The risk of occurrence of venous thrombosis: focus on protein Z

Thrombosis Research 128 (2011) 508–515

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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s

Review Article

The risk of occurrence of venous thrombosis: focus on protein Z Valeria Bafunno a, Rosa Santacroce a, Maurizio Margaglione a, b,⁎ a b

Medical Genetics, Department of Biomedical Sciences, University of Foggia, Foggia, Italy Atherosclerosis and Thrombosis Unit, Research Department, Istituto di Ricovero e Cura a Carattere Scientifico, Casa Sollievo della Sofferenza, S. Giovanni Rotondo, Foggia, Italy

a r t i c l e

i n f o

Article history: Received 18 April 2011 Received in revised form 14 July 2011 Accepted 3 August 2011 Available online 31 August 2011 Keywords: Protein Z venous thrombosis polymorphisms coagulation pregnancy risk factor

a b s t r a c t Protein Z (PZ) is a vitamin K-dependent factor identified in human plasma in 1984 characterized by an homology with other vitamin K-dependent factors. PZ acts as the cofactor of the PZ dependent inhibitor (ZPI), in the inhibition of activated factor X bound on phospholipid surface. In humans, PZ is characterized by an unusual wide distribution in plasma partly explained by a genetic control. Several PZ gene polymorphisms influencing plasma concentration have been described. In mice, the disruption of PZ gene is asymptomatic, but in association with homozygous FV Leiden produced a severe prothrombotic phenotype. This review analyzes the results obtained from different studies so far published in order to understand whether PZ deficiency could be considered as a risk factor for venous thrombosis. The roles of PZ plasma level and PZ gene polymorphisms remain debated with conflicting results. Many of these studies reported low PZ levels in association with an increased risk of venous thrombosis. On the other side, some studies did not observe an association between low levels of PZ and thrombotic events. A relationship between PZ deficiency and pregnancy complications was also described but not confirmed by all studies. These discrepancies can be explained by the heterogeneity of populations chosen as control, by the PZ interindividual variability and by the small size of the cohorts in mainly retrospective studies. Large prospective studies remain to be done to investigate its possible role in thrombosis. © 2011 Elsevier Ltd. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . PZ structure . . . . . . . . . . . . . . . . . . . . PZ gene . . . . . . . . . . . . . . . . . . . . . . PZ synthesis and plasma levels . . . . . . . . . . . Mechanism of inhibition of FXa by the PZ/ZPI complex Physiological function of PZ/ZPI complex . . . . . . PZ and venous thrombosis . . . . . . . . . . . . . PZ and other hypercoagulable states . . . . . . . . -PZ deficiency and antiphospholipid antibodies . -PZ deficiency and obstetrical pathologies . . . Conclusions . . . . . . . . . . . . . . . . . . . . Conflict of Interest Statement . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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Introduction

⁎ Corresponding author at: Medical Genetics, Department of Biomedical Sciences, University of Foggia, Viale Pinto, 71100 Foggia, Italy. Tel.: + 39 0881 733842; fax: + 39 0881 736082. E-mail address: [email protected] (M. Margaglione). 0049-3848/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2011.08.007

Protein Z (PZ) is a vitamin K-dependent (VKD) plasma glycoprotein belonged to different natural anticoagulant systems involved in the regulation of blood coagulation. Specifically, PZ acts as a cofactor in the down-regulation of coagulation by forming a complex with the PZ-dependent protease inhibitor (ZPI) inhibiting the activated factor

