Plasma Concentrations of Protein Z and Protein Z-Dependent Protease Inhibitor in Thalassemia Major Patients
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Thalassemia is the most common congenital hemolytic disorder by a partial or complete deficiency in globin chain synthesis. Recent investigations have suggested a correlation between protein Z (PZ) and protein Z-dependent protease inhibitor (ZPI) deficiency and thrombosis. So, the aim of this study was to evaluate the PZ and ZPI levels in beta (β)-thalassemia patients. In this study, 40 patients with thalassemia major and 40 control subjects were selected. PZ and ZPI serum levels were measured by sandwich ELISA technique. PZ and ZPI were significantly higher in thalassemic patient group than control group. Also, PZ levels and PLT and Hb counts were significantly higher in splenectomized thalassemic patients than non-splenectomized ones. The exact mechanism of increased PZ and ZPI have an impact on hypercoagulable state in β-thalassemia major could not be found in our study. More studies are still needed to understand these challenges in β-thalassemia major patients.
KeywordsThalassemia major Protein Z Protein Z-dependent protease inhibitor Plasma Coagulability state
Thalassemia is an inherited hemolytic disease and one of the most common genetic disorders worldwide. Thalassemia is caused by a decreased or abnormal synthesis of alpha (α) or beta (β) globin chains which results in α- or β-thalassemia respectively. It is common in the Mediterranean region, the Far East, and South America but more prevalent in Southeast Asia. Incidence rate of thalassemia is estimated at about 1 in 100,000 annually [1, 2]. However, the life expectancy has noticeably improved in thalassemic patients as a consequence of chronic blood transfusion together with iron chelation therapies in recent years [3, 4], but clinical evidence has shown that thalassemic patients have a higher incidence risk of thromboembolic events . The overall incidence rate of thromboembolic events has been reported in thalassemia major, ranging from 0.9 to 4%, while higher rates have been estimated in thalassemia intermedia patients (3.9–29%) [2, 3]. Several factors pathophysiologically are involved in thromboembolic events such as deep vein thrombosis (DVT), pulmonary embolism, and port vein thrombosis in patients with thalassemia major and intermedia .
The most important factors involved in hypercoagulability state of thalassemia are alterations in the membrane lipid components of abnormal RBCs resulting in an increased exposure of phosphatidyle serin (PS) which in turn can reinforce the conversion of prothrombin to thrombin through its procoagulant activity, especially in splenectomized patients, thrombosis following splenectomy, cardiac dysfunction, and liver dysfunction followed by reduced protein C and protein S [1, 6].
Several studies have reported the potential role of changes in protein Z (PZ) levels in hypercoagulability state [7, 8, 9]. PZ is a plasma single-chain vitamin K-dependent protein which was detected in human plasma in 1984; however, its physiologic function remained unknown until 1998. Its structure is similar to that of factor VII, IX, X, and protein C [10, 11]. Unlike these proteins, PZ is not a zymogen of a serin protease, because it lacks SER and HIS residues of catalytic triad . In vitro and in vivo studies showed that PZ, like protein C and protein S, can act as an anticoagulant . PZ functions as cofactor of protein Z-dependent protease inhibitor (ZPI) resulting in inactivation of factor Xa. ZPI is an anticoagulant protein and a member of serpin superfamily synthesized in the liver and inhibits factor Xa in the presence of PZ, Ca2+, and phospholipid, but can inactivate factor XIa in the absence of these cofactors. PZ and ZPI circulate in plasma as a complex [11, 14]. Some studies have suggested a correlation between low levels of PZ and the risk of ischemic stroke [7, 15], while another study has reported an increased risk of ischemic stroke in high levels of PZ presence . Until recently, the role of PZ and its correlation with the incidence risk of thromboembolic events have been an area of controversy. Thus, the aim of this study was to investigate PZ and ZPI levels in patients with β-thalassemia major and their relationships with coagulability state in these patients.
