Non-Proteolytic Regulation of the Multimerization of Von Willebrand Factor By ADAMTS13 Based on a Novel Cysteine Thiol Redundancy

The platelet dependent-clotting process is a highly complex pathway that needs to be tightly controlled by a variety of soluble and cellular components. Failure to regulate the activity of multimeric von Willebrand factor (VWF) by ADAMTS13 for instance causes thrombotic thrombocytopenia purpura (TTP). Regulation is achieved through the established function of ADAMTS13 as a VWF-cleaving protease; however, it has been reported that ADAMTS13 may additionally curtail VWF multimer fibrillation by forming disulfide bridges with VWF. Since the latter mechanism remained ill-defined, we aimed to directly visualize disulfide bond formation between ADAMTS13 and VWF using multimer gel electrophoresis. Covalent interaction between ADAMTS13 and VWF was time-, concentration-, temperature-, and shear-stress dependent. The covalent linkage was independent of the proteolytic activity of ADAMTS13 and could be assigned to a C-terminal fragment comprising TSP1 domains 5 to 8 plus the CUB domains. This interaction was blocked by thiol-reactive agents, indicating that association was accomplished by a thiol/disulfide exchange reaction. Furthermore, a polyclonal anti-ADAMTS13 antibody that inhibited the protease in a FRETS-VWF73 activity assay also inhibited the formation of VWF-ADAMTS13 adducts.

Using a coupled reversed phase HPLC-ESI-QTOF-MS system, several partially reduced free thiols were identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent. Together with the observation that a C1275S point mutation retained the ability to form covalent linkages with VWF, this result indicated that ADAMTS13 has disulfide plasticity. The same conditions that led to VWF complex formation also caused a hitherto undetected homo-oligomerization of ADAMTS13. An even more pronounced oligomerization was noted in the presence of thiol-sensitive agents such as glutathione.

We propose a dynamic network of redundant, partially free thiols in ADAMTS13 that undergo intra- and inter-molecular redox reactions capable not only of depolymerizing VWF, but also to prevent further VWF multimerization by facilitating the aggregation of ADAMTS13 around the monomers.