1.Identification of two novel mutations in ADAMTS13 gene in a patient with hereditary thrombotic thrombocytopenic purpura.
Fang LIU ; Jie JIN ; Ning-zheng DONG ; Yun-gui WANG ; Chang-geng RUAN
Chinese Journal of Hematology 2005;26(9):521-524
OBJECTIVETo investigate the gene mutations of ADAMTS13 in a highly suspected hereditary thrombocytopenic purpura (TTP) patient, and then make a progressive diagnosis and adjust the plan of therapy.
METHODSADAMTS13 activity and inhibitor were determined by residual-collagen binding assay during several episodes. Genomic DNA extracted from the proband's peripheral blood was used for amplification of 29 exons and exon/intron boundaries of ADAMTS13 by PCR. The PCR products were screened by direct sequencing and the gene alterations were further confirmed by direct sequencing in her family members.
RESULTThe activity of the proband's ADAMTS13 was significantly reduced while no inhibitor was found. Two novel missense mutations were found in the TSPI repeated motif domain of ADAMTS13. In both mutations, thymine substituted for cytidine, resulting in the substitution of leucine for serine in nt 2708, exon 21 (codon S903L), and tryptophan for arginine in nt 3283, exon 25(codon R1095W). These two mutations were revealed as each heterozygote in the proband's parents.
CONCLUSIONThe deficiency of ADAMTS13 caused by two homozygote missense mutations might be responsible for episode of this TTP patient.
ADAM Proteins ; genetics ; Adult ; Exons ; genetics ; Female ; Humans ; Mutation ; Purpura, Thrombotic Thrombocytopenic ; genetics
2.Biological Function of CysR Domain of ADAMTS13.
Hao WU ; Hua LI ; Chang SU ; Hong-Yan LI ; Ri-Hua CUI ; Sheng-Yu JIN
Journal of Experimental Hematology 2021;29(3):893-900
OBJECTIVE:
To investigate the biological function of Cysteine rich (CysR) domain of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) on cleavage of von Willebrand factor (vWF) and provide experimental evidence for exploring the pathogenesis of thrombotic thrombocytopenic purpura (TTP).
METHODS:
The six amino acids (EDGTLS) in ADAMTS13 CysR domain were point mutated one by one, and the mutant ADAMTS13 proteins were expressed and purified. The cleavage products of vWF polymer by wild-type or mutant ADAMTS13 under denaturing condition or shear stress were separated by 1% SeaKem HGT agarose gel and detected by Western blot.
RESULTS:
The mutant ADAMTS13 plasmids (M1: Glu515Ala; M2: Asp516Ala; M3: Gly517Ala; M4: Thr518Ala; M5: Leu519Ala; M6: Ser520Ala) were successfully constructed and the proteins of wild-type and mutant ADAMTS13 were purified. Wild-type ADAMTS13 almost completely cleaved the vWF polymer under denaturing condition, while the cleavage activity of M1 mutant was significantly reduced in the same condition (P<0.01). The cleavage activity of M1 mutant of ADAMTS13 was also significantly reduced compared with that of the wild-type under shear stress (P<0.01). The activity of M1 mutant to cleave the FRETS-vWF73 was dramatically reduced compared with that of wild-type ADAMTS13. However, the binding ability of M1 mutant to vWF was similar with that of wild-type ADAMTS13.
CONCLUSION
The CysR domain of ADAMTS13 plays an important role in the digestion of vWF under denaturing condition and shear stress. The Glu515 amino acid residue might be an important site for substrate recognition.
ADAM Proteins
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ADAMTS13 Protein/genetics*
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Humans
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Purpura, Thrombotic Thrombocytopenic/genetics*
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von Willebrand Factor/genetics*
3.Advance in the diagnosis and treatment of hereditary thrombotic thrombocytopenic purpura.
Chinese Journal of Medical Genetics 2022;39(4):442-446
Congenital thrombotic thrombocytopenic purpura, also known as Upshaw-Schulman syndrome, is a rare autosomal recessive genetic disorder. The main pathogenesis is homozygous or compound heterozygous variants of von Willebrand factor lyase (ADAMTS13) gene mapped to chromosome 9q34, which may result in severe lack of ADAMTS13 which cleaves von Willebrand factor (vWF) multimers in the plasma and increase the risk of microvascular thrombosis, leading to various complications. The advance of research on the pathogenesis of cTTP, recombinant human ADAMTS13 and gene therapy have made breakthroughs which may lead to cure of cTTP. This article has provided a review for the latest progress made in the diagnosis and treatment of cTTP.
ADAM Proteins/genetics*
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ADAMTS13 Protein/genetics*
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Homozygote
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Humans
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Purpura, Thrombotic Thrombocytopenic/therapy*
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von Willebrand Factor/genetics*
4.Thrombotic thrombocytopenic purpura in pediatric patients.
