Measurement of cleaved high-molecular-weight kininogen in patients with hereditary angioedema due to C1-inhibitor...

High-molecular-weight kininogen (HK) is a plasma protein cleaved during the activation of contact system by the enzyme kallikrein, releasing the peptide bradykinin, potent angioedema mediator. Plasma measurement of cleaved HK allows evaluating in vivo contact system activation but, when its main natural inhibitor (C1-inhibitor) is deficient, conflicting results have been reported, possibly due to in vitro activation. We collected blood samples from 18 patients with C1-inhibitor deficiency and 7 healthy controls using five different anticoagulant mixtures to find the one that could completely block in vitro activation of contact system, so that a snapshot of the in vivo scenario could be obtained. The ability to prevent contact system activation in vitro was evaluated in plasma samples measuring levels of cleaved HK by SDS-PAGE with precast gels and immunoblotting. Correlations between cleaved HK and functional C1-inhibitor were evaluated as well as assay reproducibility. In healthy subjects, no differences in cleaved HK were found for the different anticoagulants. In patients with C1-inhibitor deficiency, we observed higher levels of cleaved HK in sodium citrate samples (median 60.5 %, IQR 51.9-61.6 %) than in samples collected in a mixture of protease inhibitors (median 49.2 %, IQR 48.1-52.0 %) (P = 0.001). Only in the latter, cleaved HK levels correlated with circulating functional C1-inhibitor levels (P = 0.005). Using precast gels, intra- and inter-assay coefficients of variation were < 5 %. In conclusion, for measuring cleaved HK in plasma from patients with C1-inhibitor deficiency, the use of anticoagulant with protease inhibitors and precast gels allows reliable evaluation of in vivo contact system activation.