J Huazhong Univ Sci Technol［Med Sci］ DOI 10.1007/s11596-010-0657-4 30(6):777-780,2010 J Huazhong Univ Sci Technol［Med Sci］ 30(6):2010

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Von Willebrand Factor Antigen and ADAMTS13 Activity Assay in Pregnant Women and Severe Preeclamptic Patients* Dandan ZHANG (张丹丹)1, Juan XIAO (肖 娟)1, Haoliang HUANG (黄浩梁)2, Juanjuan CHEN (陈娟娟)1, Tao LIU (刘 涛)1, Zongzhi YIN (尹宗智)1, Danping GAO (高单萍)1, Qiong LIU (刘 琼)1, Jihui AI (艾继辉)1, Suhua CHEN (陈素华)1# 1 Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Obstetrics and Gynecology, the First Hospital of Wuhan, Wuhan 430022, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2010

Summary: The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia. Thirty healthy women of childbearing age, 22 second trimester pregnant women, 30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study. ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay. The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women, between third and second trimester pregnant women (P<0.05). The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P<0.05). Nevertheless, no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P>0.05). In conclusion, plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels. Prothrombotic state is involved in the pathogenesis of severe preeclampsia, as a result of endothelial injury. Key words: von Willebrand factor; ADAMTS13; enzyme-linked immunosorbent assay; fluorescence resonance energy transfer

Von Willebrand factor (vWF), a blood glycoprotein involved in hemostasis, is required for platelet adhesion to sites of vascular damage. It is synthesized by vascular endothelial cells and megakaryocytes, with a large propeptide at its N-terminal end. After post-translational modification, the mature circulating vWF is a multimeric protein with a molecular mass ranging from 500 kD to more than 20 000 kD. The largest vWF multimers are hemostatically most active and can effectively mediate platelet aggregation[1]. vWF deficiency or dysfunction leads to a bleeding tendency, whereas increased concentration of ultra-large vWF (UL-vWF), as in thrombotic thrombocytopenic purpura, causes excessive platelet adhesion and is associated with formation of platelet microthrombi. ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is a newly defined zinc-containing metalloprotease that specially cleaves the vWF between Tyr842 and Met843. The Dandan ZHANG, E-mail: [email protected] Corresponding author, E-mail: [email protected] * This project was supported by grants from National Natural Sciences Foundation of China (No.30672243, No.30671093) and Natural Sciences Foundation of Hubei Province (No. ZRY1271). #

proteolytic processing of large multimeric vWF by ADAMTS13 is critical for normal homeostasis[2]. Decreased activity of plasma ADAMTS13 leads to the accumulation of UL-vWF and subsequent platelet clumping and/or thrombi under high shear stress, resulting in microvascular thrombosis. Research has shown that older women and late pregnant women have a physiological decrease in ADAMTS13 proteolysis activity[3,4]. The abnormality of ADAMTS13 proteolysis activity plays an important role in the pathophysiological course of many diseases, such as complications of pregnancy-associated hypertension, inflammation and tumor. The residual collagen binding assay traditionally used to measure the activity of ADAMTS13 is difficult to operate and with poor reproducibility[5, 6]. In our previous study, FRETvWF73 assay, a more rapid method was proved to complete quantitative measurement of ADAMTS13 activity within 1 h[7, 8]. A fluorescent signal is detected when the substrate comprising 73 amino acids of the vWF A2 domain is cleaved by ADAMTS13 in the blood sample of patients. ADAMTS-13 activity of the sample detected is defined as the relative ratio of ADAMTS-13 activity of the sample/ADAMTS-13 activity in normal pooled blood. This study examined the von Willebrand factor and ADAMTS13 activity of normal pregnant women and severe preeclamptic women in order to preliminarily