Melanie STEELE ; Howard H W CHEN ; Jeremy STEELE ; Anthony K C CHAN ; Keith K LAU
Chinese Journal of Contemporary Pediatrics 2012;14(11):803-810
Although thrombotic thrombocytopenic purpura (TTP) is rarely seen in pediatric patients, failure to recognize this condition often leads to severe consequences and poor outcomes. Classic features of TTP include thrombocytopenia, microangiopathic hemolytic anemia, acute kidney injury, fever, and central nervous system involvement. However, patients suffering from this condition may not present with all of the symptoms simultaneously. Therefore, it is of utmost importance for healthcare providers to have a high index of suspicion. Laboratory investigations may reveal the presence of schistocytes on peripheral blood smear, negative Coombs test, high lactate dehydrogenase levels and severely low platelet counts. The etiology of TTP is mainly due to insufficient cleavage of the large multimers of von Willebrand factor (vWF) secondary to decreased activity of ADAMTS13 (a disintegrin and metalloprotease with Thrombospondin type 1 repeats, member 13). TTP can be broadly classified into familial TTP (Upshaw Schulman syndrome) and non-familial TTP. Familial TTP is due to a congenital deficiency of ADAMTS13. Its mainstay of therapy is initiation of plasmapheresis during the acute phase, followed by regular fresh frozen plasma (FFP) infusions. Alternatively, non-familial TTP is due to a decrease in ADAMTS13 activity secondary to the presence of anti-ADAMTS13 antibodies. Once again, the primary treatment is plasmapheresis; however, recent anecdotal data also supports the use of rituximab in select cases.
ADAM Proteins
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genetics
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ADAMTS13 Protein
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Antibodies, Monoclonal, Murine-Derived
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therapeutic use
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Child
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Humans
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Plasmapheresis
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Purpura, Thrombotic Thrombocytopenic
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etiology
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therapy
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Rituximab
5.Recurrent thrombocytopenia with hemolytic anemia in a boy aged 3 years.
Min-Hui ZENG ; Xiang-Ling HE ; Xin TIAN ; Yu-Hui HUANG
Chinese Journal of Contemporary Pediatrics 2021;23(5):524-529
A boy, aged 3 years and 8 months, had recurrent thrombocytopenia with hemolytic anemia for more than 3 years. The physical examination showed no enlargement of the liver, spleen, and lymph nodes or finger deformities. Laboratory results showed a negative result of the direct antiglobulin test, normal coagulation function, and increases in bilirubin, lactate dehydrogenase and reticulocytes. The results of von Willebrand factor-cleaving protease ADAMTS13 activity assay showed extreme deficiency, and antibody assay showed negative ADAMTS13 inhibitory autoantibodies. Next-generation sequence showed compound heterozygous mutation in the
ADAM Proteins/genetics*
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ADAMTS13 Protein
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Anemia, Hemolytic
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Autoantibodies
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Child
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Child, Preschool
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Humans
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Infant, Newborn
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Male
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Mutation
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Purpura, Thrombotic Thrombocytopenic
6.Expression of vWF73 and VWF114 fragments of von Willebrand factor A2 domain and their utilization in detecting ADAMTS13 activity.
Jing-yu ZHANG ; Zhen-ni MA ; Ning-zheng DONG ; Lu-ping HU ; Jian SU ; Zhao-yue WANG ; Chang-geng RUAN
Chinese Journal of Hematology 2011;32(5):337-341
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
7.A2 domain of human von Willebrand factor expressed in E. coli and its biological activity.
Jian SU ; Xia BAI ; Wei-Qiang GAO ; Chang-Geng RUAN
Journal of Experimental Hematology 2005;13(6):1090-1093
Von Willebrand factor (vWF) is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease (vWD) and thrombotic thrombocytopenic purpure (TTP). To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 x His tag (pQE-30), the recombinant expression vector was transformed into E. coli (strain M15) and induced by IPTG. The recombinant fragment comprising residues 718-905 of mature vWF was designated as rvWF-A2. It was purified by Ni-NTA resin column chromatography and refolded in Tris buffer containing GSH and GSSG. The results demonstrated that rvWF-A2 was expressed successfully in E. coli M15, amounting to 42% of total bacterial protein with the purity over 98%. It was identified that rvWF-A2 can be efficiently cleaved by the citrated normal plasma while no cleavage can be detected by the TTP plasma or plasma with EDTA. It is concluded that rvWF-A2 expressed efficiently in E. coli demonstrated excellent biological activity, which lays a solid foundation for establishment of method to measure quantatively the activity of ADAMTS-13.
ADAM Proteins
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metabolism
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ADAMTS13 Protein
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Escherichia coli
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genetics
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Humans
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Peptide Fragments
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biosynthesis
;
genetics
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Purpura, Thrombotic Thrombocytopenic
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diagnosis
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metabolism
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Recombinant Proteins
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biosynthesis
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isolation & purification
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von Willebrand Factor
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biosynthesis
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chemistry
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genetics