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Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis
Thrombosis Research (2008) 121, 727–734
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REGULAR ARTICLE
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis ☆ José Pardos-Gea a , José Ordi-Ros a,⁎, Silvia Serrano a , Eva Balada a , Inmaculada Nicolau b , Miquel Vilardell a a
Department of Internal Medicine, Research Unit for Autoimmune Diseases, Spain Department of Haematology, Haemostasis Unit, Vall d’Hebrón University Hospital, Universidad Autónoma de Barcelona, Barcelona, Spain
b
Received 28 February 2007; received in revised form 27 June 2007; accepted 5 July 2007 Available online 14 September 2007
KEYWORDS Anti-Protein Z; Arterial thrombosis; Protein Z; Venous thrombosis; Protein Z deficiency; Thrombophilia
Abstract Introduction: The thrombotic risk associated with protein Z (PZ) deficiency is unclear. Anti-protein Z (anti-PZ) has been described as a risk factor in unexplained embryo demise. The aim of our study was to evaluate a possible PZ deficiency and presence of anti-PZ antibodies on thrombotic diseases. Material and methods: We performed a case-control study on 114 patients with preexisting arterial or venous thrombosis (50 and 64, respectively). Thrombosis was studied based on etiology (creating factor risk subgroups) and on specific thrombotic disease. Results: PZ levels of patients were significantly lower compared to controls (1709 + −761.3 ng/mL vs. 2437 + −964.7 ng/mL P = 0.001). The high arterial risk factor subgroup showed the lowest PZ level (1267.5 + −609 ng/mL) whereas the rest of arterial and venous etiological subgroups presented similar PZ levels. Patients with peripheral artery disease had the lowest PZ level (1022 + −966 ng/mL). The rest of arterial and venous thrombotic diseases presented similar PZ levels. A significant increased risk for arterial and venous thrombosis for the lowest (b 1685 ng/mL) quartile of PZ has been founded (OR:52, P= 0.001 and OR:18, P= 0.007, respectively). Anti-PZ antibodies were negative
Abbreviations: PZ, protein Z; anti-PZ, anti-protein Z; ZPI, protein Z-dependent protease inhibitor; Xa, activated factor X; OD, optical density; AU, arbitrary unit; PBS, phosphate buffered saline; NCEP-III, National Cholesterol Education Program; ANOVA, analysis of variance; SD, standard deviation; OR, Odds ratios. ☆ Author’s contributions: José Pardos-Gea (designed research, performed research, collected data, analyzed data, wrote the paper); José Ordi-Ros (designed research, performed research, revised content); Silvia Serrano, Eva Balada, Inmaculada Nicolau (designed research, contributed vital new reagents and analytical tools); Miquel Vilardell (designed research, revised content). ⁎ Corresponding author. Department of Internal Medicine, Vall d’Hebrón University Hospital, Paseo Vall d’Hebrón 119-129, 08035 Barcelona, Spain. Tel.: +34 93 274 61 67; fax: +34 93 489 40 45. E-mail addresses: [email protected] (J. Pardos-Gea), [email protected] (J. Ordi-Ros). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.07.009
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J. Pardos-Gea et al. in the majority of patients, although mean anti-PZ IgG antibody levels in the arterial thrombosis group were significantly higher compared to venous thrombosis and control groups (P= 0.05 and P= 0.005, respectively). Conclusions: The results suggest that both arterial and venous thrombotic events are related to low PZ levels and that low PZ concentrations are associated with thrombosis in our study. In arterial thrombosis our findings strengthen previous studies that related low PZ levels to atherosclerotic disease. Anti-PZ antibodies do not seem to play a potent role in thrombosis. © 2007 Elsevier Ltd. All rights reserved.
Introduction Protein Z (PZ) was first described in humans in 1984 [1] as a vitamin K-dependent plasma protein with structure similar to those of the coagulation factors VII, IX, X and protein C [2,3]. Its physiological function remained uncertain until Han et al. [4], in 1998, described the protein Z-dependent protease inhibitor (ZPI), a human serpin, member of the superfamily of proteinase inhibitors and synthesized in the liver [5] , produces rapid inhibition of activated factor X (Xa), strictly requiring PZ, Ca ++ and phospholipids [6]. The cofactor role of PZ in factor Xa inhibition by ZPI was the first clearly identified function for human PZ [7]. To investigate the in vivo consequences of PZ deficiency, the PZ gene was disrupted in mice. PZ-null mice had a normal phenotype, but PZ deficiency increased thrombotic manifestations in mice with factor V Leiden [8]. These data led to speculation that PZ deficiency could be a risk factor for thrombosis in human beings. Studies in venous thrombosis have revealed both low [9] and normal [10,11] PZ plasma levels . In arterial thrombosis, studies have found more conflicting results. Coronary heart disease has been related to PZ deficiency [12], but also to normal PZ levels [13]. In ischemic stroke, Vasse et al. [10] were the first to report low PZ levels. Later, other authors have found similar results [14,15]. Anyway, studies relating high [16–18] and normal [19] PZ levels and stroke exist. In antiphospholipid syndrome, low PZ levels have been associated with a 7-fold increased risk of arterial thrombosis [20,21]. Finally, PZ deficiency has been found in women with early fetal demise [22] associated with the presence of antiPZ antibodies [23]. This was the first description of anti-PZ antibodies. The aim of our study was, first, to investigate the relationship between PZ levels and thrombosis in both arterial and venous systems, and second, to clarify the possible presence of anti-PZ antibodies in thrombotic processes.
