title: Simplified assay for VWF cleaving protease (ADAMTS13) activity and inhibitor in plasma.
abstract: The absence of VWF cleaving protease activity (VWFcp, ADAMTS13) has recently been identified as a central component in the pathogenesis of TTP. Several assays for the measurement of ADAMTS13 activity have been described, but most are cumbersome and employ techniques not easily adapted to routine laboratories. Thus, ADAMTS13 assays are available at only a few reference or research laboratories. Prompt identification of absent or impaired ADAMTS13 activity could prove invaluable to clinical decision-making regarding diagnosis and treatment of suspected TTP patients. We describe an assay for ADAMTS13 that uses a commercial source of VWF substrate and obviates dialysis for sample and substrate preparation. Patient and normal plasma are prepared by desalting over Micro-Spin columns, and barium is added to activate the cleaving protease. Humate-P(R) is prepared over a desalting column and reconstituted in urea prior to use as the exogenous VWF substrate. Desalted plasma is mixed with prepared substrate, and digestion is carried out at 37 degrees C. Maximum sensitivity to very low levels of enzyme activity is achieved by using undiluted plasma and allowing digestion to proceed for 14 hr. The extent of VWF multimer cleavage is then demonstrated by Western blot. We used this assay to analyze VWF cleaving protease activity in plasma samples from 30 patients treated at our institution for TTP. Plasma from patients lacking ADAMTS13 was incubated with PNP and then analyzed by the same methods to determine the presence of an inhibitor. Our results indicate that the assay is suitable for providing timely information regarding ADAMTS13 activity for the diagnosis and treatment of patients with suspected TTP.
authors.affiliation: Section on Hematology and Oncology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA. mknovich@wfubmc.edu
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Knovich
authors.forename: Karen
authors.initials: K
authors.lastname: Craver
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Matulis
authors.forename: Heather
authors.initials: H
authors.lastname: Lawson
authors.forename: John
authors.initials: J
authors.lastname: Owen
mesh_terms: D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D015153:Blotting, Western; D005169:Factor VIII / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000097:blood* / Q000172:deficiency; D011480:Protease Inhibitors / Q000097:blood*; D011697:Purpura, Thrombotic Thrombocytopenic / Q000175:diagnosis / Q000201:enzymology; D012680:Sensitivity and Specificity
chemical_list: D011480:Protease Inhibitors; D005169:Factor VIII; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human