Protein Z Research Articles

Is There a Role for MDR1, EPHX1 and Protein Z Gene Variants in Modulation of Warfarin Dosage? A Study on a Cohort of the Egyptian Population

There is considerable inter-individual variability in warfarin dosages necessary to achieve target therapeutic anticoagulation. Polymorphisms in genes, which master warfarin pharmacokinetics and pharmacodynamics, might influence warfarin dose variation. Genes encoding drug transporters, such as human multidrug resistance (MDR1), as well as epoxide hydrolase 1 (EPHX1), which is a putative subunit of the vitamin K epoxide reductase, and Protein Z (PZ), which is a vitamin K-dependent plasma glycoprotein, are among those candidate genes. The purpose of this study was to investigate the contribution of MDR1 C3435T, EPHX1 H139R and PZ A-13G gene polymorphisms in warfarin dose variation in a cohort of the Egyptian population. Eighty-four patients whose international normalized ratio (INR) was in the range of 2-3, 41 males and 43 females, with a mean (±SD) age of 40.9 (13.3) years were recruited into this study. MDR1 C3435T, EPHX1 H139R and PZ A-13G gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism. Primarily, linear regression analysis, including the variables age, gender, MDR1 C3435T, EPHX1 H139R and combined MDR1 C3435T, EPHX1 H139R and PZ A-13G genotypes, was used to assess the effective factors for warfarin maintenance dose. Secondly, the previously examined cytochrome P450 (CYP) 2C9 A1075C and vitamin K epoxide reductase complex subunit 1 (VKORC1) C1173T were added to the regression analysis. Warfarin dose/week was not influenced by each of the MDR1 C3435T, EPHX1 H139R, and PZ A-13G gene polymorphisms when examined separately. However, when these single nucleotide polymorphisms (SNPs) were combined, MDR1 TT/EPHX1 RH,RR/PZ AA subjects showed statistically significant increase in warfarin dose/week when compared with MDR1 CC/EPHX1 RH,RR/PZ AA subjects [median (25th-75th percentiles): 49.0 (42.0-59.5) vs. 35.0 (24.5-42.0)mg/week, respectively] (p=0.014). In contrast, in the presence of wild-type EPHX1 HH, there was a decrease in warfarin dose/week in MDR1 TT subjects when compared with CT and CC subjects [median (25th-75th percentiles): 22.0 (17.5-30.6), 42.0 (35.0-49.0) and 42.0 (28.0-54.3)mg/week, respectively] (p=0.005 and 0.030, respectively). Age had a significant contribution (p=0.048) to the overall variability in warfarin dose. Calculated weekly dose=52.928-(0.289×age)+(9.709×combined genotype). The multivariate linear regression equation of warfarin maintenance dose accounted for about 8% of variation in dose (R (2)=0.079), age accounted for 5% of variation, while combined genotypes added the extra 3%. However, the new regression equation accounted for 20.9% of variation in dose. Age accounted for 5%, while VKORC1 C1173T accounted for an extra 13% of variation and MDR1 C3435T accounted for the remaining 3% of variation. Calculated dose=64.909-(0.282×age)-(13.390×VKORC1)-(7.164×MDR1). Correlation analysis showed a close and significant relationship between the calculated and actual warfarin dose (r=0.457; p<0.0005). Warfarin dose/week was significantly influenced by the combined MDR1 C3435T and EPHX1 H139R gene polymorphism since no polymorphism of PZ A-13G SNP was detected in our studied Egyptian population. Future studies with larger sample size will be needed to confirm our findings before definitive conclusions can be made.