V. Bafunno et al. / Thrombosis Research 128 (2011) 508–515

X (FXa). PZ was first identified in bovine plasma by Prowse and Esnouf in 1977 and isolated from human plasma in 1984 by Broze and Miletich [1,2]. It was named Z corresponding to the last character of the alphabet because it is the last of vitamin K-dependent proteins to elute during anion exchange chromatography. Despite its initial characterization, PZ role in normal and pathologic coagulation is still somewhat controversial. In recent years different studies have suggested an association between PZ deficiency and venous thrombosis. The aim of this review is to summarize available data obtained in these studies, which suggest that this anticoagulant system might be relevant to the haemostatic system in vivo and could play a role in the pathogenesis of venous thrombosis. PZ structure PZ was purified from human plasma by a four-step procedure which included barium citrate adsorption of the VKD proteins [2]. Ichinose et al. in 1990 isolated a cDNA coding for human PZ and they found that it is synthesized with a prepro-leader sequence of 40 amino acids [3]. The mature protein has a molecular mass of 62 kDa and contains 360 residues resulting shorter than bovine PZ. This structural difference seems to be important for the interaction of human PZ with human thrombin, since it has been demonstrated that bovine PZ binds to thrombin with the residues 366 to 396 promoting the association of bovine thrombin with phospholipid vesicles. Since these residues are missing in human PZ and it binds thrombin poorly (Kd = 8.9 μM), no physiologically relevant association with thrombin and phospholipids can occur [4]. Complete amino acid sequence of human PZ shares an homology of 59% with the amino acid sequence of bovine PZ. Human PZ presents a modular organization including four domains: a Gla domain (residues 41–86) with 13 γ-carboxyglutamic acid (Gla) residues (residues 47, 48, 51, 55, 57, 60, 61, 66, 67, 70, 73, 75 and 80), two epidermal growth factor (EGF)-like domains – an EGF1 domain (residues 87–123) with a βhydroxyaspartic acid residue at 104, and an EGF2 domain (residues 125– 166) – and a serine protease-like domain (residues 175–360) with high homology to the catalytic domain of other VKD serine proteases [3,5]. PZ structure is similar to that of other VKD factors (factor VII, IX, X, protein C and protein S) but despite these structural similarities, human PZ is not the zymogen of a serine protease (SP), because it has two non-homologous residues in the putative catalytic triad of the SPlike domain: Lys178 instead of His57, and Asp313 instead of Ser195 [6]. The region around the typical activation cleavage site of coagulation factors is also absent in the human PZ. In addition, an O-glycosylation of a serine residue was found in the first EGF-like domain. Four oligosaccharide groups are attached to the Asn 59, Asn 191, Asn 289 and Thr 288 residues [7]. The thrombin cleavage sites are quite different from those described above for α-thrombin on bovine PZ, since thrombin cleaves human PZ in its NH-2 terminal, and thrombin-cleaved human PZ loses its capacity to be absorbed to barium citrate. PZ gene The gene for human PZ, PROZ, is localized to chromosome 13q34 where the genes for factors VII and X exist side by side. This genomic organization and the structure and sequence similarities of the VKD proteins have shown that these proteins may have evolved through a series of duplications and diversification of an ancestral gene at this locus, acquiring a degree of functional diversity in the blood coagulation pathway [8]. The gene spanned about 14 kb and consisted of a 389 bp promoter and nine exons, including one alternative exon, followed by 275 bp of 3` UTR. 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

509

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 VKD plasma proteins. A reporter gene assay identified a minimal promoter region (site A) and two enhancer regions (sites B and C) in the PROZ gene. Sugarawa et al. have shown that the liver-enriched transcriptional factor hepatocyte nuclear factor (HNF)-4a binds to site A, the ubiquitous transcriptional factor Sp1 to sites A and C resulting in a significant increase of PZ promoter activity [9]. PZ synthesis and plasma levels PZ is mainly synthesized in the liver and secreted into the circulation as supported by the strong correlations between PZ and other plasma proteins of liver origin, and the severe deficiency of plasma PZ found in patients with liver disease. Moreover, Vasse et al. have demonstrated, by immunological studies, that PZ was present in the endothelial cells of both normal arterial and venous vessel sections suggesting that these cells could be involved in the production of PZ [10]. Plasma concentrations of PZ vary widely among individuals (0.6-5.7 μg/mL) with an average of 2.9 μg/mL and a SD of 1.0 μg/mL (95% interval of 32% to 168% of the mean) in a sample of 455 American healthy blood donors [11,12]. Moreover, inconsistencies in mean plasma levels have been observed in control groups of different countries with values varying from 1.16 μg/ml in an Australian study [13] to 2.71 μg/ml in an Uruguayan study [14] or with higher levels observed in AfricanAmericans than in white Americans [15]. PZ increases rapidly during the first months of life followed by slow increases during childhood, with adult levels reached during puberty [12]. Genetic and environmental factors may be an important determinant of the wide normal range of PZ plasma concentrations. The human PZ gene is highly polymorphic and a series of common single nucleotide polymorphisms (SNP) in the PZ locus were found. Rice et al. identified, in 95 healthy blood donors, 14 polymorphisms, some of them with a high degree of linkage disequilibrium, and defined up to 10 different haplotypes [16]. Different studies have found a significant association between two PZ polymorphisms and plasma PZ levels: the promoter -13A N G (at the major transcription start site) and the intron F G79A polymorphisms. It has been shown that PZ concentrations were highest among subjects with the A-13 G AA genotype and with the G79A GG genotype, intermediate among those with the A-13 G AG genotype and with the G79A GA genotype, and lowest among those with the A-13 G GG genotype and with the G79A AA genotype, as assessed in control subjects of different countries [17–19]. A polymorphism in the intron C, G-42A, seems also to be linked with different plasma levels of PZ, with the lowest PZ levels for the genotype AA [20]. The mode of secretion of PZ seems to be unique as demonstrated by Souri et al. who performed structure-based examinations of PZ secretion in comparison with that of FX [21]. PZ secretion was much less efficient than FX, was totally dependent upon added vitamin K, and was highly sensitive to warfarin. The authors concluded that the difference observed in secretion patterns of PZ and FX was mainly based on the structure of their γ-carboxyglutamic acid domains. Indeed, some amino acid substitutions of the Gla-30 residue influencing the secretion of PZ have been reported. A nucleotide substitution of G by C in exon II of the PROZ gene, resulting in the replacement of Glu-30 with a Gln residue (E30Q) was identified in a patient with a severe thrombotic tendency, whose plasma PZ level was about 15% of normal. Expression studies revealed that the E30Q mutant was not released from synthesizing cells and interfered with the secretion of the wild type [21]. Similarly, the substitution of Gla-30 by a Lys residue (E30K) is also associated with a defective secretion of PZ [22]. Age is usually considered to play a minor role on PZ plasma levels, as determined in 455 normal healthy adults (aged 19 to 80 years) [12], although a significant negative correlation was recently reported by Hebb et al. [23]. Moreover, the plasma PZ levels

