Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Plaimauer, Barbara; Zimmermann, Klaus F.; Völkel, Dirk; Antoine, Gerhard; Kerschbaumer, Randolf J.; Jenab, Pegah; Furlan, Miha; Gerritsen, Helen; Lämmle, Bernhard; Schwarz, Hans; Scheiflinger, Friedrich

doi:10.1182/blood-2002-05-1397

Cited by 216 publications

(169 citation statements)

References 40 publications

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“…The samples were resolved on 8% to 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and visualized by Western blot using the anti-ADAMTS13 monoclonal antibody, 242/H2. 16 …”

Section: Transient Expression Of the Adamts13 Mutantmentioning

confidence: 99%

“…16,17 Dilutions of purified recombinant ADAMTS13 of known concentration were used for assay calibration. Proteolysis of VWF was measured by resolving reactions on 1% agarose gel electrophoresis or 8% SDS-PAGE under reducing conditions and blotting the VWF and fragments with iodinated or peroxidase-conjugated anti-VWF polyclonal antibodies (Dako, Carpinteria, CA), respectively.…”

Section: Assay Of Adamts13 Activitymentioning

confidence: 99%

See 1 more Smart Citation

Congenital thrombotic thrombocytopenic purpura in association with a mutation in the second CUB domain of ADAMTS13

Pimanda

Maekawa

Wind

et al. 2004

Blood

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IntroductionThrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by a schistocytic hemolytic anemia and consumptive thrombocytopenia with varying degrees of neurologic and renal impairment. The presence of ultralarge von Willebrand factor (VWF) multimers in the plasmas of patients with chronic relapsing TTP during remission that disappear during an attack led to the implication of these multimers in the pathogenesis of the platelet-rich, fibrin-poor thrombi that occlude arterioles and are the hallmark of this disorder. 1 The VWF-cleaving metalloproteinase, ADAMTS13, hydrolyzes the ultralarge VWF multimers as they emerge from endothelial cells and undergo conformational strain due to shear stress in arterioles and capillaries. 2 A severe deficiency of VWF-cleaving proteinase activity is associated with the pathogenesis of congenital TTP (the SchulmanUpshaw syndrome). 3,4 A number of mutations, spread throughout the ADAMTS13 gene, have been reported in association with congenital TTP. 3,[5][6][7][8][9] The CUB domains at the C-terminus of ADAMTS13 are of uncertain physiologic relevance. The findings that the CUB domains are not required for VWF-cleaving proteinase activity measured under static conditions 10,11 and that certain strains of mice have a variant form of murine ADAMTS13 that lacks the CUB domains (together with the seventh and eighth thrombospondin type 1 (TSP-1) repeats) support the premise that these domains are dispensable in vivo. 12 However, a report that peptides from the CUB domains inhibit VWF-cleaving proteinase activity under flow, but not static, conditions suggests a functional role for these domains. 13 We studied a patient with congenital TTP who is a compound heterozygote for the Thr196Ile mutation in the metalloproteinase domain and the insertion of a nucleotide (A) at 4143-4144. This insertion results in a frameshift in the second CUB domain and loss of the last 49 amino acids of the protein. The CUB mutation had little effect on the specific activity of the enzyme but its secretion from COS-7 cells was impaired. Study design Case historyWe investigated an 8-year-old white girl with congenital TTP. Diagnosis was made when she was 18 months of age; she receives a plasma infusion every 2 to 3 months when her platelet count falls below 30 ϫ 10 9 /L. Assay of plasma VWF-cleaving proteinase activityAn assay based on the collagen-binding activity of digested VWF 14 was used to measure the VWF-cleaving proteinase activity in the plasma of the patient (during remission) and her family. ADAMTS13 sequence analysisDNA was extracted from peripheral blood leukocytes and all the exons and intron-exon boundaries of the ADAMTS13 gene were amplified by polymerase chain reaction (PCR) and sequenced. 5 The study was conducted with parental consent and institutional ethics approval. Transient expression of the ADAMTS13 mutantThe 4143insA mutant of the ADAMTS13 cDNA 15 was created using PCR-based mutagenesis. The wild-type and mutant constructs were subcloned into the mam...

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Section: Transient Expression Of the Adamts13 Mutantmentioning

confidence: 99%

Section: Assay Of Adamts13 Activitymentioning

confidence: 99%

Congenital thrombotic thrombocytopenic purpura in association with a mutation in the second CUB domain of ADAMTS13

Pimanda

Maekawa

Wind

et al. 2004

Blood

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“…Since the identification of ADAMTS13 protease,24 increased attention was directed to the cloning and expression of the protease to better evaluate its function and therapeutic opportunities. Antoine et al., showed the normalization of the ADAMTS13 activity in two brothers with congenital TTP when their plasma was mixed with rADAMTS13 obtained from transfected HEK293 cells 25.…”

Section: Recombinant Adamts13 (Radamts13 Bax930)mentioning

confidence: 99%

Novel therapies in thrombotic thrombocytopenic purpura

Masias

Cataland

2018

Research and Practice in Thrombosis and Haemostasis

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Essentials The standard of care for patients with TTP remains daily plasma exchange in addition to immune suppressive therapy.Despite the improved treatment options for TTP, the acute mortality of TTP remains between 15‐20%.Caplacizumab reduces the time to platelet recovery and the exacerbation rate in acute TTP.A better understanding of the cause and treatments of long‐term complications of TTP are needed. Thrombotic thrombocytopenic purpura (TTP) is characterized by microangiopathic hemolytic anemia and a consumptive thrombocytopenia, as a result of severe deficiency of ADAMTS13. The standard of care of the acute episode is treatment with plasma exchange and immunosuppression. After the acute episode is resolved, patients face a significant risk of relapse and long‐term complications associated with significant morbidity and even mortality. Novel treatments have been under development and will be discussed in this review. Caplacizumab, a nanobody that blocks the interaction between VWF and platelets, has shown promising results in decreasing the time to recover from the acute events that will hopefully translate into long‐term clinical benefit for patients. In addition, identifying biomarkers to allow us to better predict the risk for relapse and the development of these long‐term complications in patients with TTP are a few of the challenges that require our attention moving forward.