Protein Z Variants Associated With Protein Z Plasma Levels and With Risk of Idiopathic Recurrent Miscarriage

Protein Z (PZ) deficiency due to anti-PZ autoantibodies and/or mutations in PZgene was linked with adverse pregnancy outcomes, including idiopathic recurrent miscarriage (IRM). We investigated the association of rs3024718, rs3024719, rs3024731, rs3024778, rs3024772, and rs3024735 (G79A) PZ variants and changes in PZ levels in 287 women with IRM, and 308 control women. Of the 6 single nucleotide polymorphisms (SNPs) analyzed, higher minor allele frequency of rs3024735 (G79A) and rs3024731 were seen in IRM cases than in control women. Significantly higher frequencies of rs3024735/G79A G/A and A/A (P< .001), rs3024719 G/A (P= .009), and rs3024731 A/A (P = .012), but not rs3024718 (P= .12), rs3024778 (P = .76), or rs3024772 (P= .27) genotype carriers were seen between IRM cases versus control women, respectively, and was linked with reduced PZ levels. Six-locus (rs3024718/rs3024719/rs3024778/rs3024731/rs3024735/rs3024772) PZhaplotypes analysis demonstrated increased frequency of GAGAAG and AGGTAG and reduced frequency of AGGTGC haplotypes in IRM cases, thereby conferring disease susceptibility and protective nature to these haplotypes, respectively. These results demonstrate that specific PZSNPs and haplotypes are significantly associated with IRM.

Protein Z, an anticoagulant protein with expanding role in reproductive biology

Protein Z (PZ) is a vitamin K-dependent factor characterized by its homology to other vitamin K-dependent factors (factors VII, IX, and X, protein C and protein S), but lacks any enzymatic activity. Instead, PZ acts as a cofactor for the inhibition of factor Xa through the serpin PZ-dependent protease inhibitor (ZPI). PZ deficiency is associated with a procoagulant state, highlighted by excessive FXa secretion and thrombin production, and is linked with several thrombotic disorders, including arterial vascular and venous thromboembolic diseases. A role for the PZ-ZPI complex in the regulation of physiological pregnancy has been demonstrated, highlighted by the progressive elevation in PZ levels in the first trimester of gestation, which then steadily decline toward delivery. An association between altered plasma PZ concentrations and adverse pregnancy outcomes (recurrent miscarriage, stillbirth, preeclampsia, intrauterine growth restriction, and placental abruption) has been reported. The mechanism by which PZ deficiency leads to adverse pregnancy outcomes is not clear, but it is multifactorial. It may be attributed to the anti-PZ IgG and IgM autoantibodies, which apparently act independently of classical antiphospholipid antibodies (lupus anticoagulant, anticardiolipin, and anti-β2-glycoprotein I antibodies). PZ deficiency has also been reported to be constitutional, and a number of variants in the PROZ (PZ) gene and SERPINA10 (ZPI) gene are linked with specific adverse pregnancy complications. This review summarizes the relationship between adverse pregnancy outcomes and acquired and constitutional PZ-ZPI deficiency, in order to understand whether or not PZ deficiency could be considered as a risk factor for poor pregnancy outcomes.

Residues of the 39-Loop Restrict the Plasma Inhibitor Specificity of Factor IXa

The two plasma inhibitors, protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), effectively inhibit the activity of activated factor X (FXa); however, neither inhibitor exhibits any reactivity with the homologous protease activated factor IX (FIXa). In this study, we investigated the molecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that unique structural features within residues of the 39-loop are responsible for restricting the inhibitor specificity of FIXa. This loop in FXa is highly acidic and contains three Glu residues at positions 36, 37, and 39. On the other hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. We discovered that replacing residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and TFPI. Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), negatively charged membrane vesicles, and calcium ions approached the same diffusion-limited rate (>10(7) m(-1) s(-1)) that has been observed for the PZ-dependent inhibition of FXa by ZPI. Interestingly, sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inhibition by the PZ-ZPI complex. These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.