Materials and methods Patient selection For our study, we recruited a total of 114 subjects (53 male and 61 female) who had suffered from an arterial (group A) or venous (group B) thrombotic event, during a 6-month period (between February, 2004 and July, 2004), and were followed on ambulatory visits at our hospital. Ethics Committee from our hospital approved the study. All patients gave their informed consent. Women with fetal demise or pathologic pregnancies were not included. Patients under warfarin therapy were excluded from enrollment. As oral contraceptives increase PZ levels [11], women taking contraceptive pills were excluded from the study. We recruited patients with ischemic stroke, acute myocardial infarction and peripheral arterial thrombosis as arterial thrombosis samples, and pulmonary embolism, deep vein thrombosis and portal vein thrombosis as venous thrombosis samples. Classical arterial risk factors (i.e., age N 50, male gender, hypertension, dyslipidemia, diabetes, obesity, smoking) and a previous history of arterial thrombotic events were evaluated in each patient. Diabetic subjects were identified in agreement with the American Diabetes Association definition or if the patient so confirmed by medication or chart review. Patients with dyslipidemia were identified in agreement with the National Cholesterol Education Program (NCEP-III) definition or if the patients reported taking specific medication. Hypertensive patients were identified according to guidelines of European Society of Hypertension/ Cardiology or if the patients reported taking specific medication. Obesity was defined as body mass index N 30 kg m− 2. Patients were also investigated for classical venous thrombotic risk factors (i.e., surgery fewer than 6 months earlier, immobility, neoplasm), for venous thrombotic antecedents such as deep venous thrombosis or pulmonary embolism, and for thrombophilic factors as described (essential thrombocythemia; antiphospholipid/anticofactor antibodies; dysfibrinogenemia; deficiency in antithrombin, protein C or protein S; factor V Leiden mutation; prothrombin gene mutation; high plasminogen activator inhibitor − 1 plasma levels; and hyperhomocysteinemia). To detect possible vitamin K deficiency a prothrombin time test was performed on all patients and controls. Both groups of patients (A and B) were thus studied under two points of view. First, one related to the clinical entities or thrombotic diseases they suffered from. Second, depending on the etiology of the thrombotic disease, using the risk factors previously evaluated in order to make three etiological subgroups from groups A and B; low risk factor subgroup (if one or no arterial/venous risk factor was present), high risk factor subgroup (if two or more classical arterial/venous risk factors were present or a previous arterial/venous thrombotic disease existed) and thrombophilic factor subgroup (if a thrombophilic factor existed). A control group of 82 healthy donor individuals from our hospital’s
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis Table 1
Demographic characteristics and traditional arterial and venous risk factors in patients and controls
Number (n) Male/Female (n) Median Age (years) a Male gender (n) Hypertension (n) Smoking habit (n) Dyslipidemia (n) Diabetes (n) Obesity (n) Previous arterial thr. (n) Immobility (n) Recent surgery (n) Cancer (n) Previous venous thr. (n) a
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Group A (Arterial Thr.)
Group B (Venous Thr.)
Controls
P-value
50 23/27 52 23 16 13 15 5 1 4 – – – –
64 30/34 50 – – – – – – – 15 10 8 11
82 34/48 45 34 3 5 6 0 0 0 0 0 0 0
0.3 0.6 0.09 0.002 0.01 0.01 0.03 0.18 0.04 0.001 0.002 0.01 0.008
Age at the time of thrombosis for patients and at blood sampling for controls.
blood bank were recruited, and arterial and venous classical risk factors evaluated.
Methods Blood samples were collected throughout the study a mean of 60 days after the thrombotic event as citrate anticoagulated plasma samples and were processed and stored at −80 °C. PZ concentrations were measured by means of a commercially available enzymelinked immunosorbent assay (Asserachrom Protein Z; Diagnostica Stago and Serbio, Asnières, France). Anti-PZ antibodies were determined by a homemade ELISA. Each well was coated with 62.5 ng of human PZ (Hyphen BioMed, Neuville sur Oise, France) diluted in phosphate buffered saline (PBS) or with PBS alone (to determine the non-specific binding of each sample). A blocking and dilution buffer consisted of bovine serum albumine 1% diluted with PBS-Tween20 1%. Plasmas (50 μl diluted 1:50) were added in duplicate to the wells and were incubated for 1 h and a half at room temperature. After six washes, the plates were incubated for one hour at room temperature with alkaline phosphatase-labeled goat anti-human IgG (or IgM) (1:1000 in the blocking solution) (Sigma, Madrid, Spain). After washing, color was developed by
Table 2
adding p-nitrophenylphosphate (Sigma) in its corresponding buffer (1 M diethanolamine, 0.5 mM MgCl2, 0.22 M NaCl, pH 9.5) for 15 min at room temperature. The optical density (OD) of each well was read at 405 nm in a spectrophotometer (Labsystems iEMS reader MF, Barcelona, Spain). The non-specific background OD (wells without the antigen) of each sample was substracted from the corresponding tested sample (wells with the antigen). We initially tested a panel of 50 healthy individuals together with different dilutions; the mean absorbance value in healthy individuals plus 2 standard deviations was said to correspond, by definition, to 1 arbitrary unit (AU) of anti-protein Z IgG or IgM antibodies. This represented the cut-off level of our study. The between and withinassay coefficients of variation evaluated were lower than 1.5%.
Statistical analysis A statistical analysis was performed using SPSS software for Windows (version 12.0). The Kolmogorov–Smirnov test and the Levene test were performed in patients and controls in order to determine normal or abnormal distribution of variables (PZ and anti-PZ antibodies) and homogeneity of variances, respectively. The association between continuous biomarkers was examined
Clinical entities and etiological subgroup classification of patients
Number (n) Clinical entities (n)
Etiological subgroups 1) Low Arterial or Venous risk factor subgroup. [n(%)] 2) High Arterial or Venous risk factor subgroup. [n(%)] 3) Thrombophilia subgroup [n(%)] Antithrombin III def. Factor V Leiden Hyperhomocysteinemia Antiphospholipid Antib. Prothrombin G20210A
Group A (Arterial Thr.)
Group B (Venous Thr.)
50 Ischemic stroke (20) Acute myocardial infarction (15) Peripheral arterial thrombosis (15)
64 Pulmonary embolism (24) Deep vein thrombosis (29) Portal vein thrombosis (11)
27 (53%)
28 (44%)
15 (30%)
13 (20%)
8 (17%) 1 3 2 1 1
23 (36%) 2 6 6 4 5
730
J. Pardos-Gea et al. standard deviation (SD). The relationships between the global risk of arterial or venous thrombotic event and the levels of PZ were assessed by means of logistic regression analysis, introducing into the analysis the possible confounding variables for both thrombotic diseases. Odds ratios (OR) were calculated as estimates of the relative risk with 95% confidence intervals. The PZ levels of the patients were divided into quartiles according to the distribution of protein Z of our control group (first quartile b1685 ng/mL, second quartile 1685–2478 ng/mL, third quartile 2478–3270 ng/mL; fourth quartile N3270 ng/mL).
Results The baseline characteristics of patients and controls such as age and sex were not significantly different between groups, but the presence of arterial and venous thrombosis risk factors in group A and B, respectively, were found to be significantly more prevalent with respect to control subjects (Table 1). The specific characteristics of the groups studied are shown in Table 2.
