ADAMTS13, a plasma metalloprotease, specifically cleaves von Willebrand factor (vWF). Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP). In this review, the structure and function of ADAMTS13 protease and its relationship with TTP are summarized.
ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Humans ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; pathology ; therapy

This study was to acquire recombinant protein of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thromboSpondin type 1 motifs 13), for further studies on its biological function in thrombosis and hemostasis. We transfected the Hela cells with the plasmid pSecTag-ADAMTS13 by lipofectamine. A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. We also measured the enzymatic activity of recombinant protein (rADAMTS13) by GST-His two-site ELISA assay. The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of rADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 kDa with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of rADAMTS13 activity was 6.4 U/mL, according to the normal plasma defined as 1 U/mL. In conclusion, rADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease.
ADAM Proteins ; biosynthesis ; genetics ; metabolism ; ADAMTS13 Protein ; HeLa Cells ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Transfection ; von Willebrand Factor ; metabolism

This study was aimed to explore if the intracellular transportation direction of von Willebrand factor-cleaving protease (ADAMTS13, vWF-CP) after synthesis is determined by the carboxyl terminal TSP2-8CUB1+2 domains of ADAMTS13 and to decipher the relationship between the structure and function of ADAMTS13. The recombinant plasmids pcDNA3.1-ADAMTS13 and pcDNA3.1-delTSP2-8CUB1+2 ADAMTS13 were introduced into Madin-Darby canine kidney cells (MDCK) by lipofectamine-mediated DNA transfection. Positive cell clones gained after antibiotic-screening were grown on 6-well transwell filter units with a zeolite membrane in the middle layer. The conditioned culture media in both apical and basolateral wells were collected when cells reached confluency and the tight cell monolayer formed. ADAMTS13 proteases in the conditioned media were determined by Western blot, and the direction of ADAMTS13 secretion in polarized cells was comparatively analyzed. The results showed that Madin-Darby canine kidney cells stably expressing wild-type ADAMTS13 were grown on 6-well transwell filter units, then ADAMTS13 protease was only determined in the apical area of the transwell filter units by Western blot, but the recombinant ADAMTS13 protease was determined both in the apical and basolateral area of cells in the group of expressing TSP2-8CUB-1+2 domain-deleted ADAMTS13. It is concluded that the metalloprotease ADAMTS13 is sorted apically in polarized cells, and the carboxyl-terminal TSP2-8 and CUB1+2 domains of ADAMTS13 are important for the direction of ADAMTS13 protease transportation in the cells after being synthesized.
ADAM Proteins ; biosynthesis ; ADAMTS13 Protein ; Animals ; Dogs ; Madin Darby Canine Kidney Cells ; Plasmids ; Protein Interaction Domains and Motifs ; Protein Transport ; genetics ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; secretion

OBJECTIVETo investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A.

METHODSRecombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis.

RESULTSIn vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition.

CONCLUSIONThe A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

ADAM Proteins ; genetics ; metabolism ; ADAMTS13 Protein ; Genotype ; HeLa Cells ; Humans ; Hydrolysis ; Mutation ; Recombinant Proteins ; genetics ; metabolism ; von Willebrand Disease, Type 2 ; genetics ; metabolism ; von Willebrand Factor ; genetics

BACKGROUNDA severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor (VWF) within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity.

METHODSFirst, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated.

RESULTSPlasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction (AMI), and idiopathic thrombocytopenic purpura (ITP) patients determined by the new assay were (89.75 +/- 7.93)%, (17.63 +/- 18.71)%, (68.55 +/- 18.08)%, (85.83 +/- 9.84)%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a TTP plasma sample from a non-TTP plasma sample (P < 0.01), and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient.

CONCLUSIONThis new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.

ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombotic Thrombocytopenic ; metabolism ; Young Adult ; von Willebrand Factor ; metabolism

ADAM Proteins ; biosynthesis ; genetics ; ADAMTS13 Protein ; Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; Cell Line ; Female ; Humans ; Liver Neoplasms ; metabolism ; Middle Aged ; von Willebrand Factor ; biosynthesis ; genetics

OBJECTIVETo investigate the relationship between post-stenting coronary thrombolysis in myocardial infarction (TIMI) flow and plasma von Willebrand factor (vWF) and its cleaving protease (ADAMTS-13) levels in patients with ST segment elevation myocardial infarction (STEMI).

METHODSSTEMI patients who underwent primary percutaneous coronary intervention (PCI) and stenting between September, 2007 and December, 2009 were enrolled. According to the post-stenting TIMI flow, patients were divided to TIMI ≤ 2 group (n = 43) and TIMI 3 group (n = 43). Patients with chest pain or dyspnea and normal coronary angiographic results served as control group (n = 43). The levels of vWF and ADAMTS-13 were measured by ELISA at three time points: immediately after admission, beginning of PCI and 1 week after PCI.

