Purification and functional characterization of two thrombosis-related molecules: von Willebrand Factor and ADAMTS-13

von Willebrand Factor (vWF) is secreted as the ultra-large (UL) multimeric glycoprotein by endothelial cells and megakaryocytes. It can be cleaved into small multimers by metalloprotease ADAMTS-13 following its secretion. As a member of a disintegrin and metalloproteinase with thrombospondin family, ADAMTS-13 plays a crucial role in thrombosis regulation. Many studies have revealed that unsuccessful cleavage of UL-vWF by ADAMTS-13 induced thrombosis. Here, we have purified A domain, which is the most important activity unit of vWF, and the full-length ADAMTS-13. Results of SDS-PAGE and Western blot confirmed two purified proteins have correct molecular weights. Flow cytometry analysis showed that the microspheres were functionalized via coating purified ADAMTS-13 on their surfaces. Using binding assay, microspheres coated with ADAMTS-13 can adhere on petri dishes coated with immobilized vWF A1A2A3 under static conditions, which were resulted from the binding between ADAMTS-13 and vWF A1A2A3. These results demonstrated purified ADAMTS-13 and vWF A1A2A3 have functional activities and can be used as an important tool for exploring the mechanism of interaction between vWF A1A2A3 and ADAMTS-13 in further studies for thrombosis regulations.