Z-dependent Protease Inhibitor Research Articles

The Locus Control Region Activates Serpin Gene Expression through Recruitment of Liver-Specific Transcription Factors and RNA Polymerase II

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human alpha1-antitrypsin (alpha1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3beta (HNF-3beta), HNF-6alpha, CCAAT/enhancer binding protein alpha (C/EBPalpha), and C/EBPbeta. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the alpha1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the alpha1AT gene.

Serpins in thrombosis, hemostasis and fibrinolysis.

Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin-protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure-function relationships is imperative for the development of state-of-the-art pharmaceuticals for the selective fine-tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z-dependent protease inhibitor, alpha(1)-protease inhibitor, protein C inhibitor, alpha(2)-antiplasmin and plasminogen activator inhibitor-1. We focus on the biological function, the important structural elements, their known non-hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins.

Hybridization Probe Genotyping of the R67X Nonsense Polymorphism in the Protein Z-Dependent Protease Inhibitor Reveals a New R67Q Mutation

The protein Z-dependent protease inhibitor (ZPI, gene symbol SERPINA10 , serine peptidase inhibitor, clade A, member 10) inhibits the hemostatic factors Xa and XIa, and in vitro studies indicate that it is an anticoagulant (1)(2). However, the exact role of ZPI in the coagulation system is still unknown. In a recent study, 1018 patients with an episode of venous thromboembolism and 1018 controls with no history of thromboembolic disease were screened for different ZPI polymorphisms(3). A multivariant analysis, which included the Factor V Leiden mutation and the prothombin 20210 G>A sequence alteration, showed that the R67X change is an independent risk factor for venous thrombosis. The calculated odds ratios were 3.32, 5.32, and 4.24 for ZPI-R67X, Factor V Leiden, and prothombin 20210 G>A, respectively(3). We designed an assay for rapid detection of the R67X mutation that involves real-time PCR and melting point analysis on a LightCycler 1.5 Instrument. Primers (5′-CAG CTT GCC AAG GAG AC-3′ and 5′-CTC TCT TGA TCT GGG TTT CAG T-3′; NM 016186 position 686–702 and …

Prospective study of polymorphisms of the protein Z-dependent protease inhibitor and risk of venous thromboembolism

Prospective study of polymorphisms of the protein Z-dependent protease inhibitor and risk of venous thromboembolism -

Mutations R67X and W303X of the protein Z-dependent protease inhibitor gene and venous thromboembolic disease: a case–control study in Italian subjects

The protein Z-dependent protease inhibitor (ZPI) is a single-chain glycoprotein, which appears to play a role in coagulation. In vitro studies indicate that this protein is a serpin and could act as inhibitor of coagulation [1, 2]. ZPI inhibits the activated factors X and XI by different molecular mechanisms and its inhibitory activity is enhanced by heparin. Low levels of either protein Z or ZPI have been shown to be associated with ischemic stroke [3, 4] and central retinal vein or artery occlusion [5]. No association was found between plasma levels of protein Z and ZPI and venous thrombosis [6, 7]. Nevertheless, it has been recently shown that, among 16 mutations/polymorphisms of ZPI gene, two nonsense mutations (R67X and W303X) were associated with venous thromboembolic disease (VTE) in New Zealand [8]. Considering both mutations, heterozygous subjects were 4.4% in VTE patients and 0.8% in normal controls. In that study, patients were included if they had their first thromboembolic event before the age of 60 years. Moreover, both patients and controls were European White subjects, of whom >90% of northern European ancestry, and the W303X mutation was more frequent than the R67X one. According to a different study, however, the W303X mutation was not present in any of 1018 Spanish patients with VTE, suggesting that this gene variant could be a founder mutation, restricted to specific population [9]. In that study, the R67X mutation was present in 0.9% of controls and 3% of VTE subjects. These conflicting data would indicate that the two nonsense ZPI gene mutations would play a thrombotic role only in specific ethnic groups, suggesting the relevance to test the frequency of these mutations in other populations. Therefore, aim of our study was to investigate the presence of the R67X and W303X mutations in thromboembolic Italian patients. Our study was conducted on DNA samples collected from 183 consecutive VTE patients (80 males, 103 females) with a first episode of venous thrombosis before 75 years old and 135 blood donors (64 males, 71 females) without a history of thromboembolism as normal controls. The R67X and W303X mutations were detected according to the bidirectional allelespecific PCR method published by Van de Water [8]. The R67X mutation was not present in any of the Italian VTE patients or control subjects. The W303X mutation was present in two Italian VTE patients (1.1%) and one control (0.74%). According to the v test, this difference was not statistically significant. The overall frequency of these ZPI gene mutations in control subjects was similar to that reported in the New Zealand study. However, with respect to the same study, the frequency of ZPI gene mutations in the Italian cohort of VTE patients was much lower. Among Italian VTE subjects, the two patients that D. Fabbro Istituto di Genetica, Policlinico Universitario di Udine, Udine, Italy

Association of Low Protein Z with Ischemic Stroke in Young Women.

