ADAMTS13 variant (R1206K) exhibits increased VWF-cleaving activity and inhibition of thrombus formation under flow Free

Background: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy, caused by a severe deficiency of plasma ADAMTS13, a metalloprotease that cleaves ultra-large von Willebrand factor (UL-VWF) multimers. This cleavage is essential for preventing spontaneous platelet adhesion/aggregation and thrombus formation. While wild type (WT) human recombinant ADAMTS13 (rADAMTS13) has been approved for prophylactic treatment of hereditary TTP, the functionally enhanced rADAMTS13 variant may have better therapeutic efficacy and reduces administration dosage, thus cost. Here, we describe one of such rADAMTS13 variants.

Methods: Using bioinformatics and recombinant technologies, we identified a novel gain-of-functional rADAMTS13 variant (R1206K). Proteolytic activity was measured using the FRETS-VWF73, a fluorogenic substrate and multimeric VWF substrate. Binding kinetics were performed with a modified VWF73 (L1603A) peptide that is not cleavable by rADAMTS13 using a biolayer interferometric (BLI) assay. The conformational changes of R1206K induced by lowering pH or binding to a human monoclonal antibody (scFv3-3) that targets the distal CUB domains were determined by functional assay and enzyme-linked immunosorbent assay. Finally, the anti-thrombotic activity of the variant was determined by shear induced platelet adhesion and aggregation on a fibrillar collagen-costed surface using a BioFlux system.

Results: By the FRET-73 assay, rADAMTS13 (R1206K) variant demonstrated ~4-fold increase in its catalytic efficiency (k_cat/K_m) compared to the wild-type rADAMTS13 control. BLI binding assay revealed a similar binding affinity (the association constant, KD) of approximately 5 µM for both WT-rADAMTS13 and rADAMTS13-R1206K variant to interact with the VWF73^L1603A substrate. The rADAMTS13-R1206K variant also exhibited a significantly increased proteolytic activity compared with WT-rADAMTS13 for multimeric VWF substrate under denaturing conditions. Microfluidic shear- based assay demonstrated a significantly increased anti-thrombotic activity of rADAMTS13-R1206K variant compared with WT-rADAMTS13. Moreover, lowering pH to 6.0 or adding scFv3-3 to WT-rADAMTS13 increased its specific activity towards FRETS-VWF73 substrate, but such a measure did not further increase the proteolytic activity of rADAMTS13 R1206K variant, suggesting that the variant is in its “open” confirmations.

Conclusions: Our results demonstrate that we have identified a rADAMTS13 R1206K variant that exhibits an enhanced enzymatic activity for cleaving a surrogate VWF peptide substrate and more physiological substrate such as multimeric VWF under static/denaturing and fluidic shear conditions. This rADAMTS13 variant may be further explored for the treatment of TTP and other inflammatory thrombotic disorders associated with relative deficiency of ADAMTS13 function.