OC 43.5 - Domain-specific nanobodies distinguish intact and proteolyzed forms of von Willebrand factor in congenital von Willebrand disease

Monday, July 11, 2022

3:45 PM – 4:00 PM

Location: ExCel Center, ICC Capital Suite Room 7&12

Presentation Format: In-Person

Presenting Author(s)

Claire Kizlik-Masson, PhD

Post Doc
Inserm U1176
Le Kremlin Bicetre, Ile-de-France, France

Co-Author(s)

IP

Ivan Peyron

Post-doc
Inserm U1176 - HITh
Le Kremlin-Bicetre, Ile-de-France, France
SG

Stéphane Gangnard

post-doc
INSERM U1176
Le Kremlin Bicetre, Ile-de-France, France
MC

Marie Clavel

Engineer
INSERM U1176
Le Kremlin Bicetre, Ile-de-France, France
VR

Véronique Regnault

Director of research
Inserm 1116
Nancy, Lorraine, France
EJ

Emmanuelle Jeanpierre

PharmD
Lille University Hospital, Lille, France
Lille, Nord-Pas-de-Calais, France
AD

Annabelle Dupont, Pharm D PhD

PharmD, PhD
Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, F5900 Lille, France
59000 Lille, Nord-Pas-de-Calais, France
OC

Olivier D. Christophe

Principal Investigator, Director of research
Laboratory for Hemostasis, Inflammation and Thrombosis, Unité Mixte de Recherche S1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
Paris, Ile-de-France, France
Caterina Casari, PhD

Principal Investigator
UMR_S 1176
PARIS, Ile-de-France, France
CD

Cecile Denis

Director of research
INSERM U1176
Le Kremlin Bicetre, Ile-de-France, France
Sophie Susen, MD, PhD

Professor, MD, PhD
CRC-MHC, Lille University Hospital, Lille, France
Lille, Nord-Pas-de-Calais, France
Peter J. Lenting, PhD

Principal Investigator, Director of research
Laboratory for Hemostasis, Inflammation and Thrombosis, Unité Mixte de Recherche S1176, Institut National de la Santé et de la Recherche Médicale, Université Paris-Saclay, Le Kremlin-Bicêtre, France
Paris, Ile-de-France, France

Background: Proteolysis of von Willebrand factor (VWF) in its A2 domain by ADAMTS13 results in smaller-sized multimers with lower hemostatic potential. Both increased shear stress and specific genetic variants in VWF may alter the susceptibility of ADAMTS13-mediated proteolysis.

Aims: To develop nanobodies that are selective for cleaved and uncleaved forms of VWF.

Methods: Two nanobodies were generated that recognized intact VWF (designated KB-VWF-D3.1) and ADAMTS13-cleaved VWF (KB-VWF-F1.1), respectively. Epitopes of both nanobodies were identified using purified VWF and derivatives thereof. Plasma from congenital VWD-patients were analyzed for their interaction with both nanobodies.

Results: Nanobody KB-VWF-F1.1 (interacting with A2-domain residues Met1606-Arg1668) specifically bound to unfolded and ADAMTS13-proteolyzed VWF, but not to globular VWF. KB-VWF-D3.1 bound to uncleaved VWF and lost its epitope upon ADAMTS13-cleavage. Unexpectedly, its epitope was located in the VWF-A3 domain, overlapping the collagen-binding site. Both nanobodies were used to follow ADAMTS13-mediated VWF degradation over time, resulting in increased recognition by KB-VWF-F1.1 paralleling the decreased recognition by KB-VWF-D3.1 (Fig. 1). Nanobodies were then applied to the analysis of VWD-patient samples. VWF binding to KB-VWF-D3.1 was significantly reduced relative to controls (defined as 1.0) for all types: VWD-type 1: 0.7±0.3 (n=20, p=0.0002); VWD-type 2A: 0.5±0.2 (n=46, p< 0.0001); VWD-type 2B: 0.6±0.2 (n=24, p= < 0.0001) and VWD-type 2M: 0.7±0.2 (n=13, p=0.001). Interestingly, binding was lower in VWD-type 2A(IIA) (0.4±0.2; n=31) compared to VWD-type 2A(IIE) (0.6±0.2; n=15; p=0.026). As for KB-VWF-F1.1, binding was increased 8±4-fold for VWD-type 2A(IIA) samples compared to controls (p < 0.0001), whereas no statistical significant increase in KB-VWF-F1.1 binding was detected for other VWD-types.

Conclusion(s): We identified two novel nanobodies that recognize different configurations of VWF, dependent on the degree of ADAMTS13-dependent proteolysis. Nanobody KB-VWF-D3.1 revealed the existence of ADAMTS13-induced conformational changes outside the A2-domain. Understanding how specific variants affect such changes could be insightful to better understand the corresponding phenotype of the patients.