RESULTSLevels of vWF in STEMI patients at all 3 time points were significantly higher than in control patients, and the level of vWF was significantly higher in TIMI ≤ 2 group than in TIMI 3 group [at admission: (6721.83 ± 1380.58) U/L vs. (4786.12 ± 2362.01) U/L, P < 0.05; at the beginning of PCI: (5744.65 ± 1240.71) U/L vs. (3011.33 ± 2270.40) U/L, P < 0.05 and at 1 week after PCI: (2001.48 ± 931.70) U/L vs. (1365.17 ± 724.12) U/L, P < 0.05]. ADAMTS-13 levels were similar among groups at admission and at beginning of PCI, however, the level of ADAMTS-13 at 1 week after PCI was significantly higher in TIMI ≤ 2 group than that in TIMI 3 group [(406.93 ± 101.44) mg/L vs. (270.34 ± 115.12) mg/L, P < 0.001]. Logistic regression analysis showed that both vWF at admission (OR = 1.917, P < 0.01) and vWF at the beginning of PCI (OR = 2.016, P < 0.01) were risk factors of TIMI ≤ 2.

CONCLUSIONIncreased vWF during peri-PCI periods was associated with post-stenting coronary TIMI ≤ 2 after primary PCI in STEMI patients, and the imbalance between vWF and ADAMTS-13 may thus play an important role in the development of slow flow post PCI.

ADAM Proteins ; blood ; ADAMTS13 Protein ; Aged ; Angioplasty, Balloon, Coronary ; Case-Control Studies ; Coronary Circulation ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; blood ; physiopathology ; therapy ; von Willebrand Factor ; metabolism

OBJECTIVETo investigate the ADAMTS13 antigen levels and activity in patients with acute myocardial infarction (AMI) and acute ischemic stroke (AIS), and explore its significance in these diseases.

METHODSADAMTS13 activity levels were detected by a new developed Frests-vWF73 kit, ADAMTS13 antigen levels by ELISA kit, and vWF multimers by electrophoresis.

RESULTSADAMTS13 antigen in normal control, AMI and AIS was (878 +/- 198), (618 +/- 188) and (702 +/- 155) U/L, and ADAMTS13 activity was (81.7 +/- 13.9)%, (59.2 +/- 22.1 )% and (65.4 +/- 15.8)%, respectively, being significantly decreased in AMI and AIS patients.

CONCLUSIONADMATS13 might involve in arterial infarction diseases.

ADAM Proteins ; blood ; ADAMTS13 Protein ; Aged ; Aged, 80 and over ; Brain Infarction ; blood ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; blood ; von Willebrand Factor ; metabolism

Von Willebrand factor (vWF) is a glycoprotein with a crucial role in the formation of platelet thrombi, and ADAMTS13 is the main enzyme responsible for vWF cleavage. Both are important in the relationship between diabetic nephropathy, hypercoagulability, and cardiovascular disease. This study evaluated a potential relationship between vitamin D (vitD) levels, vWF, ADAMTS13 activity, and inflammation in diabetic patients on chronic hemodialysis (HD). Blood samples from 52 diabetic patients on chronic HD were obtained to determine vitD levels, vWF, and ADAMTS13 activity, and inflammatory markers. HD patients were grouped according to 25-hydroxyvitamin D [25(OH) VitD]<25 nmol/L (n=16) or >25 nmol/L (n=36). vWF antigen and vWF activity were elevated in both groups, with an average of 214.3±82.6% and 175.8±72.6%, respectively. Average ADAMTS13 activity was within the normal range in both groups. Blood samples from the vitD <25 nmol/L group showed a positive correlation between c-reactive protein (CRP) and vWF levels (P=0.023; r=0.564; 95% confidence interval=0.095-0.828), with a negative correlation between HbA1c and 25(OH) VitD (P=0.015; r=-0.337; 95% confidence interval=-0.337-0.19). Diabetic patients on chronic HD had elevated vWF levels and activity with no significant change in ADAMTS13 activity. The correlation between CRP and vWF levels in the 25(OH) VitD<25 nmol/L group suggests inflammatory-related endothelial dysfunction in these patients.
ADAMTS13 Protein/*metabolism ; Aged ; C-Reactive Protein/analysis ; Diabetes Mellitus, Type 2/complications/*diagnosis/metabolism ; Female ; Hemoglobin A, Glycosylated/analysis ; Humans ; Male ; Middle Aged ; Renal Dialysis ; Renal Insufficiency, Chronic/complications/*diagnosis/metabolism ; Vitamin D/*analogs & derivatives/blood ; von Willebrand Factor/*metabolism

OBJECTIVETo construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.

METHODSThe DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates.

RESULTSTwo small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates.

CONCLUSIONSTwo GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

ADAM Proteins ; blood ; genetics ; ADAMTS13 Protein ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Glutathione Transferase ; metabolism ; Humans ; Male ; Purpura, Thrombotic Thrombocytopenic ; blood ; genetics ; metabolism ; von Willebrand Factor ; genetics ; metabolism