P7. Abstract Title: Mapping the Interactome of the Metalloprotease ADAMTS13 in Plasma using In-Vitro BioID

Abstract

<h3>Background</h3> Von Willebrand Factor (VWF) is a multimeric plasma protein that tethers platelets to sites of vascular damage, and is an important initiator of blood clotting. ADAMTS13 is a metalloprotease that cleaves VWF to regulate its platelet binding activity. Decreased activity of ADAMTS13 leads to larger VWF multimers resulting in pathological blood clots, such as thrombotic thrombocytopenic purpura (TTP) and stroke. Unlike other circulating proteases, ADAMTS13 is secreted as an active enzyme and is resistant to all known natural protease inhibitors. Despite this seemingly unregulated protease activity, VWF is the only known substrate for ADAMTS13. As a result, ADAMTS13 is an enigmatic protease whose role in the cardiovascular system remains poorly understood. <h3>Objective</h3> Identify novel physiologic substrates/ligands for ADAMTS13 in plasma using in-vitro BioID. <h3>Method</h3> BioID is a proximity-dependent biotinylation technique in which the protein of interest (ADAMTS13), is fused with a variant of biotin ligase (BirA*). BirA* activates biotin that can diffuse away and covalently attach to proteins within a 10 nm radius. Therefore, proteins interacting with the bait protein can be labelled with biotin. The biotinylated proteins are isolated using streptavidin pulldown and identified by LC/MS/MS. This technique has successfully been used to identify protein interaction networks in cells. <h3>Results</h3> In preliminary studies, we cloned, expressed, and characterized ADAMTS13-BirA* fusion. The recombinant fusion protein exhibits normal ADAMTS13 proteolytic activity and comparable biotinylation activity relative to BirA* alone. Preliminary LC-MS/MS data from labelling in 100 μL plasma yielded 36 unique proteins. ADAMTS13 was the most abundant protein, consistent with auto-biotinylation of the fusion protein. Other unique proteins labelled were vitronectin, PON1, and actin-B. However, the fusion protein did not label VWF possibly to due to the absence of shear stress (which is known to facilitate VWF cleavage by ADAMTS13) during labelling. Validation studies showed that both the fusion protein and BirA* alone bind to vitronectin. These data suggest that sensitivity and specificity of the assay requires improvement. We showed that ATP has a half-life of ∼4 minutes in plasma, which may limit the sensitivity of the BioID assay. While phosphatase inhibitors such as NaVO4, EDTA, IBMX or NBTI improved ATP half-life, they also blocked the activity of BirA*. Therefore, to improve labelling efficiency, we simply add ATP throughout the labelling experiment to maintain optimal ATP concentrations. These data demonstrate the viability of an in-vitro BioID experiment in human plasma, and optimized reaction conditions will yield novel substrates/ligands for ADAMTS13. <h3>Conclusion</h3> BioID may be a promising discovery tool for identifying novel protease interactions.