Vitamin K-dependent Plasma Glycoprotein Research Articles

A Case of Acute Renal Infarct Secondary to Protein S Deficiency

Renal infarction is an underdiagnosed and underreported condition with multiple etiologies. A 45-year-old man presented with acute pain in the right lumbar region, CT scan showed a wedge shaped, non-enhancing, hypodense lesion in the cortex of the upper pole of the right kidney- suggestive of infarct. A pro-thrombotic workup revealed a protein S deficiency and a heterozygous mutation for MTHFR gene. Protein S is a vitamin-K dependent plasma glycoprotein, the deficiency of which is associated with a hypercoagulable state, which in turn led to renal infarction in this patient.

Protein S Coordinates the Inhibition of Prothrombinase By Tfpiα and Activated Protein C

Background: Protein S (PS), a vitamin K-dependent plasma glycoprotein, functions as a cofactor for the anticoagulants activated protein C (APC) and tissue factor (TF) pathway inhibitor alpha (TFPIa), which inhibit factors Va (FVa) and Xa (FXa), respectively. Although it is unclear which of these functions is/are important in vivo, homozygous deficiency of PS is associated with life-threatening thrombosis shortly after birth. FVa and FXa form the prothrombinase complex, which generates thrombin, suggesting that PS has a role in the direct inhibition of thrombin production. However, neither the PS/APC nor PS/TFPIα system alone is effective at inhibiting thrombin generation by prothrombinase. In addition to its role in regulating coagulation, PS also functions as an inhibitor of the complement system. Approximately 60% of plasma PS circulates bound to complement factor C4bp, which blocks its anticoagulant activity. We sought to determine the impact of PS/APC on TFPIα function, and vice versa, using purified protein and plasma-based systems. Methods and Results: To assess the effect of the combined PS/APC and PS/TFPIα systems on thrombin generation, we supplemented plasma with thrombomodulin, which promotes APC activation. In the absence of thrombomodulin, 5nM TFPIα decreased peak thrombin by 55.1% (33.1±1.9 nM in the presence of TFPIα vs. 73.7±39.9 nM in the absence) and endogenous thrombin potential (ETP) by 35.4% (475±42 nM*min vs. 735±189nM*min). In the presence of thrombomodulin, TFPIα decreased these parameters by 65.7% (11.4 ± 2.6 nM) and 77.5% (107±22 nM*min), respectively, suggesting that APC makes TFPIα a more potent inhibitor of thrombin generation. We next sought to study each of these PS functions in a purified protein system and in plasma. To study the effect of PS/APC on TFPIα function, we produced a recombinant protein consisting of the first two epidermal growth factor-like domains of PS (EGF1-2), which contain the putative APC binding sites. In a purified protein assay, APC inhibited the rate of thrombin activation by prothrombinase by 19.62±0.01% in the absence of PS and by 34.96±0.02% in the presence of 50nM PS. EGF1-2 dose-dependently reversed the effect of PS, with 75% reversal achieved with the addition of 200nM EGF1-2. Unexpectedly, EGF1-2 had the opposite effect in plasma thrombin generation assays and potently inhibited TF-initiated thrombin generation either in the presence or absence of thrombomodulin. We hypothesize that the EGF1-2 domains also form part of the C4bp binding site, and that addition of EGF1-2 protein resulted in release of PS from C4bp and an increase in the anticoagulant PS pool. In support of this hypothesis, EGF1-2 had no effect on thrombin generation in PS-depleted plasma, which is also depleted of C4bp. When the PS-depleted plasma was supplemented with 150nM PS, EGF1-2 had the expected procoagulant activity (increasing peak thrombin from 50.4±19.9 nM to 90.4±6.0 nM). Notably, even with a saturating concentration of EGF1-2, thrombomodulin and PS significantly decreased thrombin generation, suggesting that PS-TFPIα-mediated FXa inhibition promotes APC-mediated FVa degradation, even if PS cannot directly bind APC. We similarly assessed the impact of the PS-TFPI function, using a protein from the saliva of black flies, "black fly protease inhibitor" (BFPI), which contains the TFPIα domain that inhibits FXa but lacks the domain that binds PS. BFPI inhibits free FXa similarly to TFPIα, but PS does not promote this inhibition. Like TFPIα, BFPI is a poor inhibitor of thrombin generation by prothrombinase containing thrombin-activated FVa (5 nM BFPI had no impact on thrombin generation in the presence or absence of PS). However, in the presence of APC and PS, 5nM BFPI decreased the maximum rate of thrombin generation by 17.3±3.3%. These data suggest that PS/APC-mediated degradation of FVa promotes TFPIα-mediated inhibition of FXa, regardless of whether PS is able to bind TFPIα. Conclusions: Our data suggest that the PS-APC and PS-TFPIα systems cooperatively regulate thrombin generation by prothrombinase. While maximal inhibition requires that PS act as a cofactor for both APC and TFPIα, PS-APC independently promotes TFPIα function, and PS-TFPIα separately promotes APC. Based on these data, we propose a model in which PS-APC-mediated inhibition of FVa renders FXa susceptible to TFPIα and vice versa. Disclosures No relevant conflicts of interest to declare.

