Simplified assay for VWF cleaving protease (ADAMTS13) activity and inhibitor in plasma

American Journal of Hematology
Mary Ann KnovichJohn Owen

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 dem...Continue Reading

References

Nov 26, 1998·The New England Journal of Medicine·H M Tsai, E C Lian
Aug 16, 2002·Proceedings of the National Academy of Sciences of the United States of America·Koichi KokameYoshihiro Fujimura
Jul 23, 2003·Journal of Thrombosis and Haemostasis : JTH·H-M Tsai
Jul 23, 2003·Journal of Thrombosis and Haemostasis : JTH·G Remuzzi

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Citations

Dec 26, 2006·European Journal of Pediatrics·Minola ManeaDiana Karpman
Dec 27, 2005·Best Practice & Research. Clinical Haematology·Richard K Spence
Jan 22, 2010·Journal of Thrombosis and Haemostasis : JTH·F PeyvandiS J Machin
Jul 1, 2017·Journal of Thrombosis and Haemostasis : JTH·M SahaX L Zheng

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