Structural analysis of protein Z gene variants in patients with foetal losses

The role of protein Z (PZ) in the etiology of human disorders is unclear. A number of PZ gene variants, sporadic or polymorphic and found exclusively in the serine protease domain, have been observed. Crystal structures of PZ in complex with the PZ-dependent inhibitor (PZI) have been recently obtained. The aim of this study was a structural investigation of the serine protease PZ domain, aiming at finding common traits across disease-linked mutations. We performed 10-20 ns molecular dynamics for each of the observed PZ mutants to investigate their structure in aqueous solution. Simulation data were processed by novel tools to analyse the residue-by-residue backbone flexibility. Results showed that sporadic mutations are associated with anomalous flexibility of residues belonging to specific regions. Among them, the most important is a loop region which is in contact with the longest helix of PZI. Other regions have been identified, which hold anomalous flexibility associated with potentially protective gene variants. In conclusion, a possible interpretation of effects associated with observed gene variants is provided. The exploration of PZ/PZI interactions seems essential in explaining these effects.

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

The potential role of alterations in protein Z (PZ) concentrations in the pathogenesis of coagulation has been investigated in several studies which, however, yielded conflicting results. Protein Z deficiency may induce bleeding as well as prothrombotic tendencies and it might occur as an inherited disorder. The principal aim of the present study was to explore the concentration of protein Z and protein Z-dependent protease inhibitor (ZPI) in patients with haemophilia A.In haemophilia A patients mean plasma concentrations of PZ and ZPI were significantly higher than in healthy individuals: PZ (1.87±0.68μg/mL vs 1.49±0.54μg/mL) and ZPI (5.02±1.11μg/mL vs 4.22±0.55μg/mL), with p=0.02 and p=0.03, respectively. In the subgroup with severe haemophilia A, an in-depth analysis revealed a tendency to modulating effect of the PZ (r=−0.53; p=0.072) and a statistically significant one in the case of ZPI (rho=−0.79, p=0.002) on the bleeding rate. It simultaneously disclosed a statistically significant correlation between the number of bleeds to the joints (20.18±14.1), PZ (r=−0.72; p=0.04) and ZPI (rho=−0.88, p=0.001). With reference to this particular group of patients, the study also showed some other statistically meaningful correspondences: between PZ and ZPI (rho=0.65, p=0.02), PZ and FIX (r=−0.61, p=0.04), as well as ZPI and FVIII (rho=0.78, p=0.002).In conclusion, despite the fact that FVIII deficiency is undoubtedly the main mechanism of bleeding in haemophilia A patients, the activity of PZ/ZPI complex may play some modulating role in the matter.

Protein Z Deficiency in Patients with Bleeding History. Is It a Coagulopathy?.

AbstractAbstract 2227 Introduction:Protein Z (PZ) is a vitamin K-dependent protein. In hemostasis PZ has two functions: together with Factor Xa it forms an inhibiting complex with the PZ- dependent protease-inhibitor and it enhances binding of Thrombin to phospholipid surfaces. Therefore, low PZ levels may induce thrombosis or bleeding.The latter has been questioned by Vasse 2008 and in 1995 Kemkes-Matthes reported in 58% of patients (pts) with a bleeding history PZ deficiency as the only abnormality. Aim of the study:To evaluate if low PZ levels are associated with a bleeding tendency, we reviewed data of pts referred to or laboratory. PZ determination is part of our diagnostic work up, including whole blood count, PT, PTT, VWF/FVIII-complex, FXIII and platelet function studies. Material and Methods:Over a 1 year period we investigated PZ level in 173 pts. PZ concentration was determined by ELISA (Asserachrom Protein Z; Diagnostica Stago), a sandwich immunoassay using a mouse monoclonal antibody against PZ, normal range 1600 – 3300 μg/l, mean 2600 μg/l published by Miletich. Our normal range obtained by 62 donors 990–2490, mean 1740μg/l (+/− 2SD). PZ <1000μg/l is considered to be abnormal. Results:In 75/173 (43%) pts with a bleeding history (e.g. postoperative hemorrhage, epistaxis, menorrhagia) no coagulation abnormality was detected. In 41/98 (42%) low PZ level was the only abnormality (mean PZ 642, range 195–994). 57/98 (58%) were diagnosed with vWD n=17; platelet function disorders n=10 or ASA/drugs causing platelet dysfunction n=20, others n=10. In 21/57 (37%) low PZ level was detected additionally (mean PZ 699μg/l; range 219–949) and in the remaining 36/57 (mean PZ 1689; range 1036–2842). Conclusion:Surprisingly, in 24% of pts with a bleeding history the only abnormality was PZ deficiency. Therefore, we consider PZ determination a useful parameter in patients with a bleeding history after ruling out more common disorders. Low PZ levels may cause a coagulopathy in some pts. Disclosures:No relevant conflicts of interest to declare.