Protein Z enhances inhibition of factor Xa by plasma protein Z-dependent protease inhibitor (ZPI). Several groups reported low protein Z plasma levels associated with ischemic stroke, but others found high protein Z in association with stroke. We sought to explain why our finding of low plasma protein Z in association with stroke in males (Heeb et al 2002) contrasted with some reports. We found association of low protein Z with stroke in all subgroups (including non-Hispanics) except in smokers, diabetic individuals, and women. The findings were similar in samples taken from stroke patients within 4 days of the stroke, as well as in samples taken 2 months later. Our study differed from others in that our controls were subjects with similar proportions as the stroke patients of known or possible risk factors: hypertension, hyperlipidemia, diabetes, smoking, age and gender, and 61% of our subjects were Hispanic as compared to largely European populations that were otherwise studied. Our proportion of smokers (23%) was smaller than that of European studies and could explain some of the differences in reports. High triglycerides in our controls were significantly associated with high protein Z (p=0.015). Thus, healthy controls used in other studies would be expected to include fewer subjects with high triglycerides and higher protein Z than in our studies. Most other studies did not break patients into subgroups by sex and age. Our study found a strong association of low protein Z with stroke in males (p=0.0004), but only a trend in females (p=0.13). Some studies may have missed these associations because men, nonsmokers, and nondiabetic subjects were not evaluated separately. The lack of significant association of low protein Z with stroke in women could be due to hormonal influences that vary with age. Therefore, we divided our female subjects into ages 24 to 56 and ages 57 to 90. Remarkably, the younger female stroke patients had a significant association of low protein Z levels with stroke compared to controls (mean plasma levels 2.08 versus 2.60 μg/ml, p=0.044), while the older female stroke patients did not (2.22 versus 2.18 μg/ml, p=0.823). Protein Z levels for the younger female cases and controls were nearly identical to those for men in the same age range (younger men: 2.07 versus 2.58 μg/ml, p= 0.037). Older female controls, as compared to the younger female and male controls, had significantly lower mean protein Z (p=0.023), while the older female stroke patients had significantly higher protein Z values than the older male stroke patients (2.22 versus 1.75 μg/ml, p=0.010). Thus, it is difficult to further explain why only the older female stroke patients failed to have a significantly lower mean protein Z level than their control group, even when diabetic individuals were excluded. There was not a lower proportion of older female controls with high triglycerides. In summary, these data show that subgroup analysis by age, gender, and diabetic status is important for plasma protein Z studies and that low plasma protein Z levels are found in association with stroke in males of all ages and in young women, though the association remains strongest in men over age 56 (p=0.002).

Determinants of Specificity of Factor Xa Interaction with its Physiological Inhibitors

Factor Xa (fXa) is the vitamin K-dependent serine protease of the prothrombinase complex (fXa, factor Va, negatively charged membrane, and calcium) which is responsible for the conversion of prothrombin to thrombin in the final stage of the coagulation cascade. The proteolytic activity of fXa in plasma is primarily regulated by three physiological inhibitors, antithrombin (AT), protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI). The first two inhibitors belong to the serpin family of plasma inhibitors, both of which require cofactors for their effective interaction with fXa. Thus, the AT interaction with the heparin-like glycosaminoglycans on the surface of the endothelium, and the ZPI complex formation with protein Z on membrane phospholipids is required for the physiological regulation of fXa by both serpins. On the other hand, TFPI is a slow and tight-binding, Kunitz type inhibitor that is capable of rapidly inhibiting fXa independent of a cofactor. This article will review the structural features that enable fXa to specifically interact with these three inhibitors under different conditions.

Contributions of Basic Amino Acids in the Autolysis Loop of Factor XIa to Serpin Specificity

