Unambiguous and sensitive measurement of active coagulation proteases

A. Weber, A. Engelmaier, G. Prodinger (Vienna, Austria)

Laboratory tests
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: The advent of chromogenic substrate has revolutionized protease measurements in terms of sensitivity, but has not fully resolved issues related to selectivity. When measuring closely related proteases usually the sum of activity rather than the activity of a single protease is determined. Adding specific inhibitors is a valid approach, but increases the complexity of the assay. Here we describe the use of solid phase-bound protease inhibitors to measure active proteases by formation of the immobilized inhibitor-protease complex and its immunological detection by zymogen-specific antibodies. We demonstrate the applicability of this concept for activated coagulation factors XIIa (FXIIa), factor XIa (FXIa), kallikrein (KK) and factor Xa (FXa).

Methods: The protease inhibitors corn trypsin inhibitor (CTI), C1-inhibitor (C1-inh), and tissue factor pathway inhibitor (TFPI), diluted in PBS and coated to polystyrene plates, were used to set up specific assays for measurement of FXIIa, FXIa and FXa. Detection of the formed inhibitor-protease complexes was achieved by the corresponding peroxidase-labelled polyclonal anti-zymogen antibodies, namely, anti-human FXII, anti-human FXI, and anti-human FX. Calibration curves were obtained using purified, active protease preparations.

Results: The effect of related proteases was determined by measuring of FXIIa in the presence of FXI and kallikrein, FXIa in the presence of KK and FXIIa, and FXa in the presence of plasmin, FVIIa, FXIIa, thrombin, and KK. The selectivity of the FXa measurement was confirmed by competition with the synthetic FXa inhibitor rivaroxaban. Furthermore, the FXa determination also worked in the complex matrix of an activated prothrombin complex concentrate.Sensitive calibration curves were obtained to measure the four proteases used as model systems. All mixing experiments demonstrated the assay selectivity as even high excesses of related proteases demonstrated no effect.

Conclusion: The high selectivity depends on the specificity (i) of the inhibitor and (ii) of the detection antibody used to measure the amount of complex formed on the solid phase.