C0264 Protein Z mutations in patients with fetal losses: Structural analysis

The role of protein Z (PZ) in the etiology of human disorders is unclear. A number of PZ gene variants, sporadic or polymorphic, have been observed in patients with thrombosis or fetal losses, all of them included in the serine protease domain. We previously showed that sporadic mutations within the PZ exon 8 are associated with a ~6-fold higher risk of fetal loss. Crystal structures of PZ in complex with the PZ-dependent inhibitor (PZI) have been recently obtained. A structural investigation of the serine protease PZ domain, aiming at finding common traits across disease-linked mutations, has been undertaken.

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

AbstractThe anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z–protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop–cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.

The role of Ca(2+) ions in the complex assembling of protein Z and Z-dependent protease inhibitor: A structure and dynamics investigation.

We investigated the solution structure and dynamics of the human anti-coagulation protein Z (PZ) in the complex with protein Zdependent protease inhibitor (ZPI) to order to understand key structural changes in the presence and absence of Ca2+. Structural features of the complete complex of PZ-ZPI are poorly understood due to lack of complete atomic model of the PZ-ZPI complex. We have constructed a model of the complete PZ-ZPI complex and molecular dynamics (MD) simulation of the solvated PZ-ZPI complex with and without Ca2+ was achieved for 100ns. It is consider that the Ω-loop of GLA domains interacts with negatively charged biological membranes in the presence of Ca2+ ions. The PZ exerts its role as cofactor in a similar way. However, we used solvent-equilibrated dynamics to show structural features of the PZ-ZPI complex in the presence and the absence of Ca2+ions. We observed that the distance between the interacting sites of the ZPI with the PZ and the GLA domain decreases in the presence of Ca2+ ions. Further, we postulated that the calculated distance between the dominant plane of the Ca2+ ions and Ser196 of the pseudo-catalytic triad of the PZ is similar to the equivalent distance of FXa. This suggests that the central role of the PZ in the blood coagulation may be to align the inhibitory site of the ZPI with the active site of the FXa, which is depends on the interaction of the calcium bound GLA domain of the PZ with the active membrane.

Protein Z polymorphisms associated with vaso-occlusive crisis in young sickle cell disease patients

We investigated the association of protein Z (PZ) promoter (rs3024718, rs3024719, and rs3024731) and intron (rs3024735; G79A) SNPs with sickle cell disease (SCD) vaso-occlusive crisis (VOC). Study subjects included 239 SCD patients with VOC and 138 pain-free SCD control patients. PZ genotyping was done by allelic discrimination (real-time PCR) assays. The minor allele frequency of rs3024718 (P=0.03), rs3024719 (P=0.02), rs3024731 (P<0.001), and rs3024735 (P<0.001) were higher in VOC patients than control SCD patients. Significant differences in the distribution of rs3024731 (P=0.028) and rs3024735 (P=0.045) genotypes were seen between VOC and steady-state SCD patients. This association remained significant after adjusting for gender, HbS, and HbF. Four-locus (rs3024718/rs3024719/rs3024731/rs3024735) PZ haplotypes analysis demonstrated increased frequency of GAAA (P=0.024), AGAA (P=0.011), and GGTG (P=0.002), and reduced frequency of AGTG haplotype (P=0.001) in VOC than in steady-state control patients, thereby conferring disease susceptibility and protective nature to these haplotypes, respectively. Of these, only AGTG (P(c)=0.001) and GGTG (P(c)=0.018) remained significant after applying the Bonferroni correction. In conclusion, specific PZ variants and haplotypes are significantly associated with SCD VOC.

