, renal, pancreatic, endometrial carcinoma, non-small cell lung cancer (NSCLC) as well as from glial neoplasms were obtained at surgical resection during radical treatment. The patients undergoing surgery have not received any previous antineoplastic therapy. Tumor fragments were processed acc. to AMeX method and then embedded in paraffin. Immunohistochemical studies (avidin-biotin complex-ABC - technique) were performed using a monoclonal antibody against ZPI. Expression of ZPI was observed in cancer cell bodies of colon, breast, gastric, renal, endometrial, pancreatic cancer, NSCLC and glial neoplasms. The staining intensity for ZPI was irregular: both strong and weak expression of ZPI was observed. Moreover, various percentages of ZPI-positive cancer cells were revealed in different specimens of examined tissues. Macrophages and mucin also revealed expression of ZPI in the case of colon cancer. The presence of ZPI was demonstrated in the stroma of renal cancer. The obtained results suggest that ZPI being an inhibitor of coagulation might additionally play a role in the regulation of cancer progression.

Mutations within the Protein Z-Dependent Protease Inhibitor (ZPI) Gene Are Associated with Venous Thromboembolic Disease: A New Form of Thrombophilia.

ZPI is a recently characterised inhibitory serpin present in plasma. In vitro studies have shown that ZPI inhibits both factor Xa (in the presence of protein Z) and factor XIa. Deficiency of ZPI is predicted to enhance coagulation and may be a risk factor for venous thrombosis. To test this hypothesis we carried out mutation screening within the coding region of the ZPI gene in a cohort of 150 patients with a history of DVT or PE (first event before the age of 60 years) and 150 matched controls. Five PCR products were produced for each subject. Heteroduplex analysis was performed using dHPLC and the PCR products were sequenced directly if a heteroduplex mismatch was identified.Sixteen mutations/polymorphisms were identified within the coding region of the ZPI gene. Two mutations were regarded as functionally significant as they produced stop codons at arginine 67 and tryptophan 303 and would be expected to result in the loss of ZPI activity. These stop codon mutations were found in 8 patients and 2 controls. Based on these results, a power calculation was performed that showed that an additional 100 patients and controls would be necessary to confirm that the stop codon mutations were statistically significant risk factors for thrombosis. Bidirectional allele specific PCR was developed to rapidly identify these stop codon mutations and a further 100 patients and 100 controls were examined using this technique. The stop codon mutations were found in 11 patients (W303X n=8: R67X n=3) and only 2 controls (R67X n=2) (two sided Fishers exact p=0.02) with an odds ratio = 5.7 (95%CI, 1.25 to 26.0). In addition two further non-conservative mutations were identified in two other thrombosis patients. These resulted in a serine to tyrosine (codon 122) and a phenylalanine to leucine (codon 124) change in the region homologous to the D helix of other heparin activated serpins. These changes, involving bulky aromatic amino acid residues, have the potential to disrupt the structure and function of the D helix.We identified a further 12 mutations/polymorphisms (C454G, C574T, A603G, A647G, G752A, A947T, C1276T, G1277A, G1438A, A1617C, G1789T and C1811T (italics: not previously described)). The significance of these mutations is uncertain. We have identified mutations causing stop codons, which will result in loss of ZPI function, in 4.4% of patients who present with venous thrombosis before the age of 60 years, compared with an incidence of only 0.8% in controls. Further studies are ongoing to measure the plasma concentration of ZPI in the affected individuals. Our results support an association between mutations in the ZPI gene and venous thrombosis. We propose that ZPI deficiency as a result of these mutations is potentially a new form of thrombophilia.

Mutations within the protein Z-dependent protease inhibitor gene are associated with venous thromboembolic disease: a new form of thrombophilia.

Protein Z-dependent protease inhibitor (ZPI) is a serpin that inhibits the activated coagulation factors X and XI. The precise physiological significance of ZPI in the control of haemostasis is unknown although a deficiency of ZPI may be predicted to alter this balance. The coding region of the ZPI gene was screened for mutations using denaturing high-performance liquid chromatography. 16 mutations/polymorphisms within the coding region of ZPI were identified including two mutations, which generated stop codons at residues R67 and W303. We observed nonsense mutations within the ZPI gene in 4.4% of thrombosis patients (n = 250) compared with 0.8% of controls (n = 250). The difference in distribution of stop codon mutations between thrombosis patients and controls was significant (P = 0.02) with an odds ratio of 5.7 (95% confidence interval, 1.25-26.0). Our results suggest an association between ZPI deficiency and venous thrombosis and we propose that ZPI deficiency is potentially a new form of thrombophilia.

La protéine Z : une protéine régulatrice de la coagulation au niveau artériel ?

