|Year : 2017 | Volume
| Issue : 4 | Page : 130-135
Evaluation of von Willebrand Factor-cleaving Proteinase(vWF-CP) in pediatric patients with primary glomerulonephropathy
Hasan Tarek Abd-Allah Ebrahim
Department of Pediatric, Banha Teaching Hospital, Benha, Qalubia Governate, Egypt
|Date of Web Publication||29-Dec-2017|
Dr. Hasan Tarek Abd-Allah Ebrahim
Department of Pediatric, Benha Teaching Hospital, Benha, Qalubia Governate
Source of Support: None, Conflict of Interest: None
Background and Objectives: Nephrotic syndrome (NS) is a well-known risk factor for arterial or venous thromboembolism (TE). There is a higher risk of TE in steroid-resistant nephrotic syndrome (SRNS) than in steroid-sensitive NS (SSNS). The aim of this study was to investigate serum level of von Willebrand factor-cleaving protease activity which is known as vWF-CP in children with idiopathic NS (INS) and its correlation with clinical and laboratory parameters. Patients and Methods: This study was conducted on 120 children with INS, including 40 SSNS, 40 SRNS, and 40 healthy controls. All subjects are investigated by complete blood count, 24 h collected urine analysis for urine volume, urinary proteins, total serum protein and serum albumin, total serum cholesterol, prothrombin time, partial thromboblastin time (PTT), and serum vWF-CP activity. Results: There was a highly significant decrease in serum vWF-CP activity in SSNS and SRNS groups when compared to control group while there was no significant difference in serum vWF-CP activity between SSNS and SRNS groups. Conclusions: Serum vWF-CP activity is a biomarker for endothelial dysfunction and hypercoagulable state. The decreased vWF-CP activity in different extent of nephrotic patients (SSNS and SRNS) may be one of the pathogenesis of thrombosis as a common complication of NS. Regular follow-up of nephrotic patients and estimation of serum vWF-CP level as its decreased level is a risk factor of thrombosis.
Keywords: Children, nephrotic, von willebrand factor cleaving protease
|How to cite this article:|
Abd-Allah Ebrahim HT. Evaluation of von Willebrand Factor-cleaving Proteinase(vWF-CP) in pediatric patients with primary glomerulonephropathy. J Integr Nephrol Androl 2017;4:130-5
|How to cite this URL:|
Abd-Allah Ebrahim HT. Evaluation of von Willebrand Factor-cleaving Proteinase(vWF-CP) in pediatric patients with primary glomerulonephropathy. J Integr Nephrol Androl [serial online] 2017 [cited 2018 Jan 17];4:130-5. Available from: http://www.journal-ina.com/text.asp?2017/4/4/130/222063
| Introduction|| |
Many predisposing factors for TE were reported in nephrotic patients, abnormalities in platelet activation and aggregation, activation of prothrombotic factors of coagulation system, for example, factors V, VII, VIII, X, von Willebrand factor (vWF), fibrinogen, and α 2–macroglobulin, decreased activity of fibrinolytic system such as plasminogen  and decreased endogenous anticoagulants, antithrombin III, protein C, protein S, and tissue factor pathway inhibitor resulting in local activation of the glomerular hemostasis system. vWF mediates platelet adhesion and aggregation at sites of vascular injury. It is released from the stimulated endothelium as unusually large (UL) multimer. ULvWF favor platelet aggregation and formation of microvascular thrombi. vWF-CP can cleave and thus converses ULvWF into a less active form. Reduced vWF-CP activity due to gene mutation or the presence of autoimmune IgM and IgG inhibitors  results in deficient proteolysis of ULvWF with the formation of disseminated platelet-rich thrombi in the microcirculation seen in thrombotic microangiopathies.,
The aim of this study was to investigate vWF-CP activity levels in children with idiopathic nephrotic syndrome (NS) and its relation to clinical and laboratory parameters.
| Patients and Methods|| |
This study was conducted on 120 children aged from 2½ to 17 years of both sexes in Benha Education Hospital (BEH), Department of Pediatric. The study was conducted from June 2016 to June 2017 after approval from Research Ethical Committee of BEH. A formal written consent of each child parents was taken separately after explanation and assurance of them. All participants' names were hidden and replaced by code number to maintain privacy of the participant.
