Abstract
ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies.
Highlights
ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13) is a metalloprotease enzyme primarily produced and secreted by hepatic stellate cells of the liver and endothelial cells [1,2,3]
Its primary function is to break down ultra-large multimers of von Willebrand factor (VWF), which are hyperactive in spontaneously agglutinating platelets at sites with damaged endothelium [4,5,6,7]
Comprehensive testing must be conducted to evaluate the efficacy, safety, and characteristics of rADAMTS13, which can be performed through laboratory-scale experimentation
Summary
ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13) is a metalloprotease enzyme primarily produced and secreted by hepatic stellate cells of the liver and endothelial cells [1,2,3]. Activity levels of plasma ADAMTS13 vary widely amongst healthy individuals and are assay-dependent. Plasmatic ADAMTS13 activity of 40–170% is considered normal for healthy individuals [8]. It has been demonstrated that the estimated heritability of ADAMTS13 antigen levels is between 59.1% and 83.5% [9]. Any distortion of the ADAMTS13/VWR axis leads to defects in primary hemostasis. Higher ADAMTS13 level (by 35%) has been observed in patients with type 3 von Willebrand disease (VWD) [10]
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