Multimeric analysis of von Willebrand factor by molecular sieving electrophoresis in sodium dodecyl sulphate agarose gel

Raines, Geoffrey; Aumann, Heather; Sykes, Stephen M.; Street, Alison

doi:10.1016/0049-3848(90)90181-b

Cited by 90 publications

(64 citation statements)

References 18 publications

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“…The specific cleavage of VWF subunits by ADAMTS13 was confirmed by SDS-PAGE and Western blotting as described above, but samples were reduced with 2% ␤-mercaptoethanol, and proteins were detected with polyclonal anti-VWF antibody (23).…”

Section: Methodsmentioning

confidence: 99%

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Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Zheng

Nishio

Majerus

et al. 2003

Journal of Biological Chemistry

180

236

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ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr 1605 -Met 1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His 6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more Cterminal TSP1 and CUB domains are dispensable in vitro.Thrombotic thrombocytopenic purpura (TTP) 1 is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia, and it may be accompanied by neurological dysfunction, renal failure, and fever (1-3). If untreated, the mortality can exceed 90%, but plasma-exchange therapy has reduced the mortality to less than 20% (4). Although the pathophysiology of TTP is not fully understood, a plausible model has been proposed in which the proteolysis of von Willebrand factor (VWF) plays a central role (5). VWF is a multimeric protein that binds receptors on the surface of platelets and in connective tissue, thereby mediating the adhesion of platelets to sites of vascular injury. If unchecked, the process can lead to microvascular thrombosis. A plasma VWF-cleaving protease has been described that acts on the Tyr 1605 -Met 1606 bond in the central A2 domain of the VWF subunit, and cleavage is stimulated by shear forces like those occurring at sites of thrombosis or by low concentrations of urea or guanidine (6, 7). This proteolytic reaction limits VWF-dependent platelet adhesion, and most adults with idiopathic TTP have an acquired autoantibody that inhibits the VWF-cleaving protease (8, 9). Therefore, therapeutic plasma exchange may ameliorate TTP by replacing the missing protease and removing the inhibitory antibody.The VWF-cleaving protease was recently purified and identified as a new member of the ADAMTS family of metalloproteases (10, 11), so named for the combination of a disintegrin-like and metalloprote...

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Section: Methodsmentioning

confidence: 99%

“…The multimer distribution of VWF after incubation with ADAMTS13 variants was assessed also by SDS-agarose gel electrophoresis and Western blotting with peroxidase-conjugated polyclonal anti-VWF antibody (catalog no. P0226, DAKO) as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Zheng

Nishio

Majerus

et al. 2003

Journal of Biological Chemistry

180

236

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“…Both antibodies recognize the dimer of VWF as well as fully multimeric VWF (13). VWF multimer analysis was performed as previously described (14) with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

Identification of the Regulatory Elements of the Human von Willebrand Factor for Binding to Platelet GPIb

Nakayama

Matsushita

Dong

et al. 2002

Journal of Biological Chemistry

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“…Again after several washes, the membrane is ''developed'' using a specific substrate that is converted by the conjugated enzyme to elicit a color signal. Numerous methods [13,16,18,[24][25][26][27][28][29] (Table I) have been published using vari- ous primary antibodies, secondary conjugated antibodies, and substrates.…”

Section: Detection Of Protein (Visualization)mentioning

confidence: 99%

Analysis of von Willebrand factor structure by multimer analysis

Ledford‐Kraemer¹

2010

American J Hematol

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Analysis of von Willebrand factor (VWF) structure is achieved by performing a highly specialized procedure, VWF multimer analysis. The test is reserved for the reference or specialized laboratory environment. The assay is qualitative (though under some circumstances multimers may be quantified) in that it assesses the overall size distribution of VWF multimers as well as their individual internal structure. The test is used predominantly to type or subtype von Willebrand disease. The analysis of VWF multimers generally consists of four steps: (1) electrophoresis of plasma in an agarose gel, (2) either gel fixation or transfer of the electrophoretic protein product to a membrane, (3) immunodetection of the protein, and (4) evaluation of the protein in the gel or membrane. The assay is complex, time consuming, requires specialized equipment and technical expertise, and is not standardized. Am. J. Hematol. 85:510-514, 2010. V

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Multimeric analysis of von Willebrand factor by molecular sieving electrophoresis in sodium dodecyl sulphate agarose gel

Cited by 90 publications

References 18 publications

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Identification of the Regulatory Elements of the Human von Willebrand Factor for Binding to Platelet GPIb

Analysis of von Willebrand factor structure by multimer analysis

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