Figure 1 Protein Z plasma levels in patients and controls. Bold line represents mean protein Z plasma levels. Group A: patients with arterial thrombosis; Group B: patients with venous thrombosis.
by Spearman correlation coefficients. Anti-PZ antibodies required the use of nonparametric statistics such as Mann–Whitney rank-sum test and Kruskal–Wallis analysis of variance (ANOVA) for comparisons of cases and controls. PZ concentration showed normal distribution and equality of variances in patients and controls and we used the unpaired samples t-test and ANOVA test in the statistical analysis. Results are presented as mean ±
Protein Z PZ levels in our study did not correlate with age (P = 0.85). Also, no relationship existed between PZ levels and time elapsed from thrombotic event to blood sample collection (P = 0.97). The mean (± SD) PZ plasma levels in patients were found to be significantly lower as compared to control subjects (1709 + − 761.3 ng/mL vs. 2437 + − 964.7 ng/mL; P = 0.001, unpaired samples t-test). As shown in Fig. 1, mean PZ levels from both groups (Arterial-group A: 1663.5 + − 792.3 ng/mL; Venous-group B: 1742.4 ± 742.4 ng/mL) of patients were also significantly lower than controls (P = 0.001 and P = 0.002, respectively, ANOVA test). However, with respect to group A (arterial) being compared to group B (venous), no significant differences in mean PZ level were observed.
Figure 2 Protein Z plasma levels in clinical entities (left) and etiological subgroups (right). Bold line represents mean protein Z plasma levels. Group A: patients with arterial thrombosis; Group B: patients with venous thrombosis. PE indicates pulmonary embolism; DVT, deep vein thrombosis; PVT, portal vein thrombosis; AMI, acute myocardial infarction; PAT, peripheral artery thrombosis.
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis Table 3
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Crude and adjusted relative risk according to quartiles of protein Z Patients
OR Group A Fourth quartile
1
Third quartile Second quartile First quartile
6 17 26
Group B Fourth quartile
2
Third quartile Second quartile First quartile
10 21 31
Multivariate a
Univariate
Reference group 5.4 15.4 26
Reference group 4.5 10 15
95%CI
0.6–49 1.6–116 3.2–210
0.8–23 2.1–50 3.2–73
P
OR
95%CI
P
0.132 0.015 0.002
Reference group 7.7 28.1 52.1
0.3–48 1.9–227 3.8–400
0.09 0.005 0.001
0.06 0.003 0.001
Reference group 4.5 13 18
0.5–48 1.7–120 2.5–170
0.18 0.01 0.007
Patients indicate number in each subgroup. OR indicates odds ratio; 95% CI indicates 95% confidence interval; P indicates P–value. Group A: patients with arterial thrombosis. Group B: patients with venous thrombosis. Quartile (Protein Z level). a Group A adjusted for age, gender, hypertension, smoking habit, dyslipidemia, obesity and diabetes; history of previous arterial thrombosis. Group B adjusted for immobility, recent surgery, cancer, history of previous venous thrombosis.
The comparison between arterial and venous thrombotic entities together was performed (ANOVA test), evaluating mean PZ plasma levels (Fig. 2). Patients with peripheral artery thrombosis had the lowest mean PZ level (1022 ± 966 ng/mL) , with a significant difference as compared with patients with stroke (1764 ± 738 ng/mL; P = 0.02), acute myocardial infarction (1842 ± 608 ng/mL; P b 0.05) and pulmonary embolism (1929 ± 896; P = 0.006). Among the rest of the groups no statistical differences were found. We then evaluated PZ levels in the etiological subgroups (Fig. 2). Patients from high arterial risk factor subgroup had the lowest mean PZ level (1267.5 ± 609 ng/mL) , with a significant
difference as compared with patients included in arterial low risk factor subgroup (1873.2 ± 751 ng/mL; P = 0.03) and venous high risk factor subgroup (1918.9 ±850 ng/mL; P = 0.04). No differences were found with the other groups. In order to test if decreasing levels of protein Z were a predisposing factor for the occurrence of thrombosis a logistic regression analysis was carried out. In patients (Table 3), the univariate analysis showed a significant increased risk for thrombosis for the lowest quartiles of protein Z (first and second quartiles), as compared with the highest quartile (fourth quartile) of both arterial and venous thrombosis groups. Between groups, we observed a greater risk of thrombosis for the arterial
Figure 3 Anti-PZ IgG and IgM in patients and controls. Data are given in arbitrary units (AU) as described. Bold line represents mean anti-PZ levels. Asterisks represent individual values. Group A: patients with arterial thrombosis; Group B: patients with venous thrombosis. The horizontal line represents the 1-AU level.
732 group of patients. After adjustment for confounding variables in the multivariate analysis, this association persisted.
Anti-protein Z The majority of the patients presented negative anti-PZ antibodies under cut-off level (Fig. 3). When comparing mean anti-PZ IgG levels of all the patients included in arterial thrombosis group (group A) and control group, we found significantly higher levels of anti-PZ IgG in arterial thrombosis patients (Mann–Whitney rank-sum test; P = 0.005). Between venous thrombosis (group B) and control subjects no differences existed (P = 0.372). Lastly, a higher mean rank of anti-PZ IgG antibody levels were detected in the arterial thrombosis group when compared to the venous thrombosis group B (P = 0.05). For anti-PZ IgM levels no differences between groups were observed. We finally studied anti-PZ antibodies on clinical entities and etiological subgroups, and no significant differences between groups were observed (data not shown). We found a moderate inverse correlation between anti-PZ IgG antibody levels and PZ concentration in the global analysis of controls and patients (Spearman correlation coefficient [p] of − 0.20 with P = 0.04) (data not shown). Anti-PZ IgM antibody levels were not correlated with plasma PZ concentrations .We thus looked separately at control and patient groups, finding no correlation within groups (P = 0.07 and P = 0.13, respectively). Anti-PZ IgG and IgM antibody levels were not correlated with age (P = 0.96 and 0.40, respectively).