Material and Methods
Study Group: Patients and Samples
In this study, 40 patients with thalassemia major (24 (60%) males, 16 (40%) females (5–40 years old, median age 18.2) were included in this study after morphologic and clinical examinations. Subjects receiving drugs that affect hemostasis (anticoagulants), those who were consuming alcohol, taking contraceptive drugs, and pregnant subjects were excluded from the study. Forty healthy subjects, who were proportionally sex and age matched, were selected as the control group. The control group had no clinical symptoms of thalassemia or other diseases, and all their laboratory tests were normal based on clinical examination and test results. All peripheral blood (PB) samples were obtained from the Gachsaran Shahid Rajaee Hospital during 4 months with written informed consent from the subjects. This study was approved by the local ethics committee of Ahvaz Jundishapur University of Medical Sciences (AJUMS.REC.1393.277) and was conducted within 4 months.
Blood Sampling and Sandwich ELISA Technique
Five milliliters of PB samples were collected from all subjects, immediately before transfusion in patients, in tubs containing EDTA anticoagulant and centrifuged at 3200g for 15 min within 1 h, and plasma was collected and stored at − 80° until the time of assay. Then, PZ and ZPI were measured by sandwich ELISA technique (Cusabio Kit, Behring ELISA processor instrument).
The results were expressed as mean ± standard error (SE) (range), and variations between the data of two sets were analyzed by t test. Relationship between proteins was assessed by use of the Mann-Whitney test. p value < 0.05 was considered statistically significant.
Comparison of the PZ and ZPI serum levels in thalassemia major patients and control subjects
Group 1 thalassemic
ZPI, μg/mL (mean ± 2SE)
1.53 ± 0.48
0.5 ± 0.24
p = 0.001
PZ, ng/mL (mean ± 2SE)
1031 ± 230
621 ± 156
p = 0.004
Comparison of the PZ and ZPI serum levels with PLT and Hb in splenectomized and non-splenectomized thalassemia major patients
Transfusion dependency time, mean (range)
8 months (6–14)
19 months (6–60)
PLT (mean ± 2SE)
283 ± 37.4
519.3 ± 147.8
p = 0.002
Hb, g/dL (mean ± 2SE)
8.5 ± 0.34
8.6 ± 0.44
p = 0.006
ZPI, μg/mL (mean ± 2SE)
1.28 ± 0.44
2.28 ± 1.34
p = 0.077
PZ, ng/mL (mean ± 2SE)
872 ± 252
1507 ± 410
p = 0.015
PZ is a plasma vitamin K-dependent glycoprotein acting as a cofactor of ZPI to inhibit factor Xa together with Ca2+ and phospholipid . Recent studies have represented evidences on the potential role of this protein in thromboembolic complications . Therefore, in this study, investigation of PZ and ZPI levels in patients with thalassemia major and their possible correlations with coagulability was done in these patients.
In the current study, a significant difference in PZ and ZPI levels was found in patients with thalassemia rather than the control subjects. In contrast, Del Vecchio et al. in 2007 have shown reduced levels of PZ in thalassemic patients rather than in healthy subjects by ELISA method . In addition, Gris et al. in 2002 and Bretelle et al. in 2005 have suggested that low serum levels of PZ were associated with thrombosis risk in patients with pregnancy complications by ELISA method [19, 20]. The mean difference of PZ levels in several studies was supposed to be due to PZ responses to inflammatory reactions or the influence of gene polymorphisms on gene expression values [21, 22, 23]. In confirmation of the inflammatory response hypothesis, the data published by Kobelt et al. in 2001 showed that PZ levels were increased at the time of ischemic stroke incidence . Cesari et al. in 2007 reported that PZ levels were elevated in acute phase of coronary artery diseases and it began to reduce about 3 months after an acute onset by ELISA method . In addition, Girard et al. in 2013 have precisely investigated the response of PZ and ZPI to inflammatory state, and they consequently showed that the ZPI was an acute phase protein while the elevation of PZ in inflammatory responses was derived from increased ZPI . Some studies have shown that infection and inflammation are the second leading cause of death in patients with major thalassemia . But Bolkun et al. in 2013 revealed that due to FVIII deficiency in hemophilia A patients, PZ and ZPI levels were higher than the control group by the ELISA method in their study . Therefore, although a similar technique has been used in our study and other studies and according to controversy results, PZ and ZPI levels can be increased in inflammatory and anti-thrombotic conditions, but still more studies are needed to confirm this in patients with thalassemia.