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“…Even though functionally active recombinant ADAMTS-13 has been expressed in transfected mammalian cells already in 2002 [15], and many diagnostic studies on VWF-cleaving protease activity and functionally inhibiting autoantibodies in patients with TTP, other thrombotic microangiopathies and other disorders have been reported [for review, see [16][17][18], assays of ADAMTS-13 activity in human plasma have been subject to ongoing controversy. Several ADAMTS-13 activity assays, based on the degradation of purified or recombinant multimeric VWF by patient plasma and analyzing its multimeric pattern [1,5] or disulfide-linked cleavage fragments [2,6], measuring residual collagen-binding activity or ristocetin cofactor activity of degraded VWF substrate and other functional assays have been described [for review, see 19].…”

mentioning

confidence: 99%

“…Even though functionally active recombinant ADAMTS-13 has been expressed in transfected mammalian cells already in 2002 [15], and many diagnostic studies on VWF-cleaving protease activity and functionally inhibiting autoantibodies in patients with TTP, other thrombotic microangiopathies and other disorders have been reported [for review, see 16-18], assays of ADAMTS-13 activity in human plasma have been subject to ongoing controversy. Several ADAMTS-13 activity assays, based on the degradation of purified or recombinant multimeric VWF by patient plasma and analyzing its multimeric pattern [1,5] Two multicenter studies [20,21] on several of these activity assays have been performed and generally have shown acceptable agreement concerning the detection of severe ADAMTS-13 functional deficiency ( < 5% of normal plasma activity); however, there was less concordance of the results on plasma samples with mildly decreased or normal ADAMTS-13 activity.…”

mentioning

confidence: 99%

A new tool to further explore the role of ADAMTS‐13 in health and disease

Lämmle

Hovinga

2006

Journal of Thrombosis and Haemostasis

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See also Feys HB, Liu F, Dong N, Pareyn I, Vauterin S, Vandeputte N, Noppe W, Ruan C, Deckmyn H, Vanhoorelbeke K. ADAMTS-13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences. This issue, pp 955-62.In 1996, a hitherto unrecognized protease was isolated from human plasma [1,2] that cleaved multimeric von Willebrand factor (VWF) in vitro to cleavage fragments previously reported to be generated in vivo [3]. Shortly after its discovery this VWFcleaving protease activity was found to be severely deficient in many patients diagnosed with acute idiopathic thrombotic thrombocytopenic purpura (TTP) [4][5][6], most often because of inhibitory IgG autoantibodies in acute acquired TTP [5][6][7], but sometimes also in siblings with an obviously hereditary form of TTP without inhibiting autoantibodies [4,5], a condition previously denoted as Upshaw-Schulman syndrome [8,9].In 2001, the VWF-cleaving protease was purified from human plasma to homogeneity allowing partial amino acid sequencing [10][11][12] and identification of the enzyme as a member of the ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type 1-motifs) family of metalloproteases, denoted as ADAMTS-13 [13]. Independently, genome-wide linkage analysis in pedigrees affected by hereditary TTP identified the same gene and several different ADAMTS-13 mutations were reported: double heterozygous or homozygous mutations were putatively responsible for severely deficient ADAMTS-13 activity in affected patients; heterozygous carriers of mutations were asymptomatic and showed about 50% of normal VWF-cleaving protease activity [14].Even though functionally active recombinant ADAMTS-13 has been expressed in transfected mammalian cells already in 2002 [15], and many diagnostic studies on VWF-cleaving protease activity and functionally inhibiting autoantibodies in patients with TTP, other thrombotic microangiopathies and other disorders have been reported [for review, see 16-18], assays of ADAMTS-13 activity in human plasma have been subject to ongoing controversy. Several ADAMTS-13 activity assays, based on the degradation of purified or recombinant multimeric VWF by patient plasma and analyzing its multimeric pattern [1,5] Two multicenter studies [20,21] on several of these activity assays have been performed and generally have shown acceptable agreement concerning the detection of severe ADAMTS-13 functional deficiency ( < 5% of normal plasma activity); however, there was less concordance of the results on plasma samples with mildly decreased or normal ADAMTS-13 activity. Moreover, some assays proved to be clearly unreliable for diagnostic purposes [21]. A recently developed functional assay using a truncated 73-amino acid VWF peptide encompassing the 1605 Y-1606 M cleavage site and labeled with a fluorescent and quencher group on both sites of the cleavage site, respectively, allows for rapid activity measurement of ADAMTS-13 by fluorescence resonance energy transfer and may greatly...

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Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Cited by 216 publications

References 40 publications

Congenital thrombotic thrombocytopenic purpura in association with a mutation in the second CUB domain of ADAMTS13

Congenital thrombotic thrombocytopenic purpura in association with a mutation in the second CUB domain of ADAMTS13

Novel therapies in thrombotic thrombocytopenic purpura

A new tool to further explore the role of ADAMTS‐13 in health and disease

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