A Sensitive and Specific Shear-Based Assay for Plasma ADAMTS13 Activity and Inhibitors-the Diagnostic Utility for Thrombotic Microangiopathies.

Abstract 2114

Poster Board II-91

Severe deficiency of plasma ADAMTS13 activity causes a potentially fatal thrombotic thrombocytopenic purpura (TTP). To date, functional assays for plasma ADAMTS13 activity and inhibitors rely on cleavage of von Willebrand factor (VWF) in the presence of 1.5 M urea or guanidine-HCl or small peptides derived from the central VWF-A2 domain. To develop a more physiological assay under fluid shear stress, we assessed the proteolytic cleavage of plasma-derived VWF by plasma ADAMTS13 under constant vortexing (2,500 rpm) in a PCR tube mini-mixer that mimics arterial fluid shear stress (∼30 dynes/cm²). The cleavage product was determined by 2.5% SDS-mini agarose gel electrophoresis and Western blot. Using this novel assay, we determined plasma ADAMTS13 activity, as well as the inhibition of that activity by inhibitors in plasma obtained from 23 TTP patients (10 transplantation-related TTP and 13 idiopathic TTP). We showed that under these shear conditions, proteolytic cleavage of plasma derived VWF by plasma ADAMTS13 was time and plasma concentration dependent. At 60 min of vortexing, the cleavage of VWF (150 nM) by plasma (1 μl) reached the maximal levels in the absence of zinc and/or sodium chloride. Addition of exogenous zinc ions (0.5 μM-0.5 mM) and sodium chloride (20 mM-150 mM) significantly inhibited proteolytic cleavage of VWF by plasma ADAMTS13. Concurrently, plasma ADAMTS13 activity and action of inhibitors in patient plasma were determined by an established assay measuring the cleavage of a recombinant fluorescein-labeled VWF73 peptide containing amino acid residues between Asp¹⁵⁹⁶ and Arg¹⁶⁶⁸ from the VWF-A2 domain (rF-VWF73). Overall, the plasma ADAMTS13 activity determined by the shear-based vortex assay was highly correlated with the results obtained by the rF-VWF73 cleavage assay (correlation coefficient, r=0.682). However, and more interestingly, four TTP patients (two with idiopathic TTP and 2 with bone marrow transplant-related TTP) with mild to moderate reduced plasma ADAMTS13 activity (25∼75%) by the rF-VWF73 assay showed severely reduced ADAMTS13 activity (less than 5%) in the shear-based assay, suggesting that the fluid shear-based assay may be a more accurate reflection of plasma ADAMTS13 activity in vivo. Additionally, we determined the activity of anti-ADAMTS13 inhibitors in patient plasma by mixing normal human plasma with heat-inactivated (56 °C for 60 min) patient plasma at a ratio of 50:50 for 60 min at 37 °C, followed by either the rF-VWF73 cleavage assay or our novel shear-based assay. The inhibition of vWF cleavage detected by the rF-VWF73 assay in patients with idiopathic TTP was also seen by the shear-based assay. We conclude that a sensitive, specific and physiologically relevant fluid shear stress-based assay has been developed to assess plasma ADAMTS13 activity and inhibitors. This novel assay may provide a tool for investigating structure-function and regulation of ADAMTS13 protease. It may also be applicable to establishing clinical diagnosis and monitoring therapy of hereditary and acquired TTP.