The protease ADAMTS13 (A Disintegrin and Metalloprotease with Thrombo Spondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder Thrombotic Thrombocytopenic Purpura (TTP). TTP has been in particular observed in transplant patients following treatment with Cyclosporin A (CsA), a drug which is frequently administered in patients to suppress allogeneic immune response. However, a mechanistic explanation for such a link has remained unclear. Here, we report that cyclophilin-B mediated peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is also targeted by CsA, plays an important role in the production of active ADAMTS13.
An in vitro transient expression system was used to study the effects of CsA on the synthesis and activity of ADAMTS13. We found that exposing HEK293 cells to CsA results in a dose-dependent reduction in the level of secreted ADAMTS13, but not the ubiquitously-expressed housekeeping protein Hsp70. Moreover, treatment with other immunosuppressive drugs, such as rapamycin or tacrolimus/FK506 did not impact the secretion of ADAMTS13. CsA is a known inhibitor of several cellular substrates, including cyclophilin B (CypB), which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. We show a direct, functional interaction between CypB and ADAMTS13 using co-immunoprecipitation analysis. Specifically, ADAMTS13 and CypB could be detected in association together in untreated cells, but not in those treated with CsA, where only ADAMTS13 could be detected. ADAMTS13 levels are also reduced in the setting of siRNA-mediated CypB knockdown, suggesting a direct link between CypB expression/activity and ADAMTS13 maturation. We have found that CsA leads to intracellular conformational changes in the ADAMTS13 protein, evidenced by a unique trypsin digestion pattern of ADAMTS13 from CsA-treated cells relative to untreated cells. Additionally we found that treatment of cells with N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor, resulted in about a 2.5 fold rescue in ADAMTS13 expression in the setting of CsA treatment. This observation suggests that CsA treatment leads to an enhanced proteasomal degradation of ADAMTS13, possibly due altered conformation. Although CsA did not influence the overall level of intracellular ADAMTS13, it resulted in diminished specific proteolytic activity, further suggesting improper ADAMTS13 folding. Finally, we show that plasma samples from CypB knockout mice have reduced levels of circulating ADAMTS13.
Our findings indicate that cyclophilin-mediated activity is an important factor in ADAMTS13 secretion and activity. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization process mediated by CypB in its proper folding and maturation. We suggest that CsA treatment disrupts the interaction between ADAMTS13 and CypB. Although this may not be the sole mechanism by which CsA ultimately lowers levels of secreted ADAMTS13, this work altogether provides a novel mechanistic explanation for CsA-induced TTP in transplant patients.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.