Intron F G79A Polymorphism of the Protein Z Gene in Turkish Behçet Patients

Purpose: Vasculitis is one of the major findings of Behçet’s disease (BD). Protein Z (PZ) is a glycoprotein that acts as a cofactor of PZ-dependent protease inhibitor and suppresses trombus formation by inhibiting activated factor Xa. Polymorphism of the PZ gene was mentioned as a genetic risk factor for various thrombotic events. The aim of this study is to investigate the intron F G79A polymorphism of the PZ gene in Behçet patients with and without ocular involvement.Methods: Seventy-six patients and 70 controls were included in the study. Intron F G79A polymorphism of PZ gene was determined by polymerase chain reaction based DNA analysis. The frequency of A allele and the distribution of genotypes were assessed by χ2 test and the genotype distribution and Hardy–Weinberg equilibrium were tested with the χ2 test for quality of fit.Results: The frequency of the A allele was significantly higher in overall Behçet patients than in controls (odds ratio [OR] = 6.8; 95% CI, 2.6 to 17.9; p = 0.0001). It was also significantly higher in patients with (OR = 5.3; 95% CI, 1.83 to 15.6; p = 0.0024) or without (OR = 8.2; 95% CI, 2.95 to 22.5; p = 0.0001) ocular involvement compared to controls. However, A allele frequency was not significantly different between patients with eye involvement versus patients without eye involvement (OR = 0.65; 95% CI, 0.3 to 1.4; p = 0.28).Conclusions: Although thrombosis in BD is multifactorial, intron F G79A polymorphism of PZ gene in BD may be one of the factors that contribute to this pathological process.

Protein Z Concentrations in Patients With Acute Leukemia

Protein Z (PZ) deficiency may induce bleeding as well as thrombosis. The aim of our study was to estimate the concentration of PZ in patients with acute leukemia. Plasma levels of PZ were determined in 76 patients with newly diagnosed acute leukemia ([AML], n = 50; acute lymphoblastic leukemia [ALL], n = 26) and 62 healthy participants. In the patients, mean plasma concentrations of PZ were statistically lower than in healthy individuals: AML (1.24 ± 0.11 μg/mL vs 1.58 ± 0.05 μg/mL P = .01) and ALL (1.19 ± 0.16 μg/mL vs 1.58 ± 0.05 μg/mL P = .01). Levels of PZ below the fifth percentile (0.873 μg/mL) of normal value distribution in control participants were found in 30% of patients with AML and ALL and in 3% of controls (P < .0001). In this AML subgroup, we found statistically significant correlation between episodes of bleeding and PZ level (P = .01). There was no such correlation in ALL group. The results suggest that PZ can be a cofactor associated with an increased bleeding tendency in patients with AML.

Protein-Z Deficiency and/or Protein Z Polymorphisms in First Trimester Pregnancy Complications

