OC 15.5 - The conformational opening of ADAMTS13 does not necessarily indicate preactivation of its metalloprotease domain.

Sunday, June 23, 2024

10:30 – 10:45 ICT

Room: 110 A-C

Presenting Author(s)

Quintijn Bonnez, MA

PhD Student
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium

Co-Author(s)

Magdalena Gierula

Post Doctoral Researcher
Department of Immunology and Inflammation, Imperial College London, London, United Kingdom
London, England, United Kingdom
IP

Inge Pareyn

Laboratory technician
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
FB

Febe Boudry

Master Student
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
CT

Claudia Tersteeg

Professor, Dr.
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
SD

Simon F. De Meyer

Full professor
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
JC

James T.B. Crawley

Full professor
Department of Immunology and Inflammation, Imperial College London, London, United Kingdom
London, England, United Kingdom
Karen Vanhoorelbeke

Full professor
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium

Background: Monoclonal antibodies (mAbs) targeting the ADAMTS13 Spacer and CUB1 domains, do not only uncouple the Spacer-CUB interaction to induce an open ADAMTS13 conformation but also functionally preactivate the Metalloprotease (MP) domain. An open ADAMTS13 conformation and preactivated MP domain can de be detected using mAbs recognizing cryptic epitopes in Spacer (mAb 1C4) and MP (mAb 6A6) domains. As a multidomain enzyme, it remains unknown how targeting different domains affect ADAMTS13 conformation and preactivation, which precludes a thorough understanding of the allosteric regulation of ADAMTS13.

Aims: To characterize the effects on ADAMTS13 conformation, preactivation and function when targeted by mAbs against all ADAMTS13 domains.

Methods: Using ELISA, the capacity of a panel of 48 mAbs, targeting all ADAMTS13 domains, was studied to conformationally open plasma ADAMTS13 (mAb 1C4 epitope exposure) and to preactivate the MP domain (mAb 6A6 epitope exposure). A subpanel of mAbs was functionally characterized in a VWF96 proteolysis ELISA to examine mAb-induced changes in catalytic efficiency.

Results: When targeting each ADAMTS13 domain, an open ADAMTS13 conformation could be induced as the 1C4 epitope became accessible. Unexpectedly, a subpanel of mAbs that conformationally opened ADAMTS13, could not preactivate the ADAMTS13 MP domain as the 6A6 epitope remained cryptic. The catalytic efficiencies for VWF96 proteolysis revealed some mAbs from this subpanel to functionally inhibit or functionally enhance proteolysis by ADAMTS13.

Conclusion(s): When targeting each ADAMTS13 domain, the ADAMTS13 enzyme conformationally opened. Unlike the anti-Spacer and CUB1 mAbs, a subpanel of conformationally opening mAbs did not structurally preactivate the ADAMTS13 MP domain. Intriguingly, VWF96 proteolysis revealed this subpanel to contain both functionally activating and inhibiting mAbs suggesting diverse molecular mechanisms to affect the ADAMTS13 structure and function. Currently, Michaelis-Menten studies are ongoing to stratify mAb-induced alterations in VWF substrate turnover or substrate binding and thereby provide mechanistic insights in the allosteric regulation of ADAMTS13.