Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

Qian Liang; Ernest T Parker; Gabrielle Dean; Matthew S Karpen; Yujia Wu; Xuefeng Wang; Jorge Di Paola; Cheryl L Maier; Shannon L Meeks; Pete Lollar; Robert F Sidonio; Renhao Li

doi:10.1016/j.jtha.2024.04.015

Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

J Thromb Haemost. 2024 May 3:S1538-7836(24)00235-6. doi: 10.1016/j.jtha.2024.04.015. Online ahead of print.

Authors

Affiliations

¹ Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
² Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
³ Department of Statistics, School of Statistics and Center of Statistical Research, Southwestern University of Finance and Economics, Chengdu, China.
⁴ Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
⁵ Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
⁶ Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
⁷ Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA. Electronic address: renhao.li@emory.edu.

PMID: 38704122
DOI: 10.1016/j.jtha.2024.04.015

Abstract

Background: The activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified.

Objectives: To develop an assay that can differentiate the platelet-binding activity of VWF multimers.

Methods: A novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers.

Results: Of the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab_6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab_6C11 to that of HMWMs, which is significantly higher than all other activity assays tested.

Conclusion: The VWF:GPIbNab_6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

Keywords: nanobody; platelet; ristocetin; sensitivity; von Willebrand factor.