Abstract 1221 INTRODUCTION:Protein Z (PZ) is a vitamin K-dependent coagulation factor; it is a glycoprotein that inhibits activated factor Xa, acting as a co-factor to PZ-dependent protease inhibitor, enhancing its action approximately by 1000 times. PZ levels in normal individuals vary greatly, as a result of PZ gene polymorphisms. PZ deficiency has been involved in the pathogenesis of ischemic strokes and pregnancy complications. Gris et al [Blood 2002;99(7):2606–08] first described a possible role of PZ deficiency (PZ <= 1mg/L) in women with fetal loss between the beginning of the 10th and the end of the 15th week of gestation. In a recent meta-analysis [Sofi et al, Thrombosis and Haemostasis 2010;103(4):749–56] PZ deficiency was associated with increased risk of pre-eclampsia and fetal loss, as well as with increased risk of arterial and venous thrombotic events. MATERIALS-METHODS:We studied a total of 314 women, 70 women with three or more consecutive spontaneous abortions (group A), 145 women with less than 3 early spontaneous abortions (group B) and 99 control women with at least one normal pregnancy and negative history of a thrombotic complication (group C). All women were tested for congenital and acquired thrombophilia such as antithrombin, protein C and S levels, homocysteine levels, lupus anticoagulant (PTTLa), factor V Leiden mutation, prothrombin G20210A gene polymorphism and PZ levels. We also investigated protein Z polymorphism F79A in a subgroup of our patients. Measurements were made at least 3 months apart from a thrombotic event. Differences between groups were assessed with ANOVA and chi-squared tests for continuous and categorical variables respectively. RESULTS:Statistically significant difference was found in PZ levels between the three groups. Mean PZ level was 1.23mg/dL, 1.31mg/dL και 1.61mg/dL (p<0.00001) in groups A, B, C respectively. Post-hoc Bonferroni analysis revealed a significant difference between groups A and C (p=0.0003) and between groups B and C (p=0.001). The percentage of PZ deficiency (95% condidence interval) was 40% (28%–52%), 38% (30%–46%) and 18% (11%–26%) respectively (p=0.001). Both group A (OddsRatio[OR]=3) and group B (OR=2.75) have a statistically greater PZ deficiency than control group C. The other parameters did not differ significantly between the three groups. DISCUSSION/CONCLUSIONS:Spontaneous abortions are common in women especially in first trimester. Thrombophilia has a major role in pregnancy complications. In these women that one cannot find some of the well established thrombophilic factors, searching for other possible deficiencies is necessary. The role of PZ deficiency has been investigated thoroughly in the last decade with sometimes conflicting results. To the best of our knowledge, this is the first Greek study investigating the possible role of protein Z deficiency in women with early pregnancy losses. From our study it is evident that PZ deficiency is an independent risk factor for early pregnancy losses. From our study it seems that the other thrombophic factors may play a minor role. A plausible pathophysiologic explanation is the occurrence of microthrombi due to atherosclerotic lesions soon after the development of materno-placental circulation. The role of PZ gene polymorphisms in PZ levels and in thrombotic complications remains to be investigated further. According to preliminary results from a sub-group of our patients (Topalidou et al, Thrombosis Research 2009;124:24–27), the presence of the intron F79A polymorphism was associated with significantly lower PZ levels, but was unrelated to unexplained early pregnancy losses. Disclosures:No relevant conflicts of interest to declare.

Co-localization of Protein Z, Protein Z-Dependent protease inhibitor and coagulation factor X in human colon cancer tissue: Implications for coagulation regulation on tumor cells

IntroductionSeveral hemostatic system components, including factor X (FX), contribute to cancer progression. The Protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex directly inhibits factor Xa proteolytic activity. The aim of this study was to determine the antigenic distribution of ZPI and PZ, in relation to FX, as well as indicators of blood coagulation activation (F1+2 and fibrin) in human colon cancer tissue. Materials & methodsStudies were performed on human colon cancer fragments. Immunohistochemical (IHC) ABC procedures and double staining method employed polyclonal antibodies against PZ, FX, F1+2 and monoclonal antibodies against ZPI and fibrin. In-situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either FX, PZ or ZPI mRNAs. ResultsExpression of FX, PZ and ZPI in association with colon cancer cells was observed by IHC. Moreover, the presence of both F1+2 and fibrin in association with colon cancer cells was found, which indicates that blood coagulation activation proceeds extravascularly at the tumor site. Furthermore, expression of FX and PZ was visualized in association with endothelial cells. In turn, colon cancer-associated macrophages were characterized by FX , PZ and ZPI presence. The double staining studies revealed strong FX/PZ, FX/ZPI, as well as PZ/ZPI co-localization on colon cancer cells. ISH studies revealed the presence of FX mRNA, PZ mRNA and ZPI mRNA in colon cancer cells indicating induced synthesis of these proteins. ConclusionsThe localization of PZ/ZPI and FX in colon cancer cells indicates that PZ/ZPI may contribute to anticoagulant events at the tumor site. Strong co-localization of PZ/ZPI and FX in cancer cells, and the presence of the mRNAs encoding the proteins, suggests their role in the tumor's biology. However, the presence of F1+2 and fibrin at the colon cancer site also suggests that the regulation of FXa by the PZ/ZPI complex at this site is incomplete.

