A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura.
BACKGROUND
Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either predenatured von Willebrand factor (VWF) or small peptides derived from the VWF-A2 domain. The physiologic relevance of the assay results is uncertain.


METHODS
We sought to develop a novel shear-based assay to assess plasma ADAMTS13 activity and inhibitors. We also compared this assay with a fluorogenic peptide assay.


RESULTS
We found that an incubation of purified plasma VWF with 0.5 to 1.0 µL of citrated plasma under constant vortexing at 2500 rpm for 60 minutes in the presence of 5 mmol/L CaCl(2) and 1.7 µmol/L ZnCl(2) and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear-based assay appeared to be more sensitive than the guanidine-denaturization assay for determining plasma ADAMTS13 activity.


CONCLUSIONS
Our fluid shear-based assay may be useful for investigating basic biologic function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.
Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and The University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
Yue
Y
Han
Juan
J
Xiao
Erica
E
Falls
X Long
XL
Zheng
D051722:ADAM Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D002712:Chlorides / Q000737:chemistry; D006801:Humans; D010979:Platelet Function Tests; D011697:Purpura, Thrombotic Thrombocytopenic / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013314:Stress, Mechanical; D015032:Zinc / Q000737:chemistry; D014841:von Willebrand Factor / Q000378:metabolism
D002712:Chlorides; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D015032:Zinc
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