778 explore the possible mechanism of prothrombotic state in severe preeclampsia. 1 MATERIALS AND METHODS 1.1 Patients and Samples Specimens and clinical data were obtained from 92 subjects who were admitted to or had work-up in the Department of Obstetrics and Gynecology, Tongji Hospital (China), between January 2009 and December 2009. The patients were divided into four groups: (1) normal control group consisting of 30 healthy women of childbearing age (24 to 35 years); (2) second trimester group which had 22 patients who would undergo a planned abortion [age: 22 to 34 years, mean gestational weeks: (21.7±2.9) weeks]; (3) third trimester group in which 30 patients were to be in labor, [age: 25 to 36 years, mean gestational weeks: (35.2±3.1) weeks]. All patients in the former three groups who had any other pregnancy-related complications and any systemic disorders were excluded, such as multifetal gestation, chronic hypertension, diabetes mellitus, autoimmune disease, angiopathy, renal disorder, maternal or fetal infection and fetal congenital anomaly. The healthy women of childbearing age were in the early follicular phase of the menstrual cycle (between cycle days 3 and 5), and none of them received hormonal contraception. (4) The severe preeclampsia group in which 10 patients were diagnosed with severe preeclampsia [age: 21 to 38 years, mean gestational weeks: (34.8±5.4) weeks]. The stage of severe preeclampsia was determined according to the Williams Obstetrics (22nd edition) by F Gary Cunningham, Kenneth J Leveno. Peripheral blood samples from patients were collected into sterile tubes containing no additives or sodium citrate, and were kept at room temperature until centrifugation at 1100 g for 10 min. Less than 3 h was allowed between blood collections and processing. Serum and citrated plasma aliquots were then frozen at –80ºC until assayed. Informed consent for the experimental use of the samples was obtained for all subjects and the study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (China). 1.2 Determination of vWF Antigen Levels The vWF antigen assay in the serum and plasma were carried out as previously described[3]. Briefly, a two-fold dilution series of blood sample from normal human pool (NHP) (mixed blood samples of 8 healthy blood donors) was used to give rise to standard curve. vWF antigen content corresponding to the 1:40 diluted sample was set as 100%. Next, 100 µL of each diluted standard and patient sample were incubated for 1 h at 37°C in a microplate (Costar, USA) pre-coated with rabbit anti-human vWF polyclonal antibody (Dakopatts, Glostrup, Denmark). After washing three times with TBS-T, 100 µL of horse radish peroxidase (HRP)-conjugated polyclonal rabbit anti-human vWF antibody was added to the plates and incubated for 1 h, followed by addition of ortho-phenylenediamine (OPD). The absorbance was measured at 492/630 nm. All samples were run in duplicate. Coefficient of variation for the assay inner-batch and inter-batch were 4.23% and

J Huazhong Univ Sci Technol［Med Sci］ 30(6):2010

7.19% respectively. 1.3 Determination of ADAMTS13 Activity The fluorigenic substrate, FRETS-vWF73, came courtesy of Dr. Xinglong ZHENG of the Children’s Hospital of Philadelphia, University of Pennsylvania Medical Center and was used for the determination of ADAMTS13 enzyme activity according to the protocol provided by the supplier with minor modifications[9]. Briefly, 3 µL of the test samples were added to 100 µL assay buffer (5 mmol/L Bis-Tris, 25 mmol/L CaCl2, 0.005% Tween 20, pH 6.0) and then mixed with 5 μmol/L FRETS-vWF73 substrate solution (100 μL each), in black 96-well plates (Greiner, Germany). Fluorescence was measured at 37°C every 2 min for 30 min in a Spectra Max M2 microplate reader (Bio-Tek) equipped with a 485-nm excitation and a 528-nm emission filter. The reaction rate was calculated by linear regression analysis of fluorescence over time. A dilution series of NHP was used to establish a standard curve. ADAMTS13 activity corresponding to the 3:100 diluted sample was considered to be 100%. The intra-assay coefficient of variation (CV) was 3.69%, and the inter-assay CV was 6.91%. 1.4 Statistics Analysis All the data were expressed as ±s. Student’s t-test variance was used to determine the statistical differences among the groups with the aid of SPSS version 12.0 software package. A value of P<0.05 was considered to be statistically significant. 2 RESULTS 2.1 vWF Antigen Level and ADAMTS13 Activity in the Serum and Plasma of Healthy Women of Childbearing age The serum and plasma vWF antigen levels were (107.3±4.3)% and (108.9±4.5)% in the healthy women of childbearing age (t=0.241, P>0.05); ADAMTS13 activity was (85.7±4.6)% and (89.2±5.8)% in the serum and plasma of healthy women (t=0.474, P>0.05). No significant differences were found in the vWF antigen level and ADAMTS13 activity between the plasma and serum groups in the healthy women (fig.1). 2.2 vWF Antigen Level and ADAMTS13 Activity in the Plasma in the Second and Third Trimester Pregnant Women The plasma vWF antigen level was substantially increased in the third trimester pregnant women as compared with that in the second trimester pregnant women with statistical difference being significant (mean: 103.7%, 88.8%; t=0.036, P<0.05, fig. 2). Moreover, the third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women did (mean: 84.1%, 98.9%; t=0.015, P<0.05). 2.3 vWF Antigen Levels and ADAMTS13 Activity in the Plasma in Severe Preeclampsia and Third Trimester Pregnant Women Plasma vWF antigen levels was significantly higher in severe preeclampsia women (mean 120.5%) compared to the third trimester pregnant women (mean 103.7 %) (t=0.01, P<0.05, fig. 2). Nevertheless, there were no significant differences in plasma ADAMTS13 activity be-

J Huazhong Univ Sci Technol［Med Sci］ 30(6):2010

tween severe preeclamptic patients and the third trimester pregnant women (mean: 90.7%, 84.1%; t=0.187, P>0.05, fig. 2).