The children were divided into three groups: Group 1 – forty patients with steroid-sensitive nephrotic syndrome, (SSNS), Group 2 – forty patients with steroid-resistant nephrotic syndrome (SRNS) and Group 3 – forty healthy controls matched age and sex.
SSNS and SRNS groups were diagnosed according to definitions of Bagga and Strivastava.
Other causes of generalized edema as renal, hepatic and heart failure, nutritional and allergic causes, congenital anomalies of the kidney, other causes of thromboembolic disorders and autoimmune diseases.
All children included in this study were subjected to the following:
- Complete history taking: concerning past history of recurrence, responsiveness to steroid therapy, thromboembolism
- Full clinical examination: with special emphasis on Hypertension, peritonitis, thrombosis as a complication of NS
- Laboratory investigation including: complete blood count (CBC), 24 h collected urine analysis for urine volume, urinary proteins, total serum protein and serum albumin, and total serum cholesterol, prothrombin time (PT), partial thromboplastin time (PTT), and serum vWF-CP activity.
Specimen collection and handling
A volume of 6 ml morning venous blood sample was collected under complete aseptic conditions for assessment of the level of serum vWF-CP. Morning urine samples were taken from the 60 children for complete urine analysis. We put 2 ml of the blood in EDITA tube for CBC and the remaining blood allowed for clotting, and the serum separated by centrifugation at room temperature then divided into two Eppendorf tubes. One for the routine examination which was done immediately and the other tube preserved and froze at −20°C before the assay.
Serum vWF-CP activity levels
Measured by Human vWF-CP enzyme-linked immune-sorbent assay (ELISA) Microplate Kit which based on sandwich ELISA technology in (ng/mL). The anti-human vWF-CP antibody was precoated onto 96-well plates, the Biotin conjugated anti-human vWF-CP antibody was used as detection antibodies. The standards, test samples, and biotin-conjugated detection antibody were added to the wells subsequently, and wash with wash buffer.
| Results|| |
Demographic data of studied groups are summarized in [Table 1]. Laboratory data of the studied groups were summarized in [Table 2]. There was a highly significant decrease in serum vWF-CP activity in SSNS and SRNS groups when compared to control group, whereas there was no significant difference in serum vWF-CP activity between SSNS and SRNS groups [Table 3]. There was no significant difference in serum vWF-CP activity between males and females in studied patients. There was a significant positive correlation between plasma vWF-CP activity and total serum protein in patients with SSNS [Table 4]. There was a significant positive correlation between plasma vWF-CP activity of patients with SSNS with serum albumin [Table 4]. There was a significant negative correlation between plasma vWF-CP activity and 24 h urine protein in patients with SSNS [Table 4]. There was a significant negative correlation between plasma vWF-CP activity of patients with SRNS with serum cholesterol level [Table 4].
|Table 2: Routine laboratory data of the studied patients and control groups|
Click here to view
|Table 4: Correlations between vWF-CP activity and laboratory data of studied Patients|
Click here to view
In our study, there were no patients with venous thromboembolism (VTE) in different studied patient groups, and hence, we were not able to describe VWF-CP (ADAMTS) levels in those patients. There was a significant positive correlation between plasma vWF-CP activity and both systolic and diastolic blood pressure in patients with SRNS [Figure 1] and [Figure 2].
|Figure 1: Correlations between plasma vWF-CP levels in steroid-resistant nephrotic syndrome and systolic blood pressure of patients. *Statistically significant|
Click here to view
|Figure 2: Correlations between plasma vWF-CP levels in steroid-resistant nephrotic syndrome and diastolic blood pressure of patients. *Statistically significant|
Click here to view
| Discussion|| |
Elevated plasma vWF antigen (vWF Ag) and/or decreased vWF-CP activity are associated with negative outcomes of several disorders. An imbalance between the circulating levels of vWF and vWF-CP has been reported in a number of acquired diseases in adults such as coronary artery disease and myocardial infarction, peripheral arterial disease, ischemic stroke, preeclampsia, inflammatory bowel disease, and liver cirrhosis , and these findings have been reported also in different diseases of pediatric age such as type 1 diabetes mellitus  and ESRD.
