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A nanobody-based method for tracking factor XII activation in plasma.

S de Maat1, S van Dooremalen, P G de Groot

  • 1Dr. C. Maas, University Medical Center Utrecht, Department of Clinical Chemistry and Hematology, Room G.03.550, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands, Tel.: +31 88 755 6513, Fax: +31 88 755 5418,

Thrombosis and Haemostasis
|January 26, 2013
PubMed
Summary

New nanobodies distinguish forms of activated factor XII (FXIIa), revealing distinct roles in coagulation and inflammation. This breakthrough aids in understanding FXII

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Area of Science:

  • Biochemistry
  • Immunology
  • Proteomics

Background:

  • The physiological role of plasma protein factor XII (FXII) and its pathological involvement remain unclear.
  • FXII is linked to thrombosis and inflammation, but its precise mechanisms are not fully elucidated.
  • Activated FXII (FXIIa) is implicated in both procoagulant and inflammatory pathways.

Purpose of the Study:

  • To develop and characterize nanobodies targeting the catalytic domain of activated FXII (FXIIa).
  • To investigate the differential recognition of FXIIa isoforms and activation products by these nanobodies.
  • To establish nanobody-based assays for distinguishing FXIIa species involved in distinct physiological processes.

Main Methods:

  • Generation of nanobodies against the catalytic domain of FXIIa.
  • Characterization of nanobody binding to different FXIIa isoforms (α-FXIIa and β-FXIIa).
  • Assessment of nanobody detection in plasma, with and without protease inhibitors (PPACK).
  • In vitro assays to differentiate FXII activation products using nanobodies A10 and B7.

Main Results:

  • Two nanobodies, A10 and B7, were generated, recognizing FXIIa but not FXII.
  • Nanobody A10 specifically binds α-FXIIa, while B7 binds both α- and β-FXIIa, indicating isoform-specific recognition.
  • Nanobody-based detection of FXIIa in plasma is enhanced by the protease inhibitor PPACK.
  • Distinct FXII activation products were identified: procoagulant activators yield B7-recognized species, while inflammatory activators produce B7- and then A10-recognized species.

Conclusions:

  • Nanobodies A10 and B7 provide valuable tools for studying FXIIa.
  • Differential recognition of FXIIa isoforms and activation products suggests distinct roles in coagulation and inflammation.
  • Progressive proteolysis of FXIIa generates non-procoagulant forms, while intermediate forms may trigger coagulation.
  • Nanobody development against activated enzymes offers promising avenues for investigating their roles in health and disease.