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were found to be higher in men than in women [17,23] while the opposite result was found in another [24]. Treatment with oral anticoagulant induces dramatic decrease of plasma PZ concentration. In particular, in contrast with other VKD proteins, the total PZ antigen level is extremely low in patients on stable warfarin therapy (1 to 16% of normal) [12]. Moreover, the levels of this protein in plasma was found increased with oral contraceptives [25]. Controversial results on the influence of cytokines on plasma PZ levels were described. Undar et al. in 1999 have reported that there was a significant negative correlation between the levels of PZ and IL-6 (p= 0.0084), while similar results were not seen with IL-1β or TNF-α [26]. In contrast, Cesari et al. demonstrated no direct relationship between IL-6 and PZ plasma levels during the acute phase of coronary artery disease [27] and Vasse et al. suggested a possible but weak control of PZ biosynthesis by inflammatory cytokines [28]. PZ level varies significantly in other pathological conditions [29]. Blyth et al. showed that PZ levels were reduced in patients with chronic kidney disease, and not elevated in patients on haemodialysis, arguing against a role for PZ in the bleeding diathesis of renal failure, as assessed in a previous report [30,31]. Severe diminutions of blood plasma PZ were found in patients with chronic liver diseases, disseminated intravascular coagulation (DIC), amyloidosis and thalassemia [11,29,32]. Shang et al. detected plasma levels of PZ of 80 patients with malignant tumors (MT group) and 80 healthy donors and they found that PZ significantly decreases in MT group than in control group (1,210.89 ± 251.13 ng/ml vs. 2,378.83 ± 429.51 ng/ml, p b 0.01). Moreover, the deficiency was more pronounced in the most advanced stages of the tumors [33]. Mechanism of inhibition of FXa by the PZ/ZPI complex Despite its initial isolation, the physiological function of PZ remained uncertain for a long time. It is considered as an example for the -at first sight- paradox action of coagulation proteins [34]. Studies in the 1990s suggested a relevant procoagulant role for this molecule. PZ was initially thought to act by promoting the association of thrombin with phospholipid surfaces, like bovine PZ. This concept arose from several studies investigated in patients with PZ deficiency and bleeding tendency [35,36]. However, the human form of protein binds thrombin poorly and has very little impact on the association of thrombin with phospholipids [37]. The role of PZ in the control of coagulation was clarified by the identification of ZPI. ZPI is an approximately 72-kDa serpin with a plasma concentration of 2.6-2.9 μg/mL that interacts with the active site of FXa by a covalent mechanism similar to other inhibitor serpins [38]. ZPI by itself is a poor inhibitor of FXa, but in the presence of PZ, procoagulant phospholipids, and Ca2+, it produces a rapid inhibition of FXa (t1/2 less than 10 seconds). Kinetic analyses confirmed that PZ acts catalytically to accelerate (~1000-fold) the membrane-dependent