Discussion On the comparison between arterial and venous thrombosis groups, our data demonstrate no significant differences in PZ levels. Moreover, the majority of the venous and arterial thrombotic clinical entities and etiological subgroups studied present similar PZ levels. Only one previous study [10], compared an arterial (stroke) and a venous (deep vein thrombosis) thrombotic event. In that study only patients with arterial thrombosis presented lower PZ levels than controls. Our results on venous thrombosis contradict those from previous studies. Al-Shanqeeti et al. [11] evaluated PZ levels of 426 individuals with deep vein thrombosis, discovering no differences with control group. In 2005, Martinelli et al. [9] compared the PZ levels of 443 patients with an initial episode of deep vein thrombosis versus a control group founding that low PZ levels were not an independent risk factor for deep vein thrombosis, but seemed to increase the risk associated with established causes of thrombophilia. We cannot explain our apparently conflicting results, although the inclusion of clinical entities not previously studied as pulmonary embolism and portal vein thrombosis may have influenced results. Finally to comment a recent article by Santacroce et al. [24] that founded relationship between very low PZ levels and deep vein thrombosis, in line with our results, but with a weaker association. Concerning etiology, in our subgroup analysis, the presence of either venous
J. Pardos-Gea et al. thrombosis risk factors or a thrombophilic disorder is not associated with variations in PZ levels. We could conclude that the baseline prothrombotic condition of patients does not determine plasma PZ levels. That fact makes the acute thrombotic process in itself a possible cause or effect of the low PZ levels we found in venous thrombotic pathologies, although this is only a theory and more studies are warranted. Our findings on arterial thrombosis have been in accordance with previous studies. Patients with peripheral artery disease and those with high risk factors for arterial thrombosis have had the lowest PZ levels in each subgroup. Basic research has demonstrated that PZ is present in endothelial cells and in the proliferating subendothelial space of atherosclerotic lesions of patients with different risk factors [25]. Recently, Vasse et al. [26] demonstrated that normal human endothelial cells from artery or venous vessels synthesize PZ. Clinical studies on arterial thrombosis have only investigated coronary vascular disease and stroke. In a study by Fedi et al. [12] patients with acute coronary thrombosis presented lower PZ levels than controls. The patients presented significantly more arterial classical risk factors compared to controls, and probably a more seriously chronic vascular atherosclerotic disease. Sofi et al. [27] conducted a study that included patients with stable coronary chronic disease, patients with acute or unstable coronary disease and healthy controls. They found significantly lower levels of PZ in patients with respect to controls, but no differences between patients in the stable or unstable form of coronary atherosclerotic disease. In stroke some studies that included younger-age samples without macro or microangiopathy [16,19] showed normal or high PZ levels; in contrast other studies [17,18] with older population and suspected atheromatosis presented same PZ levels. Therefore, and at least in coronary vascular disease, it would seem that chronic atherosclerotic vascular disease, and not the acute thrombotic process, would be the pathological entity related to low PZ levels. Previous studies support the fact that the acute thrombotic event does not seem to have so many influence in PZ levels in arterial thrombotic events. Staton et al. [18], found that the difference on PZ levels in stroke between the first 7 days and the follow-up period (3–6 months), was not significantly different (P= 0.07). Also Vasse [28] described no relationship between cytokines and PZ biosynthesis. We think that dysfunctional endothelium and deposits on proliferating subendothelial space of atherosclerotic lesions could be some of the reasons that explain the possible relationship between atherosclerosis and low PZ plasma levels. In our patients from arterial high risk factor thrombosis subgroup we can assume the presence of a higher degree of atherosclerotic
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis disease, and explain the low PZ levels founded. A possible mechanism to explain the low PZ levels obtained in our patients with peripheral artery disease compared to those with stroke could be that peripheral artery disease, as macroangiopathy, could reflect a more extended involvement of vessels by atherosclerosis compared to stroke. The first and only report on anti-PZ antibodies was by Gris et al. [23]. They find that anti-PZ IgG and IgM levels were higher in women with pregnancy loss compared to healthy mothers. The overall risk of pathologic pregnancy climbed with increasing antiPZ concentration. Based on the results of our study, we must say, firstly, that mean anti-PZ IgG and IgM antibody levels have been truly low and mainly below the 1 AU level. This represents a clear difference in relation to the levels seen in the study by Gris et al. [23], in which patients and controls presented higher mean antibody levels. This difference could mean that anti-PZ antibodies in systemic thrombotic processes do not play such a potent role as they do in pregnancy pathology. Nevertheless, we have found a higher number of patients with positive anti-PZ IgG antibody levels in patients with arterial thrombotic disease compared to those with venous thrombosis and the control group, and a higher mean rank of anti-PZ IgG . Despite this fact, we consider that our results indicate no relationship between anti-PZ antibodies and thrombosis. The study has some potential limitations. First, it is an observational case-control study, and thus we cannot distinguish primary from secondary effects and can document associations only. Second, the limited number of subjects interferes with the reliability of our results. Third, we have arbitrarily considered that two risk factors for arterial or venous thrombosis separate both the high from the low-risk factor population, and that point could be worthy of criticism. Fourth, we did not estimate the risk associated with presence of anti-PZ antibodies because of the low levels of autoantibodies we found, and because of the lack of a comparative population with a known high risk for thrombosis. In conclusion, our results suggest that both arterial and venous thrombotic events are related to low PZ levels and that low PZ concentrations are associated with thrombosis. Moreover, the majority of the venous and arterial thrombotic clinical entities studied presented similar PZ levels. We have observed a possible relationship between chronic atherosclerotic disease and decreased PZ levels suggesting a possible role of low levels of this protein in its pathophysiology. Lastly, anti-PZ antibodies in arterial and venous thrombotic processes do not seem to have such a relevant function in
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pathogenesis as they do in pathologic pregnancies, although anti-PZ IgG antibodies in arterial thrombotic processes warrants further investigation. Ongoing investigations should better define the possible roles of both PZ and anti-PZ antibodies in thrombosis.
Acknowledgement We would like to thank all the people in the Unit of Haemostasia of our Hospital for its contribution to the work.
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734 [16] Kobelt K, Biasiutti FD, Mattle HP, Lammle B, Wuillemin WA. Protein Z in ischaemic stroke. Br J Haematol 2001;114(1): 169–73. [17] McQuillan AM, Eilkelboom JW, Hankey GJ, Baker R, Thom J, Staton J, et al. Protein Z in ischemic stroke and its etiologic subtypes. Stroke 2003;34(10):2415–9. [18] Staton J, Sayer M, Hankey GJ, Cole V, Thom J, Eikelboom JW. Protein Z gene polymorphisms, protein Z concentrations, and ischemic stroke. Stroke 2005;36(6):1123–7. [19] Lopaciuk S, Bykowska K, Kwiecinski H, Cylonkowska A, Kucynska-Zardzewialy A. Protein Z in young survivors of ischemic stroke. Thromb Haemost 2002;88(3):536. [20] McColl MD, Deans A, Maclean P, Tait RC, Greer IA, Walker ID. Plasma protein Z deficiency is common in women with antiphospholipid antibodies. Br J Haematol 2003;120(5): 913–4. [21] Forastiero RR, Martinuzzo ME, Lu L, Broze GJ. Autoimmune antiphospholipid antibodies impair the inhibition of activated factor X by protein Z/protein Z-dependent protease inhibitor. J Thromb Haemost 2003;1(8):1764–70. [22] Gris JC, Quere I, Dechaud H, Mercier E, Pincon C, Hoffet M, et al. High frequency of protein Z deficiency in patients with unexplained early fetal loss. Blood 2002;99(7):2606–8.