Research Article Protein Z gene variants and risk of Idiopathic Recurrent Pregnancy Loss in Saudi Arabian women

Background: Protein Z (PZ) is a vitamin K-dependent plasma glycoprotein, it plays a key role in the physiologic inhibition of coagulation by acting as a cofactor in the inactivation of factor Xa. The relationship between PZ gene polymorphisms and pregnancy loss is controversial. To address this, we investigate the association between PZ (rs3024718, rs3024719, rs3024731, rs3024778 and rs3024772) single nucleotide polymorphisms (SNPs) and idiopathic recurrent pregnancy loss (IRPL) in in women from Tabuk region (North western region, Saudi Arabia). Methods: A case control study, including Sixty-three women with at least three unexplained recurrent pregnancy loss (RPL) were selected and matched with seventy-eight healthy and fertile women (controls). SNP genotyping was carried out by allelic discrimination using real time PCR. Results: The outcome was that the minor allele frequencies (MAF) are 0.23 vs 0.20, 0.24 vs 0.19, 0.02 vs 0.02, 0.26 vs 0.21, and 0.02 vs 0.01 respectively for rs3024718 A/G, rs024719 G/A, rs3024778 G/A, rs3024731 G/A, and rs3024772 A/G PZ polymorphisms. The genotype distribution was similar between women with RPL and control women (P>0.05) and none of the tested SNPs were associated with RPL under co-dominant, dominant, or recessive genetic models. The lack of association was also confirmed by haplotypes analysis showing an absence of RPL risk with the constructed haplotypes. Conclusion: It was concluded that the studied PZ polymorphisms are not associated with the risk of recurrent pregnancy loss in the studied population

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.

Membrane binding and anticoagulant properties of protein S natural variants

Introduction Protein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency. Materials and methods Binding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems. Results and conclusions Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17). In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7 ± 1.6 nM, rPS217S 146.0 ± 16.1 nM and rPSDelI203D204 234.1 ± 28.1 nM) was substantially increased by membrane oxidation (10.9 ± 0.6, 38.2 ± 3.5 and 81.4 ± 6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration. These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation.

Compound heterozygous mutations in the PROS1 gene responsible for quantitative and qualitative protein S deficiency

Protein S (PS) is a vitamin K-dependent plasma glycoprotein, which plays an important role in the blood anticoagulant system. The PS gene (PROS1) spans some 80 kb of genomic DNA, composed of 15 exons and 14 introns, and is closely linked to a highly homologous pseudogene (PROS2), whose sequence is 96% identical to that of PROS1 [1]. Congenital PS deficiency caused by PROS1 mutations is associated with an increased risk of thromboembolic disease. In this study, we investigated the molecular basis of a patient with both quantitative and qualitative PS deficiency. A 69-year-old man had a medical examination in our hospital because of progressive bilateral leg pain and swelling. He was diagnosed with deep vein thrombosis (DVT) based on the venographs of the legs. Laboratory tests showed normal values of platelet count, prothrombin time, activated partial thromboplastin time, ATIII activity and protein C activity. Test results for lupus anticoagulant and anti-cardiolipin b2GPI antibody were negative. Total PS antigen, free-PS antigen and PS activity levels were 49.0% (normal range 65‐135%), 26.7% (60‐150%) and 10% (60‐150%), respectively. From these findings, he was diagnosed as having PS deficiency.

