Abstract

ADAMTS13 proteolysis of von Willebrand Factor (VWF) generates smaller multimers that are less likely to promote blood clotting. Deficiency of ADAMTS13 leads to thrombotic thrombocytopenic purpura, a frequently fatal disease, characterized by microangiopathic hemolytic anemia and thrombocytopenia. ADAMTS13 has a characteristic domain structure that includes metalloprotease and disintegrin domains, a thrombospondin type 1 repeat (TSR), cysteine-rich and spacer domains, 7 additional TSRs, and 2 carboxyl-terminal CUB domains. The ADAMTS13 substrate, VWF, is synthesized in endothelial cells and forms large multimers within the cell. These large multimers are secreted and adhere to the endothelial cell surface where they can bind platelets flowing in blood leading to thrombosis. ADAMTS13 has been shown to cleave VWF on the surface of endothelial cells, but it is unclear if ADAMTS13 also interacts with the endothelial cell surface. We have used iodinated ADAMTS13, fluorescence-activated cell sorting (FACS), and biochemical analysis using flow conditions to demonstrate that ADAMTS13 does interact with the endothelial cell surface. Iodinated ADAMTS13 bound the endothelial cell surface at 4oC. This binding was specific since the binding was inhibited in the presence of 40-fold excess unlabeled ADAMTS13. Binding of ADAMTS13 to the cell surface was time-dependent with maximal binding occurring within two hours. The binding was also reversible; the half-time for dissociation was four hours. Binding was inhibited by heparin but not by dextran sulfate. The Kd of binding to endothelial cells was 75 nM (range 40–100 nM). FACS analysis also demonstrated binding of ADAMTS13 to endothelial cells. A fluorescein isothiocyanate labeled anti-epitope antibody bound to endothelial cells in the presence but not the absence of ADAMTS13. A polyclonal antibody to VWF inhibited binding of ADAMTS13 to VWF, but this antibody did not affect binding of ADAMTS13 to endothelial cells, suggesting that ADAMTS13 interacts with endothelial cells independently of VWF. Studies with C-terminal truncation constructs of ADAMTS13 indicated that the carboxyl-terminal TSRs are important for binding since constructs terminating with the metalloprotease domain, the first TSR, or the sixth TSR failed to compete with full-length ADAMTS13 for binding to endothelial cells, but constructs terminating with either the seventh or eighth TSR did compete for binding. Lastly, recombinant ADAMTS13 was found to be associated with endothelial cells in flow experiments. Endothelial cells were perfused with medium containing plasma concentrations of ADAMTS13 (1 μg/ml) at 10 dynes/cm2. After perfusion, the endothelial cells were washed and bound ADAMTS13 was identified from whole cell lysates through SDS-PAGE and immunoblotting with an anti-V5 epitope antibody. ADAMTS13 was found associated with endothelial cells after perfusion. Binding of ADAMTS13 to the endothelial cells prior to perfusion led to enhanced proteolysis of VWF as compared to addition of ADAMTS13 during perfusion only. This suggests that the interaction of ADAMTS13 with endothelial cells is important since it enhances the cleavage of VWF as compared to that of ADAMTS13 in solution.

Author notes

Disclosure: No relevant conflicts of interest to declare.