Identification of cysteine thiol‐based linkages in ADAMTS13 in support of a non‐proteolytic regulation of von Willebrand factor

Abstract

BackgroundADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) to regulate its function. Additionally, ADAMTS13 is thought to regulate lateral association of VWF multimers to form fibrillar structures through its free thiols.ObjectiveThe purpose of the present study is to obtain direct evidence for ADAMTS13 to engage in thiol/disulfide exchange reactions.MethodsCovalent complexes between ADAMTS13 and VWF were determined by agarose gel electrophoresis under nonreducing conditions. Free thiols in ADAMST13 were identified by a reversed phase high‐performance liquid chromatography electrospray ionization quadrupole time‐of‐flight mass spectrometry system.ResultsWe demonstrate formation of covalent linkage between ADAMTS13 and VWF, which is time, concentration, temperature, and shear dependent. This interaction is independent of proteolytic activity of ADAMTS13 but depends on the C‐terminal domains comprising the fifth through eighth thrombospondin type 1 repeats and C1r/C1s, Uegf, Bmp1 (CUB) domains. The interaction can be blocked by thiol‐reactive agents, indicating that association is accomplished through disulfide bridge formation. Several partially reduced free thiols are identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent, although a point mutation (C1275S) in ADAMTS13 does not alter its ability to form covalent linkages with VWF. This suggests functionally relevant disulfide plasticity in ADAMTS13. Interestingly, ADAMTS13 also forms homo‐oligomers under the same conditions as required for the generation of hetero‐oligomeric complexes of ADAMTS13 and VWF.ConclusionsOur results suggest that a dynamic network of free thiols in ADAMTS13 undergoing intra‐ and inter‐molecular redox reactions may add another layer of regulation to VWF function under various conditions.

Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19

“…Manufacture and characterisation of purified recombinant human ADAMTS13 (rADAMTS13, Takeda, Vienna, Austria) from a Chinese hamster ovary cell line has been described previously [ [26] , [27] , [28] ]. The rADAMTS13 product used in our investigation is the same as the investigational drug candidate identified as TAK-755, SHP655, or BAX 930 in clinical trials for hereditary thrombotic thrombocytopenic purpura ( ClinicalTrials.gov Identifier: NCT02216084 ) and for sickle cell disease ( ClinicalTrials.gov Identifier: NCT03997760 ).…”

Crystal structure of ADAMTS13 CUB domains reveals their role in global latency

“…In loop L9 in CUB1, the unpaired Cys 1275 (that we substituted to Ser) is not present in other CUB domains. Previous studies have reported the ability of Cys 1275 to undergo redox reactions that may regulate VWF function (40)(41)(42). We certainly found that, without the C1275S mutation, there appeared to be some dimerization of the recombinant CUB1-2 domains in S2 cells.…”

A human monoclonal antibody against the distal carboxyl terminus of ADAMTS‐13 modulates its susceptibility to an inhibitor in thrombotic thrombocytopenic purpura

“…mations: the open, closed, and intermediate either as a monomeric protein 15,36 or a possible oligomeric form in solution or plasma. 37 Lowering pH and binding of ADAMTS-13 to a substrate such as VWF (D4 domain) or an antibody against the various C-terminal domains of ADAMTS-13 may stabilize multiple intermediate conformations of ADAMTS-13 with a different maximal rate of catalysis 15,22,26 or convert ADAMTS-13 from its "closed" and more stable conformation to an "open" but less stable conformation. [14][15][16]18,27 The "open" ADAMTS-13 species are found to be dramatically increased in patients with acute iTTP, then reduced or normalized during clinical remission or essentially undetectable in the healthy individual.…”

“…ADAMTS‐13 is known to exhibit in multiple intermediate conformations: the open, closed, and intermediate either as a monomeric protein 15,36 or a possible oligomeric form in solution or plasma 37 . Lowering pH and binding of ADAMTS‐13 to a substrate such as VWF (D4 domain) or an antibody against the various C‐terminal domains of ADAMTS‐13 may stabilize multiple intermediate conformations of ADAMTS‐13 with a different maximal rate of catalysis 15,22,26 or convert ADAMTS‐13 from its “closed” and more stable conformation to an “open” but less stable conformation 14‐16,18,27 .…”