Protein Z levels in pregnant Omani women: correlation with pregnancy outcome

Placental insufficiency resulting in fetal loss has been recognized in women with thrombophilic predisposition. Recent studies indicate that there is a high prevalence of protein Z (PZ) deficiency in patients with unexplained fetal loss. The objective of this study was to measure the PZ levels in pregnant Omani women in the first, second and third trimesters and correlate with the pregnancy outcome. The study enrolled 126 consecutive pregnant women after an informed consent prospectively. PZ was estimated in the first, second and third trimester in 15, 97 and 66 pregnant women respectively and they were followed for pregnancy outcomes including live birth, still birth, spontaneous abortion/induced abortion, maternal complications, fetal complications and health risks/complications in the newborn. The median PZ level (Mean±SD) in the first, second and third trimester were 0.98 (1.07±0.46), 1.3 (1.36±0.61) and 1.44 (1.43±0.69) (P<0.05, Student's t-test, between first vs. second and first vs. third trimester). PZ deficiency defined as PZ level below 0.54μg/ml (below 10th centile in the Omani population) was observed in 4 (4.7%) women, but interestingly all had a normal pregnancy outcome. Amongst the 43 subjects in whom paired PZ estimations were available, reducing PZ levels were observed from baseline values in 8 (33%) with normal pregnancy outcome; 5 (55%), with diabetes; 3 (50%) with hypertension and 2 (50%) with low birth weight respectively (P<0.05, chi square test). PZ values increased progressively during the three trimesters of pregnancy. However, this increase is blunted in patients with abnormal pregnancy outcome like low birth weight babies or pregnancies associated hypertension or diabetes. Isolated PZ deficiency alone did not result in an abnormal outcome in this cohort of subjects.