J. Pardos-Gea et al. [23] Gris JC, Amadio C, Mercier E, Lavigne-Lissalde G, Dechaud H, Hoffet M, et al. Anti-protein Z antibodies in women with pathologic pregnancies. Blood 2003;101(12): 4850–2. [24] Santacroce R, Sarno M, Capucci F, Sessa F, Colaizzo D, Brancaccio V, et al. Low protein Z levels and risk of occurrence of deep vein thrombosis. J Thromb Haemost 2006;4(11): 2417–22. [25] Greten J, Kreis I, Liliensiek B, Allenberg J, Amiral J, Ziegler R, et al. Localisation of protein Z in vascular lesions of patients with atherosclerosis. Vasa 1998;27(3):144–8. [26] Vasse M, Denoyelle C, Corbière C, Litzler PY, Legrand E, Vannier JP. Human endothelial cells synthesize protein Z, but not the protein Z dependent inhibitor. Thromb Haemost 2006;95(3):519–23. [27] Sofi F, Cesari F, Vigiani S, Fatini C, Marcucci R, Giglioli C, et al. Protein Z plasma levels in different phases of activity of coronary atherosclerosis. J Thromb Haemost 2005;3(10): 2254–8. [28] Vasse M, Denoyelle C, Legrand E, Vannier JP, Soria C. Weak regulation of Protein Z biosynthesis by inflammatory cytokines. Thromb Haemost 2002;87(2):350–1.
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis ☆ José Pardos-Gea a , José Ordi-Ros a,⁎, Silvia Serrano a , Eva Balada a , Inmaculada Nicolau b , Miquel Vilardell a a
Department of Internal Medicine, Research Unit for Autoimmune Diseases, Spain Department of Haematology, Haemostasis Unit, Vall d’Hebrón University Hospital, Universidad Autónoma de Barcelona, Barcelona, Spain
b
Received 28 February 2007; received in revised form 27 June 2007; accepted 5 July 2007 Available online 14 September 2007
KEYWORDS Anti-Protein Z; Arterial thrombosis; Protein Z; Venous thrombosis; Protein Z deficiency; Thrombophilia
Abstract Introduction: The thrombotic risk associated with protein Z (PZ) deficiency is unclear. Anti-protein Z (anti-PZ) has been described as a risk factor in unexplained embryo demise. The aim of our study was to evaluate a possible PZ deficiency and presence of anti-PZ antibodies on thrombotic diseases. Material and methods: We performed a case-control study on 114 patients with preexisting arterial or venous thrombosis (50 and 64, respectively). Thrombosis was studied based on etiology (creating factor risk subgroups) and on specific thrombotic disease. Results: PZ levels of patients were significantly lower compared to controls (1709 + −761.3 ng/mL vs. 2437 + −964.7 ng/mL P = 0.001). The high arterial risk factor subgroup showed the lowest PZ level (1267.5 + −609 ng/mL) whereas the rest of arterial and venous etiological subgroups presented similar PZ levels. Patients with peripheral artery disease had the lowest PZ level (1022 + −966 ng/mL). The rest of arterial and venous thrombotic diseases presented similar PZ levels. A significant increased risk for arterial and venous thrombosis for the lowest (b 1685 ng/mL) quartile of PZ has been founded (OR:52, P= 0.001 and OR:18, P= 0.007, respectively). Anti-PZ antibodies were negative
Abbreviations: PZ, protein Z; anti-PZ, anti-protein Z; ZPI, protein Z-dependent protease inhibitor; Xa, activated factor X; OD, optical density; AU, arbitrary unit; PBS, phosphate buffered saline; NCEP-III, National Cholesterol Education Program; ANOVA, analysis of variance; SD, standard deviation; OR, Odds ratios. ☆ Author’s contributions: José Pardos-Gea (designed research, performed research, collected data, analyzed data, wrote the paper); José Ordi-Ros (designed research, performed research, revised content); Silvia Serrano, Eva Balada, Inmaculada Nicolau (designed research, contributed vital new reagents and analytical tools); Miquel Vilardell (designed research, revised content). ⁎ Corresponding author. Department of Internal Medicine, Vall d’Hebrón University Hospital, Paseo Vall d’Hebrón 119-129, 08035 Barcelona, Spain. Tel.: +34 93 274 61 67; fax: +34 93 489 40 45. E-mail addresses: [email protected] (J. Pardos-Gea), [email protected] (J. Ordi-Ros). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2007.07.009
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J. Pardos-Gea et al. in the majority of patients, although mean anti-PZ IgG antibody levels in the arterial thrombosis group were significantly higher compared to venous thrombosis and control groups (P= 0.05 and P= 0.005, respectively). Conclusions: The results suggest that both arterial and venous thrombotic events are related to low PZ levels and that low PZ concentrations are associated with thrombosis in our study. In arterial thrombosis our findings strengthen previous studies that related low PZ levels to atherosclerotic disease. Anti-PZ antibodies do not seem to play a potent role in thrombosis. © 2007 Elsevier Ltd. All rights reserved.
Introduction Protein Z (PZ) was first described in humans in 1984 [1] as a vitamin K-dependent plasma protein with structure similar to those of the coagulation factors VII, IX, X and protein C [2,3]. Its physiological function remained uncertain until Han et al. [4], in 1998, described the protein Z-dependent protease inhibitor (ZPI), a human serpin, member of the superfamily of proteinase inhibitors and synthesized in the liver [5] , produces rapid inhibition of activated factor X (Xa), strictly requiring PZ, Ca ++ and phospholipids [6]. The cofactor role of PZ in factor Xa inhibition by ZPI was the first clearly identified function for human PZ [7]. To investigate the in vivo consequences of PZ deficiency, the PZ gene was disrupted in mice. PZ-null mice had a normal phenotype, but PZ deficiency increased thrombotic manifestations in mice with factor V Leiden [8]. These data led to speculation that PZ deficiency could be a risk factor for thrombosis in human beings. Studies in venous thrombosis have revealed both low [9] and normal [10,11] PZ plasma levels . In arterial thrombosis, studies have found more conflicting results. Coronary heart disease has been related to PZ deficiency [12], but also to normal PZ levels [13]. In ischemic stroke, Vasse et al. [10] were the first to report low PZ levels. Later, other authors have found similar results [14,15]. Anyway, studies relating high [16–18] and normal [19] PZ levels and stroke exist. In antiphospholipid syndrome, low PZ levels have been associated with a 7-fold increased risk of arterial thrombosis [20,21]. Finally, PZ deficiency has been found in women with early fetal demise [22] associated with the presence of antiPZ antibodies [23]. This was the first description of anti-PZ antibodies. The aim of our study was, first, to investigate the relationship between PZ levels and thrombosis in both arterial and venous systems, and second, to clarify the possible presence of anti-PZ antibodies in thrombotic processes.
