Human monoclonal and polyclonal anti-ADAMTS13 antibodies in immune thrombotic thrombocytopenic purpura: variable mechanisms of inhibition

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by antibodies, primarily immunoglobulin (Ig) Gs, that bind the plasma metalloprotease a disintegrin-like and metalloprotease with thrombospondin type 1 repeats 13 (ADAMTS13). Whether structural differences between IgG subclasses influence their mechanisms of action in iTTP or if mechanistic effects depend on pH and temperature is unknown. OBJECTIVES: To assess how full-length antibodies and antibody subclasses mechanistically affect ADAMTS13 function, and if enzymatic assay conditions influence mechanisms. METHODS: We utilized IgG1 and IgG4 constructs derived from previously well-characterized single-chain fragments of the variable regions from patients with iTTP, plasma from patients with iTTP, and native ADAMTS13 in normal human plasma in Michaelis-Menten-based enzymatic assays in different temperature and pH conditions. ADAMTS13 antigen in patients with iTTP samples was quantified using 2 methods. RESULTS: ADAMTS13 substrate recognition was more affected than catalytic turnover in a diagnostic condition, regardless of IgG subclass. In a more physiological condition, ADAMTS13 catalytic turnover was more affected. A mixture of effects was observed when both stimulatory and inhibitory IgG's were present, and mechanisms varied in plasma from patients with iTTP. A physiological condition yielded higher iTTP plasma ADAMTS13 activity than a routine diagnostic condition, even when antigen was low. CONCLUSIONS: We demonstrated, for the first time, that inhibitory mechanisms depend on reaction environment and differ among patients. Results support hypotheses that IgG-mediated inhibition of ADAMTS13 may have multiple mechanisms, antigen depletion may not be the primary mode causing ADAMTS13 deficiency in most patients, and current diagnostic tools may not reflect iTTP incidence. Delineation of the clinical significance of these findings requires further studies.