ZPI-FXa reaction [39]. The cofactor action of PZ presumably involves its ability both to bind and bring ZPI to the phospholipid surface, as well as its ability to interact with FXa at this surface [40]. Two potential pathways for PZ-dependent FXa inhibition by ZPI are described. PZ and FXa first form a complex at the phospholipid surface and this complex is subsequently recognized by ZPI. On the other hand, a preformed PZ-ZPI complex binds to the phospholipid surface and interacts with FXa. The final result of either pathway is the formation of a Ca 2+-dependent complex at the phospholipid surface that contains PZ, FXa and ZPI (Fig. 1) [41]. However, as normal plasma contains an excess of ZPI relative to PZ, it is more probable that all PZ was bound in a complex with ZPI [40]. Therefore, the pathway on the right presumably reflects the inhibitory mechanism that occurs in the plasma. Recently, different studies focused on the interaction between PZ and ZPI for the inhibition of FXa. By using chimeric mutants in Gladomain of PZ, Rezaie et al. showed that the ZPI interactive site is located within the C-terminal part of PZ. Moreover, a specific interaction between the Gla domain of PZ and FXa contributes ~6 fold to the acceleration of the ZPI inhibition of FXa on phospholipid membranes, as assessed by binding and kinetic assay [42]. Analysis of the crystal structure of the PZ-ZPI complex confirmed that the specific interaction of the Gla-domains of PZ and FXa optimizes the inhibition of membrane-associated FXa by ZPI and revealed that the interface of PZ and ZPI was stabilized by nine additional hydrogen bonds [43]. By mutagenesis experiments it was established that ZPI Glu-313 residue is crucial in FXa binding, contributing ~5-10 fold to rate acceleration of FXa and FXI inhibition. To understand why ZPI has a Tyr residue instead of an Arg residue in the reactive center, Huang et al. examined an Arg variant of ZPI and they concluded that “unfavourable” Tyr in the reactive center impairs its spontaneous interaction with FXa protecting from the proteolysis by FXa. Moreover, it ensure that the reaction with FXa is significant only when PZ binds and localizes ZPI and FXa on cellular membranes [44]. Like other serpins, ZPI and PZ regulate the activity of FXa with the formation of an acyl-intermediate and the subsequent cleavage of ZPI. Kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (KM 53 ± 5 nm) followed by conversion to a stable ZPI-FXa complex. Then, PZ dissociated from ZPI, whereas ZPI-FXa complex dissociated with a rate constant that showed a pH dependence [45]. The complex between ZPI and FXa is less stable than other serpinproteases complexes and is unable to fully inactivate the activity of FXa, probably due to the inability of ZPI to produce a FXa distortion needed for a complete inhibition. Recent data have shown that heparin is an important cofactor of ZPI anticoagulant function producing 20-100-fold accelerations of ZPI reactions with FXa and FXIa. Heparin binds to a distinct site on ZPI and

Fig. 1. Inhibition of FXa by PZ/ZPI complex. Two mechanisms are proposed to explain the inhibition of FXa by PZ/ZPI: on the left, a pre-formed circulating PZ/ZPI complex binds to the phospholipids and interacts with FXa; on the right, PZ and FXa form a complex at the phospholipids surface and ZPI is fixed thereafter.

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activates ZPI in its physiologically relevant complex with PZ. These results suggest that whereas PZ, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit FXa that escape from a membrane site [46]. Physiological function of PZ/ZPI complex Although the mechanism of inhibition of FXa by the PZ/ZPI complex is being elucidated, the physiological role of this complex remains to be clarified. Combination of PZ and ZPI dramatically delays the initiation and reduces the rate of thrombin generation prior to the formation of the prothrombinase complex. The physiological role of PZ/ZPI complex in the control of coagulation was demonstrated by the analysis of the phenotype of PZ and ZPI deficient mice. In particular, one study evaluated the in vivo consequences of PZ deficiency in a mouse knockout model. Although PZ deficient mice have a normal phenotype, the disruption of the PZ gene in association with homozygous FV Leiden produced a severe prothrombotic phenotype, intrauterine and perinatal thrombosis that lead to mortality in almost all cases. Milder combinations (FV Leiden in heterozygous status and complete PZ deficiency or FV Leiden homozygous and heterozygous PZ deficiency) produced smaller, although significant reductions in survival [47]. In 2008 Zhang et al., using murine gene-deletion models, showed that PZ and ZPI deficiency enhances thrombosis following arterial injury and increases mortality from pulmonary thromboembolism following collagen/epinephrine infusion. On a FV Leiden genetic background, ZPI deficiency produces a significantly more severe phenotype than PZ deficiency, implying that FXIa inhibition by ZPI is physiologically relevant [48]. The studies in mice suggest that human PZ and ZPI deficiency could increase the severity of other thrombotic risk factors, with ZPI deficiency producing a more severe phenotype. As reviewed by Vasse in 2011, the PZ/ZPI complex, apart the anticoagulant activity, may have an anti-inflammation role, like other physiological anticoagulants [49,50]. In addition, some studies have shown that PZ and/or ZPI are expressed by normal kidney and different cancer cells, suggesting that the complex could play a role in avoiding local tissue deposition of fibrin [49]. PZ and venous thrombosis Based on the data from the mouse knockout model by Yin et al. [47], it has been speculated that PZ deficiency could be a modest thrombotic risk factor in humans. The potential role of PZ levels and PZ polymorphisms in the pathogenesis of thrombotic diseases have been investigated in different clinical studies but they have produced conflicting results. Although a functional test for plasma ZPI was recently described [51], no functional assay is available to quantify the cofactor activity of PZ. Therefore, the studies reviewed here used PZ immunological assays. Many of these studies reported low PZ levels in association with an increased risk of thrombosis in several types of vascular diseases, such as ischemic stroke, coronary heart disease, venous thromboembolic disease and fetal loss. On the other side, some studies either did not observe an association or reported an association between high levels of PZ and thrombotic events [52]. Sofi et al. performed a systematic meta-analysis of the available studies and selected 28 case-control studies, including 4,218 patients with thrombotic diseases and 4,778 controls. The overall analysis showed that low PZ levels were associated with an increased risk of thrombosis (odds ratio [OR] 2.90, 95% confidence interval [CI] 2.05– 4.12; p b 0.00001). By separating the studies into three different clinical outcomes, a significant association was found between low PZ levels and arterial vascular diseases (OR 2.67, 95%CI 1.60–4.48; p = 0.0002), pregnancy complications (OR 4.17, 95%CI 2.31–7.52; p b 0.00001), and venous thromboembolic diseases (OR 2.18, 95% CI 1.19–4.00; p = 0.01) [52]. Rather than reviewing the studies on the