Materials and methods Patient selection For our study, we recruited a total of 114 subjects (53 male and 61 female) who had suffered from an arterial (group A) or venous (group B) thrombotic event, during a 6-month period (between February, 2004 and July, 2004), and were followed on ambulatory visits at our hospital. Ethics Committee from our hospital approved the study. All patients gave their informed consent. Women with fetal demise or pathologic pregnancies were not included. Patients under warfarin therapy were excluded from enrollment. As oral contraceptives increase PZ levels [11], women taking contraceptive pills were excluded from the study. We recruited patients with ischemic stroke, acute myocardial infarction and peripheral arterial thrombosis as arterial thrombosis samples, and pulmonary embolism, deep vein thrombosis and portal vein thrombosis as venous thrombosis samples. Classical arterial risk factors (i.e., age N 50, male gender, hypertension, dyslipidemia, diabetes, obesity, smoking) and a previous history of arterial thrombotic events were evaluated in each patient. Diabetic subjects were identified in agreement with the American Diabetes Association definition or if the patient so confirmed by medication or chart review. Patients with dyslipidemia were identified in agreement with the National Cholesterol Education Program (NCEP-III) definition or if the patients reported taking specific medication. Hypertensive patients were identified according to guidelines of European Society of Hypertension/ Cardiology or if the patients reported taking specific medication. Obesity was defined as body mass index N 30 kg m− 2. Patients were also investigated for classical venous thrombotic risk factors (i.e., surgery fewer than 6 months earlier, immobility, neoplasm), for venous thrombotic antecedents such as deep venous thrombosis or pulmonary embolism, and for thrombophilic factors as described (essential thrombocythemia; antiphospholipid/anticofactor antibodies; dysfibrinogenemia; deficiency in antithrombin, protein C or protein S; factor V Leiden mutation; prothrombin gene mutation; high plasminogen activator inhibitor − 1 plasma levels; and hyperhomocysteinemia). To detect possible vitamin K deficiency a prothrombin time test was performed on all patients and controls. Both groups of patients (A and B) were thus studied under two points of view. First, one related to the clinical entities or thrombotic diseases they suffered from. Second, depending on the etiology of the thrombotic disease, using the risk factors previously evaluated in order to make three etiological subgroups from groups A and B; low risk factor subgroup (if one or no arterial/venous risk factor was present), high risk factor subgroup (if two or more classical arterial/venous risk factors were present or a previous arterial/venous thrombotic disease existed) and thrombophilic factor subgroup (if a thrombophilic factor existed). A control group of 82 healthy donor individuals from our hospital’s
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis Table 1
Demographic characteristics and traditional arterial and venous risk factors in patients and controls
Number (n) Male/Female (n) Median Age (years) a Male gender (n) Hypertension (n) Smoking habit (n) Dyslipidemia (n) Diabetes (n) Obesity (n) Previous arterial thr. (n) Immobility (n) Recent surgery (n) Cancer (n) Previous venous thr. (n) a
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Group A (Arterial Thr.)
Group B (Venous Thr.)
Controls
P-value
50 23/27 52 23 16 13 15 5 1 4 – – – –
64 30/34 50 – – – – – – – 15 10 8 11
82 34/48 45 34 3 5 6 0 0 0 0 0 0 0
0.3 0.6 0.09 0.002 0.01 0.01 0.03 0.18 0.04 0.001 0.002 0.01 0.008
Age at the time of thrombosis for patients and at blood sampling for controls.
blood bank were recruited, and arterial and venous classical risk factors evaluated.
Methods Blood samples were collected throughout the study a mean of 60 days after the thrombotic event as citrate anticoagulated plasma samples and were processed and stored at −80 °C. PZ concentrations were measured by means of a commercially available enzymelinked immunosorbent assay (Asserachrom Protein Z; Diagnostica Stago and Serbio, Asnières, France). Anti-PZ antibodies were determined by a homemade ELISA. Each well was coated with 62.5 ng of human PZ (Hyphen BioMed, Neuville sur Oise, France) diluted in phosphate buffered saline (PBS) or with PBS alone (to determine the non-specific binding of each sample). A blocking and dilution buffer consisted of bovine serum albumine 1% diluted with PBS-Tween20 1%. Plasmas (50 μl diluted 1:50) were added in duplicate to the wells and were incubated for 1 h and a half at room temperature. After six washes, the plates were incubated for one hour at room temperature with alkaline phosphatase-labeled goat anti-human IgG (or IgM) (1:1000 in the blocking solution) (Sigma, Madrid, Spain). After washing, color was developed by
Table 2
adding p-nitrophenylphosphate (Sigma) in its corresponding buffer (1 M diethanolamine, 0.5 mM MgCl2, 0.22 M NaCl, pH 9.5) for 15 min at room temperature. The optical density (OD) of each well was read at 405 nm in a spectrophotometer (Labsystems iEMS reader MF, Barcelona, Spain). The non-specific background OD (wells without the antigen) of each sample was substracted from the corresponding tested sample (wells with the antigen). We initially tested a panel of 50 healthy individuals together with different dilutions; the mean absorbance value in healthy individuals plus 2 standard deviations was said to correspond, by definition, to 1 arbitrary unit (AU) of anti-protein Z IgG or IgM antibodies. This represented the cut-off level of our study. The between and withinassay coefficients of variation evaluated were lower than 1.5%.
Statistical analysis A statistical analysis was performed using SPSS software for Windows (version 12.0). The Kolmogorov–Smirnov test and the Levene test were performed in patients and controls in order to determine normal or abnormal distribution of variables (PZ and anti-PZ antibodies) and homogeneity of variances, respectively. The association between continuous biomarkers was examined
Clinical entities and etiological subgroup classification of patients
Number (n) Clinical entities (n)
Etiological subgroups 1) Low Arterial or Venous risk factor subgroup. [n(%)] 2) High Arterial or Venous risk factor subgroup. [n(%)] 3) Thrombophilia subgroup [n(%)] Antithrombin III def. Factor V Leiden Hyperhomocysteinemia Antiphospholipid Antib. Prothrombin G20210A
Group A (Arterial Thr.)
Group B (Venous Thr.)
