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-Pâ 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 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. Am.

Thrombotic thrombocytopenic purpura: pathogenesis, diagnosis and potential novel therapeutics

“…ADAMTS‐13 assay for diagnostic purpose is mostly fluorescence resonance energy transfer (FRET) based , followed by ELISA , mass spectrometry , and coagulation analyzer‐based assays. Each has its advantages and disadvantages, but will produce ADAMTS‐13 activity results within hours.…”

Transplant-associated microangiopathy (TAM) in recipients of allogeneic hematopoietic stem cell transplants

“…This deficiency may be due to autoantibodies or congenital defects. [21][22][23][24][25][26][27] Other forms of thrombotic microangiopathy are associated with enteropathogenic Escherichia coli infections (hemolytic uremic syndrome), drugs, including CsA, or occur after transplant, that is, TAM. Reduced metalloprotease activity is not the pathophysiologic mechanism in TAM 25, 28 and plasma exchange is generally ineffective.…”

ADAMTS‐13 assays in thrombotic thrombocytopenic purpura

“…Since 1998, several assays for measuring ADAMTS-13 activity have been developed [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. They are based on the degradation of purified, plasma-derived or recombinant VWF multimers or of synthetic VWF peptides, by ADAMTS-13 in patient plasma and the direct or indirect detection of the VWF cleavage products ( Table 1).…”