A Small Fragment of ADAMTS13 Enhances Cleavage of von Willebrand Factor by the Intact Enzyme.

Abstract

ADAMTS13 regulates hemostasis by cleaving von Willebrand Factor (VWF) to smaller less thrombogenic fragments. Absence of ADAMTS13 activity leads to thrombotic thrombocytopenic purpura, an often fatal disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia and microvascular thrombosis especially in renal and central nervous system vasculature. ADAMTS13 like other ADAMTS family members has a characteristic domain structure consisting of a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), a cysteine-rich domain, a spacer domain, 7 additional TSRs and 2 carboxyl terminal CUB domains. Regions in the spacer and C-terminal thrombospondin type 1 repeats (TSR) and CUB domains have been shown previously to be important for the interaction of ADAMTS13 and VWF. To further characterize the interaction of ADAMTS13 and VWF, we generated a construct consisting of the spacer domain and the second TSR of ADAMTS13. This construct is called ST2 for Spacer and Tsr2. Although many constructs of ADAMTS13 fail to be secreted because of poor folding, ST2 is folded since it is secreted from human embryonic kidney 293 cells. Previous work has shown that the spacer domain alone can bind VWF under static conditions, therefore ST2 was included in VWF cleaving assays. Unexpectedly, ST2 was found to enhance the cleavage of VWF by ADAMTS13 by approximately 10-fold in assays using either guanidine or urea-denatured VWF. To understand how ST2 enhanced the cleavage of VWF by ADAMTS13, interactions of ST2 with ADAMTS13 and VWF were studied. ST2 was found to specifically co-immunoprecipitate with VWF but not with ADAMTS13. A Kd calculated from these co-immunoprecipitation experiments was 8.7 nM and the stoichiometry of binding was approximately 1 mole/mole. One explanation for the enhanced cleavage of VWF by ADAMTS13 in the presence of ST2 is that ST2 may induce a conformational change in VWF that makes the cleavage site more accessible. To test this hypothesis, VWF in the presence and absence of ST2 was subjected to trypsin digestion at different concentrations of trypsin. Differential cleavage of VWF was seen in the presence and absence of ST2 at all concentrations of trypsin tested. This is consistent with induction of a conformational change in VWF by ST2. These studies provide additional information on the structure-function relationship of these two key proteins in the control of hemostasis.