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arterial vascular diseases (mainly ischaemic stroke and atherosclerotic vascular diseases), we will focus on the role of PZ deficiency in venous thromboembolism (VTE) and other hypercoagulable states. The clinical studies evaluating the role of PZ in venous thromboembolism and related disorders are summarized in Table 1. Different case–control studies reported that low levels of PZ in patients with venous thromboembolic history are not an independent risk factor for venous thrombosis (VT). In particular, Vasse et al. found that the mean concentration of PZ in the 56 patients with previous history of deep vein thrombosis (DVT) did not differ significantly from the control group [2,11 mg/L (SD = 0,79) and 2,29 mg/L (SD = 0,64), respectively] [25]. In 2005, Al-Shanqeeti et al. measured PZ plasma levels in 426 individuals with VT and 471 control individuals participating in the Leiden Thrombophilia Study but no relationship between the level of PZ and VT was detected in the over-all casecontrol study [54]. Martinelli et al. conducted a large case–control study on 443 unrelated patients with a documented episode of VTE and 394 healthy individuals. Patients and controls presented a similar distribution of PZ levels [1.95 μg/mL vs. 1.79 μg/ mL, p = 0.07] [54]. Accordingly, similar PZ levels were found in 197 Italian patients with DVT (1.44 μg/mL [SD 0.96]) and in 197 age-matched and sex-matched controls (1.44 μg/mL [SD 0.63]) [20]. Two genetic studies have been performed to evaluate the association between functional PZ polymorphisms and DVT. In 2001, Rice et al. identified in PZ gene fourteen novel polymorphisms including the promoter A-13 G polymorphism at the major transcription start site, Arg255His in exon 8, and an insertion or deletion at the 3' end of intron C. These three polymorphisms plus the intron F G79A, were genotyped in 564 DVT patients and 492 age and sex matched controls. There were no differences in genotype frequency between patients and controls. The authors also examined haplotype distribution and linkage disequilibrium within the gene but no association were identified with DVT or pulmonary embolism [16]. Similarly, Santacroce et al. found that frequencies of the intron A G-103A, the intron C G-42A and the intron F G79A polymorphisms were similar in 197 patients with DVT and in 197 controls, and were not associated with very low PZ levels [20]. In contrast with previous studies, the same authors have shown that very low PZ plasma concentrations, below the 5.0 or the 2.5 percentile, occur more frequently among patients with documented DVT (10.2% and 8.5%, respectively) than in controls [4.1% (OR 2.7, 95% CI 1.2–7.3), and 2.0% (OR 4.6, 95% CI 1.5–13.9), respectively] [20]. Pardos-Gea et al. performed a case-control study on 64 patients with VT and they found that the mean PZ plasma levels in patients were significantly lower as compared to control subjects (1,71 ± 0.76 μg/mL vs. 2,44 ± 0.96 μg/mL; p = 0.001). A significant increased risk for VT for the lowest (b1685 ng/mL) quartile of PZ has been found (OR:18, P = 0.007) [55]. Regarding interactions between PZ levels and thrombophilia, a thrombotic synergism between PZ deficiency and FV Leiden was observed in mice [47]. Different studies suggested that low PZ levels may be a mild prothrombotic risk factor, but its procoagulant consequences might increase when combined with additional risk factors. In 2002, Kemkes-Matthes et al. have measured PZ concentrations in 46 consecutive adult FV Leiden patients with thromboembolic complications and in 46 healthy subjects. The median PZ concentration was lower in patients (1.57 μg/mL) than in controls (1.87 μg/mL). Moreover, patients with FV Leiden mutation and low PZ levels experienced first thromboembolic event earlier than FV Leiden patients with higher PZ levels [56]. The same authors reported that the missense change Arg255His in the PZ gene was present in 5-10% of 134 patients carrying FV Leiden who displayed low PZ levels and reported early onset of thromboembolism [57]. This results was not confirmed in a functional analysis performed by Ndonwi et al. in 2008, who showed that the Arg225His substitution seems not affect PZ expression and secretion [58].

