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

The physiological role of the plasma protein factor XII (FXII), as well as, its involvement in human pathology, is poorly understood. While FXII is implicated in thrombotic pathology as a coagulation factor, it can contribute to inflammatory conditions without triggering coagulation. We recently generated nanobodies against the catalytic domain of activated FXII (FXIIa). Here, we describe two of these nanobodies, M and B7, both of which do not recognise FXII. Nanobody A10 recognises the catalytic domain of purified alpha-FXIIa (80 kDa), but not that of purified beta-FXIIa (28 kDa), whereas nanobody B7 recognises both. This suggests minute differences in the catalytic domain between these isoforms of FX11a. The detection of FXIIa by these nanobodies in plasma can become compromised through inactivation by serine protease inhibitors. This effect can be efficiently countered through the addition of the small-molecular protease inhibitor PPACK. Finally, we show that