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

J Thromb Haemost. 2020 Sep;18(9):2155-2168. doi: 10.1111/jth.14940.


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 and KB-PN1-02). Despite their specificity, none displayed inhibitory activity toward 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, half-maximal inhibitory concentration values for hPN-1 were 50 ± 10, 290 ± 30, and 960 ± 390 nmol/L, for B11, F06, and A08, respectively, and 1580 ± 240, 560 ± 130, and 2880 ± 770 nmol/L 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.

Keywords: phage display; protease nexin-1; serpin; single-domain antibody; thrombin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal
  • Cell Surface Display Techniques
  • Humans
  • Mice
  • Serpin E2 / genetics
  • Single-Domain Antibodies*
  • Thrombin*


  • Antibodies, Monoclonal
  • Serpin E2
  • Single-Domain Antibodies
  • Thrombin