OC 26.5 - Regulation of VWF Cleavage is Mediated by Coupling ADAMTS13 D- and C- Domain Binding Sites

Sunday, June 25, 2023

15:45 – 16:00 ET

Room: 510

Session Available As: On-Demand

Presenting Author(s)

Frida Z. Miranda

Graduate Student, Graduate Research Assistant
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma, United States

Co-Author(s)

KS

Kathryn L. Smith

Medical Student
Oklahoma State University Center for Health Sciences, College of Medicine
Tulsa, Oklahoma, United States
AP

Anna Platt

Graduate Student, Graduate Research Assistant
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma, United States
J. Cameron Barton

Medical Student
Oklahoma State University Center for Health Sciences, College of Medicine
Tulsa, Oklahoma, United States
VM

Veera Manukonda

Research Associate
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma, United States
Karen Vanhoorelbeke, PhD

Prof., Dr.
KU Leuven
Kortrijk, West-Vlaanderen, Belgium
Joshua Muia, PhD

Prof., Dr.
Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma
Tulsa, Oklahoma, United States

Background: The metalloprotease ADAMTS13 cleaves the highly hemostatic active von Willebrand factor (VWF) to limit VWF-induced platelet aggregation. ADAMTS13 consists of metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by seven T domains and two CUB domains. ADAMTS13 activity is regulated by blood shear stress and substrate-induced activation (allosteric regulation). We previously demonstrated that the capacity for ADAMTS13 to undergo allosteric activation differs across different vertebrate species.

Aims: To characterize the sequence-determinants of substrate-induced activation of ADAMTS13.

Methods: Multiplex FRET ADAMTS13 assays were performed using 10 fluorogenic substrates derived from VWF A2 domains of two vertebrate species (Fig 1A). Substrates P3-P10 are chimeric substrates between Cattle VWF (C-71) and Human VWF (H-71). Plasmas and recombinant proteins from various vertebrates have been characterized and reported before. Assays were performed as previously described and mean results are reported.

Results: Compared to C-71 (P2), only the chimeric P3 and P6 exhibited reduced rates of cleavage by plasma ADAMTS13 from various vertebrates (Fig 1B). The P3 substrate substituted the D binding residues from H-71 into C-71, whereas P6 substituted the C binding residues from H-71 into C-71. For P3, all ADAMTS13 assayed showed ~30-100% reduction in the cleavage rate except armadillo and dog plasma. Likewise, P6 was cleaved ~0-60% slowly relative to P2 by all ADAMTS13. There was no major change in cleavage rates when D- and C- VWF binding sites were of the same species or when the pH was adjusted from 7.4 to 6.

Conclusion(s): ADAMTS13 D- and C- binding sites on VWF A2 function in tandem to modulate ADAMTS13 activity. Although more work is needed, we postulate that the coupling of the disintegrin-like with Cys-rich domains may prevent the activation of ADAMTS13 metalloprotease by off-target substrates.

Learning Objectives:

Recognize that ADAMTS13 and VWF proteins have not evolved to be optimal enzyme-substrate pairs.
Determine the sequence determinants of substrate-induced activation of ADAMTS13.
Appreciate the role of synergetic coupling of the Disintegrin-like with Cys-rich domains to regulate ADAMTS13 activity.