The study results showed that there is no significant difference between studied groups regarding platelet count. Eneman et al. found that NS is associated with a significantly increased risk of thrombotic events. Alterations in plasma levels of pro-and anti-coagulant factors are involved in the pathophysiology of venous thrombosis in NS. However, the fact that the risk of both venous and arterial thrombosis is elevated in NS points to an additional role for blood platelets. Increased platelet counts and platelet hyperactivity have been observed in nephrotic children. Platelet hyperaggregability increased the release of active substances, and elevated surface expression of activation-dependent platelet markers have been documented. The mechanisms underlying platelet alterations are probably due to changes in plasma levels of platelet-interfering proteins and lipid changes, as a consequence of nephrosis.
Anand et al. clarify the importance of coagulation profile in nephrotic syndrome as a high index of suspicion for thromboembolic complications, especially in patients with thrombocytosis.
Our results showed a significant decrease in total serum protein and serum albumin in both steroid sensitive, steroid resistant NS when compared to control group. This is in agreement with US National library of medicine that defined the nephrotic syndrome. and Mulukala et al. who stated that NS is manifested by hyperproteinuria, low total serum protein, hypoalbuminemia, and edema. While there is no difference in serum albumin and total serum protein between SSNS and SRNS groups.
In our study, there is no significant difference in PT) and PTT between studied groups. This is in agreement with Yalçinkaya et al. who stated that PT, PTT as well as platelet count and mean plasma Protein C activity were similar in the NS group when compared with the control group and in addition no remarkable difference was found in the mean plasma Protein C activity between the steroid sensitive and resistant NS groups. In contrast, the mean plasma antithrombin III (AT III) activity was significantly reduced in patients with NS when compared to controls correspondingly, it was directly correlated with serum albumin and inversely correlated with proteinuria.
However, our findings regarding PT and PTT are not in agreement with Anand et al. who stated that thromboembolic complications of NS should be suspected in patients with thrombocytosis, hyperfibrinogenemia, prolonged activated partial thromboplastin time and in children with low levels of antithrombin-III, protein C and protein S.
Limited studies of vWF-CP have been done on pediatric nephritic patients. The study results showed that there was a highly significant decrease in serum vWF-CP activity in SSNS and SRNS studied groups when compared to control group. This is in agreement with, LiQion J who stated that vWF-CP activities of both steroid sensitive group and steroid-resistant group are decreased when compared with normal control group.
In this work, there was no significant difference in serum vWF-CP activity between studied SSNS and SRNS groups. This is in agreement with LiQion J stated that no differences are observed in vWF-CP activity among steroid resistant nephrotic syndrome group when compared to steroid sensitive nephrotic syndrome group.
Our results showed that there was no significant difference between studied males and females as regards vWF-CP activity in studied patients.
In our study, there were no patients with venous thromboembolism (VTE) in different studied patient groups, and hence, we were not able to describe VWF-CP (vWF-CP) levels in those patients.
Correlation analysis of our study showed that there that there is a significant positive correlation between plasma vWF-CP activity of patients and total serum protein and serum albumin in patients with SSNS, whereas there is a significant negative correlation between plasma vWF-CP activity of patients and serum cholesterol level in SRNS group. This correlation results are in agreement with LiQion J who stated that plasma vWF-CP antigen of patients with NS is positively correlated with serum albumin (r = 0.385, P < 0.01) and negatively correlated with total blood cholesterol (r = −0.317, P < 0.01).
Correlation analysis of our study showed that there is a significant negative correlation between plasma vWF-CP activity and 24 h urine protein of patients with SSNS. This is in agreement with LiQion J who stated that vWF-CP activity of patients with nephrotic syndrome is negatively correlated with the quantitative measurement of 24 h urinary protein (r = −0.242, P < 0.05).
| Conclusions|| |
It is recommended for regular follow-up of children with nephrotic syndrome and early estimation of reduced serum vWF-CP level to control this risk factor of thrombosis. It may be possible to re-engineer vWF-CP protease to improve specific activity, which may offer preventive and therapeutic benefits to nephrotic patients in pediatric age with thromboembolic complications.
Regular follow-up of nephrotic patients and estimation of serum vWF-CP level as its decreased level is a risk factor of thrombosis.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zumberg M, Kitchens CS. Consultative Hemostasis and Thrombosis. 2nd
ed. Philadelphia: Saunders Elsevier; 2007.