50 Ischemic stroke (20) Acute myocardial infarction (15) Peripheral arterial thrombosis (15)
64 Pulmonary embolism (24) Deep vein thrombosis (29) Portal vein thrombosis (11)
27 (53%)
28 (44%)
15 (30%)
13 (20%)
8 (17%) 1 3 2 1 1
23 (36%) 2 6 6 4 5
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J. Pardos-Gea et al. standard deviation (SD). The relationships between the global risk of arterial or venous thrombotic event and the levels of PZ were assessed by means of logistic regression analysis, introducing into the analysis the possible confounding variables for both thrombotic diseases. Odds ratios (OR) were calculated as estimates of the relative risk with 95% confidence intervals. The PZ levels of the patients were divided into quartiles according to the distribution of protein Z of our control group (first quartile b1685 ng/mL, second quartile 1685–2478 ng/mL, third quartile 2478–3270 ng/mL; fourth quartile N3270 ng/mL).
Results The baseline characteristics of patients and controls such as age and sex were not significantly different between groups, but the presence of arterial and venous thrombosis risk factors in group A and B, respectively, were found to be significantly more prevalent with respect to control subjects (Table 1). The specific characteristics of the groups studied are shown in Table 2.
Figure 1 Protein Z plasma levels in patients and controls. Bold line represents mean protein Z plasma levels. Group A: patients with arterial thrombosis; Group B: patients with venous thrombosis.
by Spearman correlation coefficients. Anti-PZ antibodies required the use of nonparametric statistics such as Mann–Whitney rank-sum test and Kruskal–Wallis analysis of variance (ANOVA) for comparisons of cases and controls. PZ concentration showed normal distribution and equality of variances in patients and controls and we used the unpaired samples t-test and ANOVA test in the statistical analysis. Results are presented as mean ±
Protein Z PZ levels in our study did not correlate with age (P = 0.85). Also, no relationship existed between PZ levels and time elapsed from thrombotic event to blood sample collection (P = 0.97). The mean (± SD) PZ plasma levels in patients were found to be significantly lower as compared to control subjects (1709 + − 761.3 ng/mL vs. 2437 + − 964.7 ng/mL; P = 0.001, unpaired samples t-test). As shown in Fig. 1, mean PZ levels from both groups (Arterial-group A: 1663.5 + − 792.3 ng/mL; Venous-group B: 1742.4 ± 742.4 ng/mL) of patients were also significantly lower than controls (P = 0.001 and P = 0.002, respectively, ANOVA test). However, with respect to group A (arterial) being compared to group B (venous), no significant differences in mean PZ level were observed.
Figure 2 Protein Z plasma levels in clinical entities (left) and etiological subgroups (right). Bold line represents mean protein Z plasma levels. Group A: patients with arterial thrombosis; Group B: patients with venous thrombosis. PE indicates pulmonary embolism; DVT, deep vein thrombosis; PVT, portal vein thrombosis; AMI, acute myocardial infarction; PAT, peripheral artery thrombosis.
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis Table 3
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Crude and adjusted relative risk according to quartiles of protein Z Patients
OR Group A Fourth quartile
1
Third quartile Second quartile First quartile
6 17 26
Group B Fourth quartile
2
Third quartile Second quartile First quartile
10 21 31
Multivariate a
Univariate
Reference group 5.4 15.4 26
Reference group 4.5 10 15
95%CI
0.6–49 1.6–116 3.2–210
0.8–23 2.1–50 3.2–73
P
OR
95%CI
P
0.132 0.015 0.002
Reference group 7.7 28.1 52.1
0.3–48 1.9–227 3.8–400
0.09 0.005 0.001
0.06 0.003 0.001
Reference group 4.5 13 18
0.5–48 1.7–120 2.5–170
0.18 0.01 0.007
Patients indicate number in each subgroup. OR indicates odds ratio; 95% CI indicates 95% confidence interval; P indicates P–value. Group A: patients with arterial thrombosis. Group B: patients with venous thrombosis. Quartile (Protein Z level). a Group A adjusted for age, gender, hypertension, smoking habit, dyslipidemia, obesity and diabetes; history of previous arterial thrombosis. Group B adjusted for immobility, recent surgery, cancer, history of previous venous thrombosis.
The comparison between arterial and venous thrombotic entities together was performed (ANOVA test), evaluating mean PZ plasma levels (Fig. 2). Patients with peripheral artery thrombosis had the lowest mean PZ level (1022 ± 966 ng/mL) , with a significant difference as compared with patients with stroke (1764 ± 738 ng/mL; P = 0.02), acute myocardial infarction (1842 ± 608 ng/mL; P b 0.05) and pulmonary embolism (1929 ± 896; P = 0.006). Among the rest of the groups no statistical differences were found. We then evaluated PZ levels in the etiological subgroups (Fig. 2). Patients from high arterial risk factor subgroup had the lowest mean PZ level (1267.5 ± 609 ng/mL) , with a significant
difference as compared with patients included in arterial low risk factor subgroup (1873.2 ± 751 ng/mL; P = 0.03) and venous high risk factor subgroup (1918.9 ±850 ng/mL; P = 0.04). No differences were found with the other groups. In order to test if decreasing levels of protein Z were a predisposing factor for the occurrence of thrombosis a logistic regression analysis was carried out. In patients (Table 3), the univariate analysis showed a significant increased risk for thrombosis for the lowest quartiles of protein Z (first and second quartiles), as compared with the highest quartile (fourth quartile) of both arterial and venous thrombosis groups. Between groups, we observed a greater risk of thrombosis for the arterial
Figure 3 Anti-PZ IgG and IgM in patients and controls. Data are given in arbitrary units (AU) as described. Bold line represents mean anti-PZ levels. Asterisks represent individual values. Group A: patients with arterial thrombosis; Group B: patients with venous thrombosis. The horizontal line represents the 1-AU level.
732 group of patients. After adjustment for confounding variables in the multivariate analysis, this association persisted.
Anti-protein Z The majority of the patients presented negative anti-PZ antibodies under cut-off level (Fig. 3). When comparing mean anti-PZ IgG levels of all the patients included in arterial thrombosis group (group A) and control group, we found significantly higher levels of anti-PZ IgG in arterial thrombosis patients (Mann–Whitney rank-sum test; P = 0.005). Between venous thrombosis (group B) and control subjects no differences existed (P = 0.372). Lastly, a higher mean rank of anti-PZ IgG antibody levels were detected in the arterial thrombosis group when compared to the venous thrombosis group B (P = 0.05). For anti-PZ IgM levels no differences between groups were observed. We finally studied anti-PZ antibodies on clinical entities and etiological subgroups, and no significant differences between groups were observed (data not shown). We found a moderate inverse correlation between anti-PZ IgG antibody levels and PZ concentration in the global analysis of controls and patients (Spearman correlation coefficient [p] of − 0.20 with P = 0.04) (data not shown). Anti-PZ IgM antibody levels were not correlated with plasma PZ concentrations .We thus looked separately at control and patient groups, finding no correlation within groups (P = 0.07 and P = 0.13, respectively). Anti-PZ IgG and IgM antibody levels were not correlated with age (P = 0.96 and 0.40, respectively).