Definite diagnosis in Japanese patients with protein C deficiency by identification of causative PROC mutations

Protein C is a vitamin K-dependent plasma glycoprotein that functions as an important regulator of blood coagulation, and its deficiency is known to be a risk factor for thrombosis [1]. Congenital protein C deficiency is an autosomally inherited disorder, and has been classified into 2 types: a quantitative deficiency (type I), and qualitative deficiency (type II) [2]. Heterozygous patients with inherited protein C deficiency are mildly affected, and are either symptomatic (1 in 16,000 of the general population [3]) or asymptomatic (1 in 500 of the healthy population [4]). Thus, protein C deficiency in itself is thought to be a relatively mild risk factor for thromboembolism, and it is suggested that thrombosis-prone protein C deficient families might carry additional genetic factors that increase the risk, such as FV Leiden mutation and prothrombin 20210G [ A mutation in Caucasian populations [5]. Severe congenital protein C deficiency is a much rarer disease, most often caused by a homozygous (or compound heterozygous) protein C gene (PROC) mutation(s). Some homozygous subjects develop purpura fulminans or skin necrosis and intravascular disseminated coagulation at birth [6–8], while heterozygous deficiency predisposes to venous thrombosis in adulthood. This clinical heterogeneity could reflect a variety of molecular mechanisms. The prevalence of protein C deficiency in the general Japanese population was also estimated similarly to be approximately 1 in 500 [9]. In this study, we investigated the molecular defects of protein C deficiency in 6 Japanese patients, and defined respective causative mutations in the PROC. All patients were diagnosed as having protein C deficiency (Table 1) [10]. After informed consents were obtained, genomic DNAs were isolated from peripheral blood leukocytes of the patients and studied under approval of the study from the Ethics Committee of the Nagoya University School of Medicine. We amplified each of the 8 exons including the exon/intron boundaries of the PROC by polymerase chain reaction (PCR), and directly sequenced them as described previously [7]. We identified 6 distinct heterozygous PROC mutations in the 6 Japanese patients with protein C deficiency: 5 missense mutations (p.Arg42Ser [c.124C [ A], p.Met406Ile [c.1218G [ A], p.Cys147Tyr [c.439G [ A], p.Arg211Trp [c.631C [ T], A. Takagi R. Tanaka D. Nakashima Y. Fujimori T. Yamada K. Okumura T. Murate T. Kojima Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan

The Detection of Large Deletions of PROS1 - a Common Cause of Protein S Deficiency

