Assays of ADAMTS-13 activity.
Various assays for determination of ADAMTS-13 activity in plasma have been developed, all comprising two steps. The first step consists of proteolyzing a substrate by ADAMTS-13. Substrates were either exogenous von Willebrand factor (VWF) (purified from human plasma concentrates or recombinant VWF [rVWF]), purified VWF fragments, or endogenous VWF (from the tested plasma sample). All assays required a step in which substrate unfolding is performed using either urea or guanidine. Test plasmas were used at various dilutions and ADAMTS-13 was activated in most cases by divalent cations. The second step consists of quantifying the digestion products or the residual VWF remaining after proteolysis. Cleavage of VWF was thus estimated using electrophoresis (generation of proteolytic fragments or decrease of the size of multimers), functional methods (decrease of the collagen binding activity or of ristocetin cofactor activity), or immunological methods (enzyme-linked immunosorbent assay [ELISA] or immunoradiometric assay (IRMA) using selected monoclonal antibodies to VWF). Since 1998, all of these assays have been used to demonstrate the relevance of a deficient ADAMTS-13 activity in thrombotic thrombocytopenic purpura (TTP). However, improvements are required, as these methods remain cumbersome, time-consuming, and too remote from physiology to be routinely helpful for rapid laboratory diagnosis.
Veyradier
Agnès
A
INSERM U.142, Hôpital de Bicêtre, Paris, France.
Girma
Jean-Pierre
JP
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D014841:von Willebrand Factor / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D014841:von Willebrand Factor; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human