Heightened Proteolysis of Plasma ADAMTS13 Antigen in Patients with Thrombotic Thrombocytopenic Purpura.

A total of 140 subjects with thrombotic thrombocytopenic purpura (TTP)/hemolytic-uremic syndrome (HUS), in which 25 were Upshaw-Schulman syndrome (USS, belonged to 20 families), 107 were acquired TTP (96 idiopathic and 11 drug-induced), and 8 were acquired O157:H7-associated HUS, were analyzed on their plasma ADAMTS13 antigen (:AGN), using 3 novel anti-ADAMTS13 murine monoclonal IgG antibodies (A10, C7, and WH2-1–11). Epitope of these antibodies resided on the disintegrin-like, ^7th/8th thrombospondin-1, and ^4th thrombospondin-1 domains, respectively. The WH2-11–1 alone was immunoreactive on western blot (WB) under reducing conditions. Further, in a static assay system, A10 totally and C7 partially inhibited ADAMTS13 activity (:ACT), but WH2-11–1 did not. A sandwich ELISA using C7 as the ^1st antibody and biotinylated A10 as the ^2nd antibody revealed a significant reduction of plasma ADAMTS13:AGN level in patients with acquired TTP, but a considerable variation in those with USS, suggesting that the quantitative ELISA has a limited diagnostic value.

A quantitative and semi-qualitative WB analysis using A10 and WH2-11-1, however, demonstrated that normal plasmas had a 190/180 kD-doublet band under non-reducing conditions. A size exclusion chromatography of normal plasma was able to separate a 190 kD-protein from a 180 kD-band, and the former turned to be a single 200 kD-band on WB under reducing conditions, but in the latter such protein band was completely invisible, indicating that the 180 kD-band had undergone proteolytic modification. All the patient plasmas with USS and a roughly half of those with acquired TTP had a broad and fuzzy ~190/180 kD-band on WB before reduction, but it almost entirely disappeared after reduction. In patient plasmas with acquired HUS, however, the pattern of WB analysis was indistinguishable from that of the normal subjects.

Aforementioned WB analysis was then performed on a girl of acquired TTP with high-titer inhibitors against ADAMTS13, using the sequential plasma samples obtained during the past 3 years, and confirmed complete normalization of the abnormal ADAMTS13:AGN patterns in accord to the increase of plasma ADAMTS13:ACT, together with her clinical improvements. Protease(s) responsible for this heightened cleavage is currently under investigation.