In our study, PZ levels were significantly higher in splenectomized patients than non-splenectomized ones, but there was no significant difference in ZPI levels between these two groups. In addition, a significant difference in PLT and Hb count was found between non-splenectomized and splenectomized patients. Schettini et al. in 1987 reported that hemostatic system in patients with thalassemia major was changed and protein C levels and also PLT count were increased in splenectomized patients with thalassemia major by ELISA technique . But, Angchaisuksiri et al in 2007, confirmed the platelet activation, inflammation, impaired fibrinolysis with elevated levels of factors such as thrombinantithrombin, beta2 thromboglobulin, C-reactive protein (CRP), tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in splenectomized patients with E/β-thalassemia and anticoagulants factors such as protein C, S, anti-thrombin, and fibrinogen were significantly decreased in these patients in comparison with non-splenectomized patients by ELISA technique . In addition, Hashemieh et al. in 2016 showed that protein C, S, fibrinogen, and factor V activity were decreased in patients with thalassemia intermedia, in comparison with the control group, and splenectomy has no significant effect on hemostatic factors except activity of D-dimer in these patients . Therefore, the results of our study and other studies have approximately confirmed the coagulability challenges, but still, more studies are needed to understand its exact mechanism in thalassemia patients.
There is also evidence of oxidation of membrane components contributing in the pathophysiology of β-thalassemia. The excess of free α-globin chains can induce ROS formation via binding to iron and heme resulting in oxidative damage of lipids, proteins, and nucleic acid. Lipid peroxidation and protein oxidation cause loss of membrane lipid organization and deformity of the cell. The consequence of these alterations is the exposure of PS . It has been suggested that PS acts as a recognition signal of damaged RBCs by phagocytes of reticuloendothelial system, their removal, and apoptosis . Therefore, these damaged cells remain in circulation in splenectomized subjects. Thalassemic RBCs enhance thrombin production with negatively charged PSs on their surface, and consequently, anticoagulants such as PZ are increased to prevent thrombotic events.
In brief, the results of our study did not show the exact influence of PZ and ZPI on provoking the hyperactivity of coagulative system and increased risk of vascular thromboembolism in patients with major thalassemia, but PZ can be suggested to decrease thrombotic events in splenectomized patients.
In this study, PZ and ZPI were significantly higher in thalassemic patients. Also, PZ levels, PLT, and Hb count were significantly higher in splenectomized thalassemic patients. The results of our study cannot exactly show the PZ and ZPI influence on coagulative system, but can suggest that PZ and ZPI are increased in inflammatory and anti-thrombotic conditions to prevent thrombotic events. But still, more studies are required to confirm the exact mechanism of PZ and ZPI in coagulation and thrombotic challenges in β-thalassemia major patients.
Z.T.A have conceived the manuscript and revised it. M.Gh and M.A. J wrote the manuscript. M.A. J and S. S provided the clinical data, pathological diagnoses, and information. M.Gh performed the technical tests.
This paper is issued from the thesis of Majid Ghazanfari, MSc student of hematology and blood banking. This work was financially supported by grant th93/26 from vice chancellor for research affairs of Ahvaz Jundishapur University of Medical Sciences.
Compliance with Ethical Guidelines
Conflicts of Interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of local ethics committee of the Ahvaz Jundishapur University of Medical Sciences (AJUMS.REC.1393.277) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
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