Proteolytic Cleavage of Endothelial Cell-Bound Von Willebrand Factor Polymers by ADAMTS13 in the Absence of Flow Shear Stress

Degradation of newly released unusually large (UL)-vWF on endothelial cells by plasma ADAMTS13 metalloprotease is considered to be critical for maintaining normal hemostasis. An inability to degrade the cell bound string-like UL-vWF multimers by ADAMTS13 may result in excessive platelet adhesion and aggregation on cell surface, leading to thrombotic thrombocytopenic purpura (TTP). However, the requirement for shear stress and potential cofactors in this process is not fully defined. By immunofluorescent microscopy, we showed that upon histamine stimulation the newly released UL-vWF rapidly formed strings and bundles on human umbilical vein endothelial cells (HUVECs). These strings and bundles were removed within 2~5 minutes by plasma-derived and recombinant ADAMTS13 at concentrations of between 2.5 and 10 nM in 20 mM HEPES buffer, pH 7.5 containing 150 mM NaCl and 1 mM CaCl. This process did not appear to depend upon flow shear stress and coagulation factor VIII that has been shown to markedly increase the proteolytic cleavage of soluble vWF under high shear stress. The removal of the cell-bound UL-vWF strings and bundles correlated with the increases of vWF antigen and proteolytic cleavage fragments (176K and 140K) in the conditioned medium as determined by an enzyme-linked immunoassay and Western blot, respectively. The proteolytic cleavage of the cell bound UL-vWF by ADAMTS13 was time- and concentration-dependent, with a half maximal concentration of ADAMTS13 of approximately 5 nM. No cleavage product was detected when the histamine-stimulated HUVECs were incubated with the buffer alone or plasma from patients with acquired TTP and inhibitors or with heat-inactivated recombinant ADAMTS13. These results suggest that the degradation of cell bound UL-vWF is specific for ADAMTS13. Unexpectedly, the vWF multimers in the conditioned medium of ADAMTS13-treated endothelial cells were quite similar in sizes to those in the endothelial cells treated with histamine in the absence of ADAMTS13, indicating that the proteolytic cleavage of UL-vWF by ADASMTS13 occurs at the site proximal to cell membrane. Scanning electron microscopy clearly demonstrated that the breaking point of UL-vWF strings and bundles by ADAMTS13 was located at approximately 2~5 μm from the cell membrane. Structure-function analysis further demonstrated that the Cys-rich and spacer domains of ADAMTS13 were required for proteolytic cleavage of the cell-bound UL-vWF. The more distal carboxyl-terminal domains including TSP1 2–8 and CUB domains of ADAMTS13 appeared to be dispensable. We conclude that the cell bound UL-vWF polymers adopt an open conformation that is sensitive to ADAMTS13 proteolysis, analogous to those observed under the denaturing conditions. We hypothesize that membrane binding and/or interaction of UL-vWF bundles with certain scaffold proteins may provide an access of ADAMTS13 metalloprotease to the cleavage (Tyr¹⁶⁰⁵-Met¹⁶⁰⁶) bond at the central A2 domain of the vWF proximal to cell surface. These findings provide novel insight into the biological function of ADAMTS13 in processing UL-vWF and help understand the pathogenesis of TTP.