Fig. 1 vWF antigen level and ADAMTS13 activity in healthy women of childbearing age

Fig.2 Plasma ADAMTS13 activity and vWF antigen levels of pregnant women and severe preeclamptic patients

3 DISCUSSION The 250 kD mature vWF subunit consists of 11 domains (D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2)[10]. ADAMTS13 cleaves the Tyr 1605-Met 1606 bond in the A2 domain of vWF[1,11,12]. This proteolysis plays an important role in patho-physiological processes of the diseases such as complications of pregnancy-associated hypertension, inflammation and tumor[13]. In recent years, relationships between vWF level, ADAMTS13 activity and pregnancy-associated diseases have captured more attention[14–16]. Some studies revealed that reduced ADAMTS13 activity has been observed previously in pregnant women with HELLP syndrome (haemolysis, elevated liver enzymes, and low platelet count)[17]. Investigating the relationship of ADAMTS13 activity and severe preeclampsia can make further study on the mechanism of complications of pregnancy-associated hypertension. The original ADAMTS13 activity assay method was developed by Furlan et al and Tsai et al separately [18, 19] . At present the assay method commonly used of ADAMTS13 activity is residual collagen-binding assay. Whole purified plasma vWF as a substrate is comprised of non-uniform multimers with multiple cleavage sites[20, 21] . In contrast, FRETS-vWF73 is a monomeric molecule with a single cleavage site, facilitating the determination of cleavage kinetic parameters. No denaturants are re-

779 quired for the reaction, making this assay more closely reflect the physiological conditions. The FRETs-vWF73 is applicable to fluorescent indicators for the proteolysis activity of ADAMTS13[22, 23]. In the present study, FRETS-vWF73 assay, conducted within 1 hour period by using 96-well plates, is more accurate and reproducible. Therefore, FRETs-vWF73 is the model substrate for the ADAMTS13 activity assay in patients compared with the traditional substrate, which provided a good basis for further research. The mechanisms involving ADAMTS13 activity are not completely understood at present, particularly in pregnancy. vWF and its cleaving protease appear to interact to form a fine-tuned vWF/ADAMTS13 system. The increase in vWF antigen level and decrease in ADAMTS13 activity are conducive to the prevention of postpartum hemorrhage during late pregnancy. The most remarkable observation of our study is that, despite the significantly higher plasma vWF antigen levels, plasma ADAMTS13 activity is normal in severe preeclampsia. In several conditions associated with an increased plasma vWF antigen level, ADAMTS13 activity was found to be low previously[4, 16]. However, the biological significance of these observations is uncertain. Our results were consistent with those reported by Molvarec et al that no significant difference in plasma ADAMTS13 activity was found between preeclamptic patients and healthy pregnant, non-pregnant women, whereas plasma vWF antigen levels were significantly higher in the preeclamptic group compared with the healthy pregnant and non-pregnant groups[3]. The increased vWF antigen and unchanged ADAMTS13 activity in severe preeclampsia patients may be a result of a primary endothelial injury, leading to a micro-thrombus state. As the disease progresses, it may further exacerbate vascular endothelial cell injury and severe coagulopathy which develops into HELLP syndrome. In conclusion, no significant difference was noted in the plasma ADAMTS13 activity between severe preeclamptic patients and the third trimester pregnant women, whereas plasma vWF antigen levels were significantly higher in the severe preeclamptic group compared with the third trimester group. FRETS-vWF73 used in the present study to test the ADAMTS13 activity in pregnant women and severe preeclamptic patients helps to examine the possible mechanism of a prothrombotic state in severe preeclampsia. However, due to the limited size of this study further research involving a larger sample size and a more diverse study group is required in order to determine whether a decrease in plasma ADAMTS13 activity precedes the development of HELLP syndrome in severe preeclamptic patients. REFERENCES 1 Sadler JE, Moake JL, Miyata T, et al. Recent advances in thrombotic thrombocytopenic purpura. Hematologyo (Am Soc Hematol Educ Program), 2004, 2004(1):407-423 2 Fujikawa K, Suzuki H, McMullen B, et al. Purification of human von willebrand factor–cleaving protease and its identification as a new member of the metalloproteinase family. Blood, 2001,98(6):1662-1666 3 Molvarec A, Rigo J, Boze T, et al. Increased plasma von Willebrand factor antigen levels but normal von Wille-

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