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Table 1 Clinical studies evaluating the role of PZ in venous thromboembolism and related disorders. Disease

PZ Levels

SNPs

Patients/ controls (n)

Population

Odds

Results

ratio

Vasse et al. 2001 [53]

VTE

Yes

No

56/88

French

1.20

Al-Shanqeeti et al. 2005 [25] Kemkes-Matthes et al. 2002 [56] Martinelli et al. 2005 [54] Santacroce et al. 2006 [20]

VTE

Yes

No

426/471

American

1.30

VTE

Yes

No

46/46

German

7.76

VTE

Yes

No

443/394

Italian

0.80

VTE

Yes

I A G-103A

197/197

Italian

2.70

I C G-42A I F G79A Pardos-Gea et al. 2008 [55] Rice et al. 2001 [16]

VTE

Yes

No

64

Spanish

18.00

VTE

No

564/492

English

-

Eroglu et al. 2009 [59]

Cancer

No

-

Behçet's disease

No

55 VTE/ 115 no VTE 176/134

Turkish

Ghinoi et al. 2009 [60]

Italian

-

Ozturk et al. 2003 [61]

Behçet's disease

Yes

A-13 G R255H I F G79A I C Ins/del Intron F G79A A-13 G I F G79A No

24/24

Turkish

-

Koren-Michowitz et al. 2005 [63] Lichy et al. 2006 [64]

CRVO

Yes

No

36/42

Israeli

3.00

CVT

No

I F G79A

77/203

German

-

Le Cam-Duchez et al. 2008 [65]

CVT

No

54/100

Caucasian

2.57

Grandone et al. 2009 [62]

VTE

Yes

I F G79A A-13 G I A G-103A I C G-42A

125/102

Italian

7.25

Gris et al. 2002 [70]

FL

Yes

No

450/200

French

6.70

Bretelle et al. 2005 [72]

PC

Yes

No

61/34

French

1.60 (PE) 3.36 (FL)

Paidas et al. 2005 [73]

PC

Yes

No

106/103

American

4.25

Grandone et al. 2004 [76]

FL

Yes

No

124/60

Italian

1.20

Grandone et al. 2008 [77] Erez et al. 2007 [75]

FL

Yes

I C G-42A

7/104

Italian

9.88

PE

Yes

No

130/71

22.60

Topalidou et al. 2009 [79]

FL

Yes

I F G79A

51/47

African-American Caucasian Caucasian

5.88

Similar PZ in patients and controls Similar PZ in patients and controls PZ deficiency and FV Leiden increases VT Similar PZ in patients and controls Similar PZ in patients and controls PZ b 5% and 2% increases VT SNPs are not genetic risk factors Low PZ levels increase the risk of VT SNPs are not genetic risk factors

SNP is not a genetic risk factor SNPs are not genetic risk factors Low PZ levels complicate prothrombotic state Low PZ levels increase the risk of CRVO SNP is not a genetic risk factor SNP is a genetic risk factor

SNP may be associated with pulmonary embolism. PZ deficiency is associated with fetal loss PZ deficiency is associated with fetal loss but not pre-eclampsia PZ deficiency is associated with pregnancy complications PZ deficiency is not associated with fetal loss SNP is a genetic risk factor for FL PZ deficiency is associated with pre-eclampsia PZ deficiency is associated with fetal loss SNP is not a genetic risk factor

VTE, venous thrombolism; I, intron; CRVO, central retinal vein occlusion; CVT, cerebral venous thrombosis; FL, fetal loss; PC, pregnancy complications; PE, pre-eclampsia.