Protein S is a vitamin-K dependent plasma glycoprotein which regulates coagulation by acting as a non-enzymatic co-factor for activated protein C during the inactivation of FVa and FVIIIa. Protein S circulates in the plasma as either free protein S or in association with C4b binding protein but only free protein S has anticoagulant properties. Protein S deficiency is an autosomal dominant disorder associated with an increased risk of venous thrombosis. The PROS1 gene is located on the long arm of chromosome 3 near the centromere, in close association with a highly homologous pseudogene PROSP. More than 200 PROS1 mutations have been described to date, but a large cohort of patients exists in whom no mutations have been detected. Several recent studies have suggested that large deletions of PROS1 are common in protein S deficient patients and should be considered when conventional methodologies fail to identify the causative mutation. Therefore there is a requirement for robust and reliable tests capable of rapidly detecting and defining deletions which can be used in a diagnostic setting. We have developed an approach which combines haplotype analysis at several SNPs and simple sequence repeats located throughout the PROS1 genomic sequence which runs in parallel with conventional mutation analysis, followed by confirmation of DNA copy number by quantitative PCR using an ABI7500 Real Time PCR System and more recently multiplex ligation-dependent probe amplification (MLPA) using the SALSA MLPA P112 Kit (MRC Holland). We have used this approach to identify and define large PROS1 deletions in three of six patients referred with protein S deficiency, and in whom mutations had not been detected. Patient 1 presented with recurrent thrombosis and was found to have a severe type I protein S deficiency. PROS1 sequence was normal, but analysis of parental DNA at Pro626 identified the presence of a null allele. Real-time PCR and MLPA revealed that one whole copy of the gene was deleted in this patient. A homozygous mutation in exon 2 was identified in patient 2 who presented with severe protein S deficiency. Analysis of DNA from his son, who also presented with protein S deficiency, showed exon 2 to be normal indicating that this sequence change was hemizygous. A partial deletion involving exons 1 to 8 was subsequently detected in this family. No phenotypic data was available for family 3, which comprised two sisters, but our combined approach identified a deletion of over 50Kb involving the 5′UTR and exons 1 and 2. This highlights the usefulness of this methodology for mutation screening when phenotypic data is unavailable, which is often the case when patients are on continuous anticoagulant therapy. None of the remaining three patients had a family history of protein S deficiency and phenotypic data was inconclusive. Analysis failed to identify any genetic abnormalities in these patients. Our analyses confirm that deletions of PROS1 are a common cause of thrombosis. Further when deletions are not detected using this methodology the information may be useful as supporting evidence for clinicians when reaching a diagnostic decision as to the cause of thrombosis. In summary our approach offers a rapid and reliable approach to the detection of large protein S deletions which would otherwise be missed by routine technologies.

Functional analysis of protein Z (Arg255His) and protein Z‐dependent protease inhibitor (Lys25Arg and Ser40Gly) polymorphisms

Functional analysis of protein Z (Arg255His) and protein Z‐dependent protease inhibitor (Lys25Arg and Ser40Gly) polymorphisms

Role of protein Z in stroke

Protein Z (PZ) is a vitamin K-dependent plasma glycoprotein that acts as a cofactor for PZ-dependent protease inhibitor to inhibit coagulation factor X(a). Studies in mice suggest that that decreased blood PZ levels lead to reduced inhibition of blood coagulation, thereby predisposing to thrombosis; however, clinical studies in humans have yielded conflicting results. Among patients with stroke, some epidemiologic studies have reported that reduced PZ levels increase the risk of stroke, whereas others have reported no association between PZ levels and stroke, or that elevated PZ levels increase stroke risk. Polymorphisms involving the gene for PZ can influence the PZ concentration and some polymorphisms (eg, intron G79A AA allele) may be protective against stroke, particularly among younger individuals. Although the association between PZ levels and stroke appears to be stronger in younger patients and in patients who do not have conventional vascular risk factors, it remains unclear whether the link between PZ levels and stroke is confounded or causal or whether blood levels of PZ are altered as a consequence of the acute stroke event.

The gene for human protein Z is localized to chromosome 13 at band q34 and is coded by eight regular exons and one alternative exon.

Human protein Z is a vitamin K-dependent plasma glycoprotein, deficiency of which leads to a mild bleeding tendency. Protein Z appears to assist hemostasis by binding thrombin and promoting its association with phospholipid vesicles. In this study, to characterize the gene for protein Z, its organization and structure were determined by a combination of PCR amplification of leukocyte DNA and isolation of phage clones from a human genomic library. The gene spanned about 14 kb and consisted of 9 exons including one alternative exon. It was of note that the gene organization was essentially identical to that of other vitamin K-dependent proteins, such as factors VII, IX, and X and protein C. The nucleotides in introns at exon/intron boundaries for eight regular exons were the consensus GT-AG sequences. In contrast, the sequence at an optional exon/intron junction was found to be GC rather than GT. The extra exon inserts a unique peptide consisting of 22 amino acids in the prepro-leader sequence. A similar situation was previously observed in factor VII, but not in other vitamin K-dependent plasma proteins. We also assigned the gene for protein Z to chromosome 13 by PCR amplification of genomic DNAs from human/hamster cell hybrids. Fluorescence in situ hybridization, employing a genomic clone coding for human protein Z, further localized the gene to band q34, where the genes of three other vitamin K-dependent proteins are clustered. These genes may have evolved via duplication of an ancestral gene at this locus.

