Cleavage of ultralarge multimers of von Willebrand factor by C-terminal-truncated mutants of ADAMTS-13 under flow.

A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.