Discussion On the comparison between arterial and venous thrombosis groups, our data demonstrate no significant differences in PZ levels. Moreover, the majority of the venous and arterial thrombotic clinical entities and etiological subgroups studied present similar PZ levels. Only one previous study [10], compared an arterial (stroke) and a venous (deep vein thrombosis) thrombotic event. In that study only patients with arterial thrombosis presented lower PZ levels than controls. Our results on venous thrombosis contradict those from previous studies. Al-Shanqeeti et al. [11] evaluated PZ levels of 426 individuals with deep vein thrombosis, discovering no differences with control group. In 2005, Martinelli et al. [9] compared the PZ levels of 443 patients with an initial episode of deep vein thrombosis versus a control group founding that low PZ levels were not an independent risk factor for deep vein thrombosis, but seemed to increase the risk associated with established causes of thrombophilia. We cannot explain our apparently conflicting results, although the inclusion of clinical entities not previously studied as pulmonary embolism and portal vein thrombosis may have influenced results. Finally to comment a recent article by Santacroce et al. [24] that founded relationship between very low PZ levels and deep vein thrombosis, in line with our results, but with a weaker association. Concerning etiology, in our subgroup analysis, the presence of either venous
J. Pardos-Gea et al. thrombosis risk factors or a thrombophilic disorder is not associated with variations in PZ levels. We could conclude that the baseline prothrombotic condition of patients does not determine plasma PZ levels. That fact makes the acute thrombotic process in itself a possible cause or effect of the low PZ levels we found in venous thrombotic pathologies, although this is only a theory and more studies are warranted. Our findings on arterial thrombosis have been in accordance with previous studies. Patients with peripheral artery disease and those with high risk factors for arterial thrombosis have had the lowest PZ levels in each subgroup. Basic research has demonstrated that PZ is present in endothelial cells and in the proliferating subendothelial space of atherosclerotic lesions of patients with different risk factors [25]. Recently, Vasse et al. [26] demonstrated that normal human endothelial cells from artery or venous vessels synthesize PZ. Clinical studies on arterial thrombosis have only investigated coronary vascular disease and stroke. In a study by Fedi et al. [12] patients with acute coronary thrombosis presented lower PZ levels than controls. The patients presented significantly more arterial classical risk factors compared to controls, and probably a more seriously chronic vascular atherosclerotic disease. Sofi et al. [27] conducted a study that included patients with stable coronary chronic disease, patients with acute or unstable coronary disease and healthy controls. They found significantly lower levels of PZ in patients with respect to controls, but no differences between patients in the stable or unstable form of coronary atherosclerotic disease. In stroke some studies that included younger-age samples without macro or microangiopathy [16,19] showed normal or high PZ levels; in contrast other studies [17,18] with older population and suspected atheromatosis presented same PZ levels. Therefore, and at least in coronary vascular disease, it would seem that chronic atherosclerotic vascular disease, and not the acute thrombotic process, would be the pathological entity related to low PZ levels. Previous studies support the fact that the acute thrombotic event does not seem to have so many influence in PZ levels in arterial thrombotic events. Staton et al. [18], found that the difference on PZ levels in stroke between the first 7 days and the follow-up period (3–6 months), was not significantly different (P= 0.07). Also Vasse [28] described no relationship between cytokines and PZ biosynthesis. We think that dysfunctional endothelium and deposits on proliferating subendothelial space of atherosclerotic lesions could be some of the reasons that explain the possible relationship between atherosclerosis and low PZ plasma levels. In our patients from arterial high risk factor thrombosis subgroup we can assume the presence of a higher degree of atherosclerotic
Protein Z levels and anti-protein Z antibodies in patients with arterial and venous thrombosis disease, and explain the low PZ levels founded. A possible mechanism to explain the low PZ levels obtained in our patients with peripheral artery disease compared to those with stroke could be that peripheral artery disease, as macroangiopathy, could reflect a more extended involvement of vessels by atherosclerosis compared to stroke. The first and only report on anti-PZ antibodies was by Gris et al. [23]. They find that anti-PZ IgG and IgM levels were higher in women with pregnancy loss compared to healthy mothers. The overall risk of pathologic pregnancy climbed with increasing antiPZ concentration. Based on the results of our study, we must say, firstly, that mean anti-PZ IgG and IgM antibody levels have been truly low and mainly below the 1 AU level. This represents a clear difference in relation to the levels seen in the study by Gris et al. [23], in which patients and controls presented higher mean antibody levels. This difference could mean that anti-PZ antibodies in systemic thrombotic processes do not play such a potent role as they do in pregnancy pathology. Nevertheless, we have found a higher number of patients with positive anti-PZ IgG antibody levels in patients with arterial thrombotic disease compared to those with venous thrombosis and the control group, and a higher mean rank of anti-PZ IgG . Despite this fact, we consider that our results indicate no relationship between anti-PZ antibodies and thrombosis. The study has some potential limitations. First, it is an observational case-control study, and thus we cannot distinguish primary from secondary effects and can document associations only. Second, the limited number of subjects interferes with the reliability of our results. Third, we have arbitrarily considered that two risk factors for arterial or venous thrombosis separate both the high from the low-risk factor population, and that point could be worthy of criticism. Fourth, we did not estimate the risk associated with presence of anti-PZ antibodies because of the low levels of autoantibodies we found, and because of the lack of a comparative population with a known high risk for thrombosis. In conclusion, our results suggest that both arterial and venous thrombotic events are related to low PZ levels and that low PZ concentrations are associated with thrombosis. Moreover, the majority of the venous and arterial thrombotic clinical entities studied presented similar PZ levels. We have observed a possible relationship between chronic atherosclerotic disease and decreased PZ levels suggesting a possible role of low levels of this protein in its pathophysiology. Lastly, anti-PZ antibodies in arterial and venous thrombotic processes do not seem to have such a relevant function in
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pathogenesis as they do in pathologic pregnancies, although anti-PZ IgG antibodies in arterial thrombotic processes warrants further investigation. Ongoing investigations should better define the possible roles of both PZ and anti-PZ antibodies in thrombosis.
Acknowledgement We would like to thank all the people in the Unit of Haemostasia of our Hospital for its contribution to the work.
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