Welch TR. Nephrosis and clots. J Pediatr 2009;155:A2.
Karim F, Adil SN, Afaq B, Ul Haq A.
Deficiency of ADAMTS-13 in pediatric patients with severe sepsis and impact on in-hospital mortality. BMC Pediatr 2013;13:44.
Arya M, Anvari B, Romo GM, Cruz MA, Dong JF, McIntire LV, et al.
Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: Studies using optical tweezers. Blood 2002;99:3971-7.
Austin SK, Starke RD, Lawrie AS, Cohen H, Machin SJ, Mackie IJ, et al.
The VWF/ADAMTS13 axis in the antiphospholipid syndrome: ADAMTS13 antibodies and ADAMTS13 dysfunction. Br J Haematol 2008;141:536-44.
Mannucci PM, Peyvandi F. TTP and ADAMTS13: When is testing appropriate? Hematology Am Soc Hematol Educ Program 2007;(1):121-6.
Tsai HM. ADAMTS13 and microvascular thrombosis. Expert Rev Cardiovasc Ther 2006;4:813-25.
Turner N, Nolasco L, Tao Z, Dong JF, Moake J. Human endothelial cells synthesize and release ADAMTS-13. J Thromb Haemost 2006;4:1396-404.
Claus RA, Bockmeyer CL, Budde U, Kentouche K, Sossdorf M, Hilberg T, et al.
Variations in the ratio between von Willebrand factor and its cleaving protease during systemic inflammation and association with severity and prognosis of organ failure. Thromb Haemost 2009;101:239-47.
Bagga A, Srivastava RN, editors. Nephrotic syndrome. In: Pediatric Nephrology. 4th
ed. New Delhi: Jaypee; 2005. p. 159-200.
Hyun J, Kim HK, Kim JE, Lim MG, Jung JS, Park S, et al.
Correlation between plasma activity of ADAMTS-13 and coagulopathy, and prognosis in disseminated intravascular coagulation. Thromb Res 2009;124:75-9.
Bongers TN, de Bruijne EL, Dippel DW, de Jong AJ, Deckers JW, Poldermans D, et al.
Lower levels of ADAMTS13 are associated with cardiovascular disease in young patients. Atherosclerosis 2009;207:250-4.
Molvarec A, Rigó J Jr., Bõze T, Derzsy Z, Cervenak L, Makó V, et al.
Increased plasma von willebrand factor antigen levels but normal von willebrand factor cleaving protease (ADAMTS13) activity in preeclampsia. Thromb Haemost 2009;101:305-11.
Skeppholm M, Kallner A, Kalani M, Jörneskog G, Blombäck M, Wallén HN, et al.
ADAMTS13 and von Willebrand factor concentrations in patients with diabetes mellitus. Blood Coagul Fibrinolysis 2009;20:619-26.
Shen L, Lu G, Dong N, Jiang L, Ma Z, Ruan C, et al.
Von Willebrand factor, ADAMTS13 activity, TNF-α and their relationships in patients with chronic kidney disease. Exp Ther Med 2012;3:530-4.
Eneman B, Levtchenko E, van den Heuvel B, Van Geet C, Freson K. Platelet abnormalities in nephrotic syndrome. Pediatr Nephrol 2016;31:1267-79.
Anand NK, Chand G, Talib VH, Chellani H, Pande J. Hemostatic profile in nephrotic syndrome. Indian Pediatr 1996;33:1005-12.
Silberberg C. Nephrotic Syndrome. USA: U.S. National Library of Medicine; 2013.
Mulukala SK, Nishad R, Kolligundla LP, Saleem MA, Prabhu NP, Pasupulati AK, et al. In silico
structural characterization of podocin and assessment of nephrotic syndrome-associated podocin mutants. IUBMB Life 2016;68:578-88.
Yalçinkaya F, Tümer N, Gorgani AN, Ekim M, Cakar N. Haemostatic parameters in childhood nephrotic syndrome. (Is there any difference in protein C levels between steroid sensitive and resistant groups?) Int Urol Nephrol 1995;27:643-7.
Li Qion J. Variation of Von Willebrand Factor Cleaving Protease in Plasma of Patients with Nephrotic Syndrome and Expression as Well as Influencing Factors in Human Renal Tubular Epithelial Cells; Internal Medicine 2011.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]