Protein Z is present in human breast cancer tissue

Thromboembolic complications are common findings in breast cancer patients [1, 2]. Hemostatic system components play a role in tumor progression, independent of their role in hemostasis [1, 2]. Since the report of multifactorial activation of factor X in cancer patients [1], inhibitors which control the activity of this factor have attracted attention. Protein Z (PZ), a vitamin K-dependent plasma nonpeptidase glycoprotein [3, 4], is involved in one mechanism responsible for the direct inhibition of factor Xa. PZ circulates in plasma complexed with PZ-dependent protease inhibitor (ZPI) and enhances the rate of ZPImediated factor Xa inhibition [5, 6]. The presence of ZPI in breast cancer tissue was previously reported [7], while information on PZ in tumor malignant tissue is lacking. The purpose of the present study was to evaluate the localization of PZ (both protein and mRNA) at the primary site of human breast cancer. Tumor fragments were obtained during the surgical treatment of 13 previously untreated breast cancer patients (clinical stage T1-2N0M0). Studies were performed on 4 cases of G2 and 9 cases of G3 invasive ductal carcinomas. Immunohistochemical studies (IHC) were performed (Vectastain Kits, Vector Laboratories, Burlingame, CA, USA) [7] employing a polyclonal antibody against homogeneous, plasma-derived human PZ (prepared in rabbits, and purified from immune sera by protein A-Sepharose chromatography). The antibody did not cross-react with all other purified human plasma vitamin K-dependent proteins including prothrombin, protein C, protein S, factor VII, factor X, and factor IX by ELISA. Controls consisted of omission of the primary antibody from the procedure. The control fragments of normal breast tissues obtained from neoplasm-free surgical margins were processed simultaneously as well. Antigen staining was detected by the dark brown reaction product. A semiquantitative analysis of PZ IHC expression in cancer cells (based on both the percentage of cancer cells with PZ positive staining and the intensity of staining) was performed [7]. Immunoreactive score (IRS) values of 1–4 were interpreted as weak, 5–8 as medium, and 9–12 as strong PZ expression. In situ hybridization (ISH) method employed a biotin-labeled 25-nucleotide single-stranded DNA probe (probe sequence: 50 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 30) specific for PZ mRNA. The probe was synthesized by Sigma-Aldrich, Poznan, Poland. The ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed. Hybrids were detected with rabbit anti-biotin monoclonal antibodies according to the IHC ABC technique associated with ImmunoMax amplification technique described in detail elsewhere [7]. Negative controls included hybridization without addition of the molecular probe and incubation of slides in a RNase A solution (R&D Systems, Minneapolis, MN, USA) before hybridization. Reaction results appeared as dark brown staining. E. Sierko M. Z. Wojtukiewicz (&) Department of Oncology, Medical University, 12 Ogrodowa St, Bialystok, Poland e-mail: mzwojtukiewicz@gmail.com

Relationship of the Protein Z Intron F G79A and IL6 C634G Gene Polymorphisms With the Risk of Recurrent Pregnancy Loss in Egyptian Women

PurposeTo investigate the relationship between recurrent pregnancy loss and single nucleotide polymorphisms in the protein Z (PZ) intron F G79A and the promoter region of the IL6 C634G genes in...

Participation of protein Z-dependent protease inhibitor and protein Z system in the pathomechanism of thrombotic complications

Thrombotic complications of unknown etiology remain a serious diagnostic and therapeutic problem. Occurrence of the inherited polymorphisms of genes encoding proteins involved in the coagulation cascade is one of the possible causes of these complications. In recent years, protein Z (PZ) and PZ-dependent protease inhibitor (ZPI) have been added to the list of prothrombotic factors. PZ is a glycoprotein serving as a cofactor of ZPI, which is responsible for the inhibition of prothrombinase. Expression of the PZ gene is under the control of many transcriptional factors; several polymorphisms alternate the rate of gene expression. The present article describes the significance of the ZPI-PZ system in venous and arterial thrombosis, adverse pregnancy outcomes and antiphospholipid syndrome complications.

High Frequency of Anti-Protein Z IgM and IgG Autoantibodies in Women with Idiopathic Recurrent Spontaneous Miscarriage

Protein Z (PZ) system is an anticoagulant pathway involved in the physiologic regulation of coagulation, and PZ deficiency reportedly enhances prothrombophilic mechanisms, including those implicated with idiopathic recurrent miscarriage (RSM). We investigate plasma anti-PZ IgM and IgG levels in RSM women and in multiparous control women. Anti-PZ IgM and IgG levels were measured in 265 RSM women and 283 age-matched control women by ELISA. Elevated anti-PZ IgG (P < 0.001) and IgM (p < 0.001) titers were seen in patients. The areas under the curves for ROC curve for anti-PZ IgM (0.898 ± 0.044) and IgG (0.898 ± 0.042) demonstrated no variation in diagnostic capacity. Multivariate analysis confirmed the association of elevated anti-PZ IgM [adjusted odds ratio, aOR (95% CI) = 6.46 (2.44-17.11)] and IgG [aOR (95% CI) = 7.44 (2.54-21.79)] as independent predictors of RSM after adjusting for confounding covariates and demonstrated a clear gradation of increasing RSM risk associated with increased antibody titers. The presence of anti-PZ IgM and IgG antibodies are risk factors for RSM.