Protein Z (PZ) is a vitamin K dependent factor identified in human plasma in 1984 whose physiological function was poorly understood. It was recently shown that protein Z is implicated in the down-regulation of coagulation by forming a complex with a plasma proteinase inhibitor called protein Z-dependent protease inhibitor (ZPI) which inhibits activated factor Xa on phospholipid surfaces. In the absence of an additional challenge, the disruption of PZ gene in mice is asymptomatic, but the association with the factor VLeiden mutation leads to a near complete mortality during the neonatal period with microvascular thrombosis. Unexpectedly, in humans, a relationship between protein Z deficiency and ischemic strokes, was firstly evidenced, but not confirmed by all the epidemiological study. Additional studies suggest that protein Z deficiency could be also a risk factor for acute coronary syndromes, early fetal losses, and increased the arterial risk in antiphospholipid syndrome. This review analyzes the different studies so far published and discusses the different results obtained in order to understand whether or not protein Z deficiency could be considered as an arterial ischemic risk factor.

Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C‐deficient family

Earlier studies found strong support for a genetic basis for regulation of coagulation factor levels and measures of a prethrombotic state (d-dimer, prothrombin fragment 1.2). Estimation of how much of the variation in the levels of coagulation factors and measures of a prethrombotic state, including measures of protein C activation and inactivation, could be attributed to heritability and household effect. Blood samples were collected from 330 members of a large kindred of French-Canadian origin with type I protein C deficiency. Heritability and common household effect were estimated for plasma concentrations of prothrombin, factor (F)V, factor VIII, factor (F)IX, fibrinogen, von Willebrand factor (VWF), antithrombin, protein C, protein S, protein Z, protein Z-dependent protease inhibitor (ZPI), fibrinopeptide A (FPA), protein C activation peptide (PCP), activated protein C-protein C inhibitor complex (APC-PCI), activated protein C-alpha1-antitrypsin complex (APC-alpha1AT), prothrombin fragment 1.2 (F1.2) and d-dimer, using the variance component method in sequential oligo-genic linkage analysis routines (SOLAR). The highest heritability was found for measures of thrombin activity (PCP and FPA). High estimates were also found for prothrombin, FV, FIX, protein C, protein Z, ZPI, APC-PCI and APC-alpha1AT. An important influence of shared household effect on phenotypic variation was found for VWF, antithrombin, protein S and F1.2. We found strong evidence for the heritability of single coagulation factors and measures of a prethrombotic state. Hemostatic markers with statistically significant heritability constitute potential targets for the identification of novel genes involved in the control of quantitative trait loci.

Low Protein Z Levels and Risk of Ischemic Stroke: Differences by Diabetic Status and Gender

ABSTRACT Protein Z was recently shown to act as an essential cofactor for protein Z-dependent protease inhibitor, a potent downregulator of coagulation Factor Xa. Thus, deficiency of protein Z is hypothesized to lead to a prothrombotic state, but two publications reported opposing results for the relationship of protein Z levels with ischemic stroke in young European subjects (mean age 33–40 years). We performed a study of stroke in a different ethnic population of greater mean age (57 years) to further clarify this issue. An ELISA was developed to measure protein Z antigen in 154 patients with ischemic stroke and in 206 controls in a largely Hispanic population. Low plasma protein Z values were significantly associated with ischemic stroke except in diabetic subjects and females. The mean protein Z value was significantly lower in stroke cases than in controls for nondiabetic subjects [1.78 ± 0.77 (S.D.) versus 2.28 ± 0.88 μg/ml, P < 0.0001] and for males (1.90 ± 0.90 versus 2.42 ± 0.99 μg/ml, P = 0.0004). Stroke risk was higher in subjects with protein Z levels at or below the fifteenth percentile of controls (≤1.46 μg/ml). The odds ratios were: 2.6 for all subjects (95% C.I. = 1.5–4.3); 3.8 for nondiabetic subjects (95% C.I. = 2.2–6.8); and 3.6 for males (95% C.I. = 2.0–6.4). This study also revealed high protein Z levels associated with high triglyceride levels in controls ( P = 0.015). Protein Z is suggested to be a physiologic downregulator of blood coagulation and low protein Z levels are associated with increased risk of ischemic stroke, particularly in males and in the absence of diabetes.

Protein Z influences the prothrombotic phenotype in Factor V Leiden patients

Protein Z enhances the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). Thus, diminution of protein Z should induce prothrombotic tendency due to lowered cofactor activity for ZPI. In Factor V Leiden mice, prothrombotic tendency of severe diminution or lack of protein Z was demonstrated. We here present first studies in humans, indicating that diminution of protein Z in factor V Leiden patients aggravates thromboembolic risk.

