Skip to Main content Skip to Navigation
Journal articles

Development and characterization of single‐domain antibodies neutralizing protease nexin‐1 as tools to increase thrombin generation: Single-domain antibodies against protease nexin-1

Abstract : Background: Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with Plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin. Objectives: Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1. Methods: PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays. Results and Conclusions: By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 & KB-PN1-02). Despite their specificity, none displayed inhibitory activity towards hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06 and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, IC50-values for hPN-1 were 50±10 nM, 290±30 and 960±390 nM, for B11, F06 and A08, respectively, and 1580±240 nM, 560±130 nM and 2880±770 nM for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1.
Document type :
Journal articles
Complete list of metadatas

Cited literature [41 references]  Display  Hide  Download

https://www.hal.inserm.fr/inserm-02934843
Contributor : Cécile Denis <>
Submitted on : Wednesday, September 9, 2020 - 4:44:51 PM
Last modification on : Wednesday, September 16, 2020 - 5:52:14 PM

File

 Restricted access
To satisfy the distribution rights of the publisher, the document is embargoed until : 2020-12-04

Please log in to resquest access to the document

Identifiers

Citation

Charlotte Kawecki, Karen Aymonnier, Stephen Ferrière, Laurence Venisse, Véronique Arocas, et al.. Development and characterization of single‐domain antibodies neutralizing protease nexin‐1 as tools to increase thrombin generation: Single-domain antibodies against protease nexin-1. Journal of Thrombosis and Haemostasis, Wiley, 2020, 18, pp.2155-2168. ⟨10.1111/jth.14940⟩. ⟨inserm-02934843⟩

Share

Metrics

Record views

68