In 2005, Martinelli et al. found a synergic effect on the risk of VTE when low PZ levels (in the lowest quartile) were associated with FV Leiden (18-fold increased risk than for low PZ levels alone), prothrombin G20210A, and hyperhomocysteinemia (four- and sixfold increased risk). Such interactions were still present when very low PZ levels were considered (below the 10th percentile), except that for prothrombin G20210A [54]. VTE is a well-recognized complication of cancer but its pathogenesis is not entirely established. Eroglu et al. investigated for the first time whether G79A SNP in intron F of PZ gene is associated with risk of VTE in cancer patients. No differences were seen in genotype and allele frequencies of PZ gene between a group of 55 cancer patients who developed thrombosis and 115 cancer patients without VTE (pN 0.05). When the analysis was also made after excluding FV Leiden positive patients, the same insignificant result was found [59]. Similarly, Ghinoi

et al. have investigated potential associations between A-13 G and G79A SNPs and VT in Italian patients with Behçet's disease (BD). No association was found with these SNPs in Italian BD patients and, furthermore, these SNPs seemed not to increase the risk of DVT due to FV Leiden or prothrombin gene G20210A mutations [60]. In contrast, Ozturk et al. showed that mean plasma concentration of PZ were significantly lower in BD patients than in healthy controls (107.8 ng/mL vs. 141 ng/mL, p b 0.05) suggesting that alterations of PZ concentration could complicate the pathobiology of the prothrombotic state of BD [61]. Pregnancy-associated VTE is one of the leading single cause of maternal death in many European Countries, and is an important cause of long-term maternal morbidity. Grandone et al. evaluated the prevalence of the G-42A gene variant and the presence of sporadic mutations in a cohort of 125 women with a history of pregnancyrelated VTE (34 with superficial vein thrombosis, 81 with DVT and 10

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with pulmonary embolism) compared to a group of 102 women with uneventful pregnancies. Results of the investigation showed that the G-42A gene variant is significantly associated only with pulmonary embolism. Sporadic mutations within the exon 8 are present in women with fetal loss, but not in those with pregnancy-related VTE (OR 7.2, 95% C.I. 1.05-50.0) [14,62]. Different studies have been performed in order to highlight the role of PZ in other thrombotic conditions, i.e. the central retinal vein occlusion (CRVO) and cerebral venous thrombosis (CVT). Koren-Michowitz et al. found that patients with no risk factors for retinal vessel occlusion had significantly lower PZ levels than controls (1379±682 vs. 2010± 603 ng/mL, p =0.022) [63]. In 2006, Lichy et al. investigated the G79A SNP in intron F in 77 consecutive patients with CVT and in 203 controls selected from the same region of Southern Germany. For this SNP, the frequency of the A-allele in CVT patient was not significantly different from controls (19.5% vs. 24.6%, p =0.31) [64]. Different results have been obtained from Le Cam-Duchez et al. who performed a retrospective genetic study comparing 100 healthy controls to 54 patients with CVT analyzing the distribution of three PZ gene polymorphisms, A-13 G, G79A and G-103A. Among these, only the G79A SNP was significantly more frequent in patients than in controls (OR 2.57, 95% CI 1.23–5.34; p= 0.012) [65]. Nevertheless, these results have to be confirmed by a prospective study including plasma PZ evaluation. PZ and other hypercoagulable states -PZ deficiency and antiphospholipid antibodies McColl and co-workers found significantly lower PZ levels in women with increased levels of antiphospholipid (aPL) antibodies but not in controls and patients with VTE [66]. Similar results have been obtained in patients having lupus anticoagulant (LA) activity, with or without anticardiolipin antibodies [14]. Forastiero and team performed a case-control study to measure plasma levels of PZ and ZPI in 66 patients with autoimmune aPL and 152 normal controls. The prevalence of low PZ levels (below the 5th percentile of controls) was significantly greater in 37 patients with definite antiphospholipid syndrome (APS) (24.3%) but not in the 29 aPL patients not fulfilling the criteria for APS (10.3%). Moreover, they also showed, in vitro, that aPL antibodies impair the inhibition of FXa by the Z/ZPI complex [67]. As anti-PZ antibodies may be associated with low PZ levels, Sailer et al. measured anti-PZ antibodies in 102 LA-patients (69 with and 33 without thrombosis) and 33 healthy volunteers. Elevated anti-PZ antibody levels, especially the IgM subtype, were more prevalent among LA-patients than among controls. However, anti-PZ antibodies are not associated with thrombosis in LA [68]. -PZ deficiency and obstetrical pathologies Changes in the coagulation and fibrinolytic systems during pregnancy lead to a higher risk of thromboembolism. These changes include the increase of many clotting factors, as well as a significant fall in activity of fibrinolytic proteins. In normal pregnancies PZ levels increase of 20% (p= 0.006) from first trimester to delivery and decrease of 30% (pb 0.0001) 6 to 12 weeks after delivery. The normal increase of PZ during pregnancy may balance the increase of clotting factors to protect pregnant women from thrombosis [69]. Different studies have analyzed the role of PZ deficiency, and anti-PZ antibodies, on pregnancy complications including: fetal loss, pre-eclampsia, fetal demise and miscarriage and a small for gestational age (SGA) neonate. Gris et al. reported a high frequency (34.8%) of PZ deficiencies in women with a episode of early fetal loss between the beginning of the 10th and the end of the 15th week of gestation (OR 6.7 [3.1-14.8], p b 0.001) [70]. The same authors described the presence of anti-PZ antibodies (both IgG and IgM) in women with unexplained primary early fetal losses and early fetal death [71]: these antibodies may