Protein Z Circulates in Plasma in a Complex with Protein Z-Dependent Protease Inhibitor

Protein Z (PZ) is a vitamin K-dependent plasma protein that forms a Ca++-dependent complex with factor Xa at phospholipid surfaces. This interaction between PZ and factor Xa enhances by >1,000-fold the inhibition of factor Xa by the serpin called protein Z-dependent protease inhibitor (ZPI). These experiments show that PZ also binds ZPI in a process that does not require Ca++ or phospholipids. In pooled normal plasma, which contains excess ZPI relative to PZ, all the PZ appears to be bound in a complex with ZPI. The binding of PZ to ZPI reduces the rate and extent of factor XIa inhibition produced by ZPI. During the course of these studies, it was noted that a PZ purification procedure, that included NaSCN (2.0 M) elution of PZ from an immunoaffinity column, produced aggregated, inactive forms of PZ.

Protein Z-Dependent Regulation of Coagulation

Protein Z (PZ) is a 62 kDa vitamin K-dependent plasma protein that serves as a cofactor for the inhibition of factor Xa by protein Z-dependent protease inhibitor (ZPI). ZPI is a recently identified 72 kDa member of the serpin superfamily of proteinase inhibitors that contains a tyrosine at its reactive center. PZ circulates in plasma in a complex with ZPI. Inhibition of factor Xa by ZPI in the presence of phospholipids and Ca++ is enhanced 1000-fold by PZ, but ZPI also inhibits factor XIa in a process that does not require PZ, phospholipids or Ca++. ZPI activity is consumed during coagulation through proteolysis mediated by factor Xa with PZ and factor Xla. Concomitant PZ deficiency dramatically increases the severity of the prothrombotic phenotype of factor VLeiden mice. Studies to determine the potential roles of PZ and ZPI deficiency in human thrombosis are in progress.

Mouse Protein Z-Dependent Protease Inhibitor cDNA

Protein Z-dependent protease inhibitor (ZPI) is plasma proteinase inhibitor in the serpin superfamily that produces rapid inhibition of factor Xa in the presence of phospholipids, Ca++ and protein Z (PZ). Mouse ZPI cDNA was isolated and cloned from mouse liver RNA using RT-PCR. The cDNA contains 100 nucleotides 5' of a translation initiation codon and an open reading frame of 1344 nucleotides followed by a 163 nucleotide 3' untranslated sequence with a poly (A) tail. The cDNA predicts a signal peptide containing 21 amino acids and a mature protein of 427 residues with 8 potential sites for N-linked glycosylation. The oligonucleotide and predicted amino acid sequences of mouse ZPI are 72% and 81% homologous with those of human ZPI. Like human ZPI, mouse ZPI contains tyrosine-serine (P1-P1') at its reactive center in contrast to the rat molecule which contains tyrosine-cysteine. By Northern analysis, mouse ZPI mRNA is 1.6 kb in size and, similar to both human and rat, it is detectable in liver, but not in heart, brain, spleen, lung, kidney, skeletal muscle or testes.

Characterization of the protein Z–dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a 72-kd member of the serpin superfamily of proteinase inhibitors that produces rapid inhibition of factor Xa in the presence of protein Z (PZ), procoagulant phospholipids, and Ca++ (t1/2 less than 10 seconds). The rate of factor Xa inhibition by ZPI is reduced more than 1000-fold in the absence of PZ. The factor Xa–ZPI complex is not stable to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but is detectable by alkaline–polyacrylamide gel electrophoresis. The combination of PZ and ZPI dramatically delays the initiation and reduces the ultimate rate of thrombin generation in mixtures containing prothrombin, factor V, phospholipids, and Ca++. In similar mixtures containing factor Va, however, PZ and ZPI do not inhibit thrombin generation. Thus, the major effect of PZ and ZPI is to dampen the coagulation response prior to the formation of the prothrombinase complex. Besides factor Xa, ZPI also inhibits factor XIa in the absence of PZ, phospholipids, and Ca++. Heparin (0.2 U/mL) enhances the rate (t1/2 = 25 seconds vs 50 seconds) and the extent (99% vs 93% at 30 minutes) of factor XIa inhibition by ZPI. During its inhibitory interaction with factor Xa and factor XIa, ZPI is proteolytically cleaved with the release of a 4.2-kd peptide. The N-terminal amino acid sequence of this peptide (SMPPVIKVDRPF) establishes Y387 as the P1 residue at the reactive center of ZPI. ZPI activity is consumed during the in vitro coagulation of plasma through a proteolytic process that involves the actions of factor Xa with PZ and factor XIa.