The autolysis loops (amino acids 143-154, chymotrypsinogen numbering) of plasma serine proteases play key roles in determining the specificity of protease inhibition by plasma serpins. We studied the importance of four basic residues (Arg-144, Lys-145, Arg-147, and Lys-149) in the autolysis loop of the coagulation protease factor XIa (fXIa) for inhibition by serpins. Recombinant fXIa mutants, in which these residues were replaced individually or in combination with alanine, were prepared. The proteases were compared to wild-type fXIa (fXIa-WT) with respect to their ability to activate factor IX in a plasma clotting assay, to hydrolyze the chromogenic substrate S2366, and to undergo inhibition by the C1-inhibitor (C1-INH), protein Z dependent protease inhibitor (ZPI), antithrombin (AT), and alpha(1)-protease inhibitor (alpha(1)-PI). All mutants exhibited normal activity in plasma and hydrolyzed S2366 with catalytic efficiencies similar to that of fXIa-WT. Inhibition of mutants by C1-INH was increased to varying degrees relative to that of fXIa-WT, with the mutant containing alanine replacements for all four basic residues (fXIa-144-149A) exhibiting an approximately 15-fold higher rate of inhibition. In contrast, the inhibition by ZPI was impaired 2-3-fold for single amino acid substitutions, and fXIa-144-149A was essentially resistant to inhibition by ZPI. Alanine substitution for Arg-147 impaired inhibition by AT approximately 7-fold; however, other substitutions did not affect it or slightly enhanced inhibition. Arg-147 was also required for inhibition by alpha(1)-PI. Cumulatively, the results demonstrate that basic amino acids in the autolysis loop of fXIa are important determinants of serpin specificity.

A nonsense polymorphism in the protein Z-dependent protease inhibitor increases the risk for venous thrombosis

The protein Z-dependent protease inhibitor (ZPI) is a hemostatic serpin with anticoagulant activity. As for antithrombin, deficiency of ZPI could have relevant thrombotic consequences. We have studied 6 genetic modifications affecting the ZPI gene, identifying 5 haplotypes. Haplotype H5 is featured by a stop codon at position 67. The relevance of these genetic modifications and haplotypes in venous thrombosis was evaluated in a case-control study including 1018 patients and 1018 age- and sex-matched controls. Surprisingly, the H5 haplotype was found in 0.9% of controls, supporting that the Arg67Stop change is a low frequency nonsense polymorphism. The prevalence of this haplotype increased significantly in patients (3.0%), one of whom was in a homozygous state. Multivariate analysis confirms that carriers have a 3.3-fold risk of developing venous thrombosis (P = .002; 95% CI: 1.5-7.1). Moreover, we observed a significant association of this polymorphism with familial history of thrombosis (P < .001). Our study supports that the ZPI Arg67Stop nonsense polymorphism might be an independent genetic risk factor for venous thrombosis. This polymorphism has slightly lower prevalence but similar thrombotic risk than the FV Leiden or prothrombin 20210A. Although further studies are required, all available data support that the ZPI is a candidate to play a significant role in thrombosis and should be evaluated in thrombophilic studies.

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.

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 -

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.

Association of Protein Z Deficiency with Retinal Artery and Vein Occlusion.

Central retinal vein occlusion (CRVO) and central retinal artery occlusion (CRAO) are common disorders mainly associated with traditional risk factors for atherosclerosis. There is no clear correlation between thrombophilia and CRVO. However it was suggested that hyperhomocysteinemia and the presence of antiphospholipid antibodies are risk factors for the development of CRAO. Protein Z is a vitamin K dependent cofactor for the inactivation of activated factor X (Xa) by the protein Z dependent protease inhibitor (ZPI). Protein Z deficiency presumably promotes thrombosis, in particular arterial thrombosis, as suggested by an association between low plasma protein Z levels and ischemic stroke. The similarity between risk factors for ischemic stroke and CRVO / CRAO and the fact that low protein Z levels are associated with an increased risk for ischemic stroke has led us to assess protein Z levels in patients with CRVO and CRAO. Plasma samples from 29 patients with CRVO and 7 patients with CRAO (total study group= 36, mean age 60±11) were screened for protein Z levels by ELISA. Study group was also screened for the presence of lupus anticoagulant (LAC) by the kaolin clotting time (KCT) index and the dilute Russells' viper venum test (DRVVT), for activated protein C resistance (APCR) and anticardiolipin antibodies (ACA). Levels of protein Z were compared with a control group of 42 healthy individuals (mean age 43±11). Patients with CRAO and history of ischemic stroke or an embolic source were excluded. Thirty patients in the study group had traditional risk factors for atheroscalerosis including hypertension, diabetes mellitus, hyperlipidemia and smoking and 6 patients had none. There was no significant difference in protein Z levels between the whole study group patients and controls (1995±810 vs. 2010±603 ng/ml p=0.922). However, patients with no risk factors for CRVO/CRAO had significantly lower protein Z levels than controls (1379±682 vs. 2010±603 ng/ml p=0.022) and patients with risk factors (1379±682 vs. 2118±785 ng/ml p=0.04). In the study group three patients had abnormal APCR and 6 had positive LAC. There was no correlation between low protein Z levels and the presence of LAC, APCR or ACA, or a correlation between low protein Z levels and the specific diagnosis (CRVO or CRAO) or age. The data show that low protein Z levels may be an additional risk factor for CRVO and CRAO in patients without traditional risk factors for these disorders. The association should be explored in a larger group of patients.

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.