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enhance the known maternal hypercoagulability as a possible cause of miscarriage during the 8th and 9th weeks of pregnancy. Anti-PZ antibodies were not correlated with other autoantibodies and their levels did not correlate with plasma PZ concentrations in healthy controls or patients [71]. In 2005, Bretelle et al. conducted a prospective case-control study over a 2-year period to evaluate the prevalence of PZ deficiency in pregnancy complications. Compared with normal pregnancy, the frequency of decreased PZ was significantly higher in cases of intrauterine growth restriction and in intrauterine fetal demise (relative risk [RR] 1.96, 95% CI 1.16-3.32; p = 0.041 and RR 3.36, 95% CI 1.65-6.8; p = 0.0031, respectively), but not in preeclampsia (RR 1.6, 95% CI 0.92.8; p = 0.23). [72]. The association with low PZ levels and different pregnancy complications (growth restriction, intrauterine fetal demise, intra-uterine bleeding) was confirmed in other studies [73–75]. Erez et al. performed a cross-sectional study included normal pregnant women (N= 71), patients with pre-eclampsia (PE) (N= 130), patients who delivered a SGA neonate (N= 58), and patients with fetal demise (N= 58). The authors found that PE, but neither SGA nor fetal demise, is associated with significantly lower maternal median plasma concentration of PZ than normal pregnancy [75]. In contrast, Grandone et al. evaluated PZ levels in a selected group of 124 women (without inherited or acquired thrombophilia factors) with unexplained fetal losses and compared theme with those of a control group of 60 women with uneventful pregnancies. No significant difference in PZ values was observed among two groups (1.37 ± 0.73 μg/mL vs. 1.43 ± 0.76 μg/mL) [76]. Moreover, the authors investigated whether PZ gene polymorphisms or sporadic mutations could be present in 7 women of the same group who showed PZ levels under the 5th percentile (i.e. 0.52 μg/mL). They found that the G-42A variant in intron C of PZ is significantly associated with the occurrence of fetal loss. No association was found for intron A G-103A and intron F G79A. Interestingly, a previously unreported sporadic missense mutation within exon 8 (Leu264Pro) is described in two patient with very low PZ levels [77]. In contrast with these results, a recent investigation analyzing the G79A polymorphism observed that the isolated presence of 79A allele as well as the combination with known thrombophilic risk factors was protective against recurrent pregnancy loss between the 8th and 12th weeks of gestation [78]. However, in a recent study of 51 women with recurrent early pregnancy loss versus 47 control women with at least one successful pregnancy and no spontaneous abortions, this finding was not confirmed [79].

Conclusions Despite extensive clinical investigations performed so far, the role for PZ in the pathogenesis of haemostatic disorders in humans remains to be established. No clear link was evidenced between PZ deficiency, PZ polymorphisms and venous thrombosis and PZ measurement is certainly not useful for this clinical condition. However, the consequences of a PZ deficiency on venous thrombotic disease or obstetrical complications cannot be discarded at this time. The results obtained in different studies and reviewed here, both for plasma PZ levels and polymorphisms, appear often controversial. Some elements may explain these conflicting data: the genetic variation of different control populations, the wide variation of plasma PZ levels in healthy subjects, the limited number of individuals enrolled and the choice of the control groups (healthy individuals or sex and age-crossed controls). Therefore, after reviewing the literature, we suggest that PZ deficiency in a patient with venous thromboembolism cannot currently be considered a biological risk factor for thrombophilia. Consequently, further studies in larger patient cohorts are needed to adequately evaluate the potential risk of venous thrombosis in individuals with PZ deficiency.

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