Abstract

ADAMTS13 is a plasma metalloprotease that is essential for the regulation of von Willebrand factor (VWF) function, mediator of platelet recruitment to sites of blood vessel damage. ADAMTS13 function is dynamically regulated by structural changes induced by VWF binding that convert it from a latent to active conformation. ADAMTS13 global latency is manifest by the interaction of its C-terminal CUB1-2 domains with its central Spacer domain. We resolved the crystal structure of the ADAMTS13 CUB1-2 domains revealing a previously unreported configuration for the tandem CUB domains. Docking simulations between the CUB1-2 domains with the Spacer domain in combination with enzyme kinetic functional characterization of ADAMTS13 CUB domain mutants enabled the mapping of the CUB1-2 domain site that binds the Spacer domain. Together, these data reveal the molecular basis of the ADAMTS13 Spacer-CUB interaction and the control of ADAMTS13 global latency.

Highlights

  • Von Willebrand factor (VWF) is produced by both endothelial cells and platelets and secreted upon stimulation as disulfide-linked multimers containing up to ~100 VWF units [1]

  • We fused CUB1-2 (C1275S) at its N terminus to maltose-binding protein (MBP). This was expressed in S2 cells and enabled expression of uniformly monomeric and monodisperse MBP-CUB1-2

  • Purified MBP-ADAMTS13 CUB1-2 was crystallized with the structure determined to a resolution of 2.8 Å (Fig. 1 and Table 1)

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Summary

Introduction

Von Willebrand factor (VWF) is produced by both endothelial cells and platelets and secreted upon stimulation as disulfide-linked multimers containing up to ~100 VWF units [1]. Following vessel damage, plasma VWF binds via its A3 domain to exposed collagen in the subendothelial milieu. This tethered VWF undergoes a conformational transition in response to the shear forces exerted by the flowing blood that causes exposure of the previously hidden GPIb binding sites in its A1 domains [2]. This process enables specific capture of platelets from blood under high shear and, in turn, facilitating platelet plug formation. Low VWF levels or function are associated with elevated risk of bleeding [6]

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