Enhancing protein C interaction with thrombin results in a clot-activated anticoagulant.

Human protein C is a vitamin K-dependent plasma glycoprotein that circulates as an inactive zymogen. At the endothelial cell surface, thrombin in complex with the integral membrane protein thrombomodulin converts protein C to its active form by specific cleavage of an activation peptide. The activated form of protein C has potent anticoagulant activity as a feedback regulator of thrombin generation (reviewed in refs 4-6), and also has profibrinolytic, anti-ischaemic and anti-inflammatory properties. Protein C is effective in the treatment of model and human thrombotic diseases but, except when it has been used to treat genetic or acquired deficiencies and microvascular thrombosis, it is administered as the activated enzyme, which has a short biological half-life. We have altered two putative inhibitory acidic residues near the thrombin cleavage site, which results in a 30-fold increase in substrate utilization by alpha-thrombin. We combined these changes with a genetically altered glycoform to generate a zymogen protein C with a 60-fold increased cleavage rate by free alpha-thrombin, independent of its cofactor thrombomodulin. We show that this 'proform' of protein C, unlike the natural circulating zymogen, can be activated by thrombin generated in clotting human plasma, resulting in an inhibition of further clot formation. Our data therefore show that we have engineered a site-activated agent, which only has anticoagulant activity when significant amounts of thrombin are being generated.

Human plasma and recombinant factor VII. Characterization of O-glycosylations at serine residues 52 and 60 and effects of site-directed mutagenesis of serine 52 to alanine

Factor VII is a multidomain, vitamin K-dependent plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. Earlier studies demonstrated a novel disaccharide (Xyl-Glc) or trisaccharide (Xyl2-Glc) O-glycosidically linked to serine 52 in human plasma factor VII (Nishimura, H., Kawabata, S., Kisiel, W., Hase, S., Ikenaka, T., Shimonishi, Y., and Iwanaga, S. (1989) J. Biol. Chem. 264, 20320-20325). In the present study, human plasma and recombinant factor VII were isolated and subjected to enzymatic fragmentation. Peptides comprising residues 48-62 of the first epidermal growth factor-like domain of each factor VII preparation were isolated for comparative analysis. Using a combined strategy of amino acid sequencing, carbohydrate and amino acid composition analysis, and mass spectrometry, three different glycan structures consisting of either glucose, glucose-xylose, or glucose-(xylose)2 were detected O-glycosidically linked to serine 52 in plasma and recombinant factor VII. Approximately equal amounts of the three glycan structures were observed in plasma factor VII, whereas in recombinant factor VII the glucose and the glucose-(xylose)2 structures predominated. In addition to the O-linked glycan structures observed at serine 52, a single fucose was found to be covalently linked at serine 60 in both human plasma and recombinant factor VII. Carbohydrate and mass spectrometry analyses indicated that the fucosylation of serine 60 was virtually quantitative. Metabolic labeling studies using [14C]fucose confirmed the presence of O-linked fucose at serine 60. In order to assess whether the carbohydrate moiety at serine 52 contributes to the biological activity of factor VII, we have constructed a site-specific mutant of recombinant factor VII in which serine 52 has been replaced with an alanine residue. Mutant factor VIIa exhibited approximately 60% of the coagulant activity of wild-type factor VIIa in a clotting assay. The amidolytic activity of mutant factor VIIa was indistinguishable from that observed for recombinant wild-type factor VIIa. In addition, the ability of mutant factor VIIa in complex with either purified relipidated tissue factor apoprotein or tissue factor on the surface of a human bladder carcinoma cell line (J82) to activate either factor X or factor IX was virtually identical to that observed for wild-type factor VIIa. These results indicate that the carbohydrate moiety O-glycosidically linked to serine 52 does not appear to be involved either in the interaction of factor VIIa with tissue factor, or the expression of its proteolytic activity toward factor X or factor IX following complex formation with tissue factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino acid sequence of human protein Z, A vitamin K-dependent plasma glycoprotein

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.