Alpha1-Antichymotrypsin Present in Therapeutic C1-Inhibitor Products Competes with Selectin - Sialyl LewisX Interaction

BACKGROUND: The plasma protein C1-inhibitor (C1-inh), belongs to the serpin superfamily and is the major inhibitor of the proteases of the complement and contact phase pathways. Hereditary or acquired deficiency of functional C1-inh results in angioedema episodes in affected individuals due to uncontrolled contact pathway activation and therapeutic C1-inh  products are effective treatment for these patients. Therapeutic C1-inh products have also been shown to attenuate neutrophil activation and infiltration in various inflammatory conditions. This ‘novel’ anti-inflammatory effect of C1-inh is attributed to its non-serpin N-terminal domain. This domain is thought to express the tetrasaccharide, sialyl Lewis^x (SLe^X), through which C1-inh can interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain small but significant amounts of co-purified proteins, the major one being the glycoprotein α1- antichymotrypsin (ACT), which is also an anti-inflammatory serpin. The potential influence of the glycans of ACT on SLe^X- selectin interactions is not clear.

METHOD: We investigated the presence of SLe^X-like epitopes on C1-inh and ACT from commercially available therapeutic C1-inh preparations using western blotting and mass-spectrometry. The influence of the products and separated C1-inh and ACT on SLe^X-selectin interaction was investigated in an a model system where SLe^X-beads were rolled on immobilized E-selectin molecules.

RESULT: We do not find any evidence of SLe^X on C1-inh using either western blotting with anti-SLe^X antibodies or by mass spectrometric analysis of C1-inh N-glycans. C1-inh products show modest but significant interference in SLe^X-selectin interaction but surprisingly this is not observed for ‘pure C1-inh’ obtained from gel-filtration of the commercial product. On the contrary, ACT, also from the C1-inh product, shows the presence of SLe^X-like epitopes, as detected by the antibody HECA-452 on western blot. In addition, at concentrations present in C1-inh products (20 -150 μg ACT/ mg active C1-inh), ACT can interfere with SLe^X-selectin interactions, in a sialic acid dependent manner. These concentrations of ACT can be achieved in vivo with a dose of  as low as 2000 U of a C1-inh product, suggesting that ACT can contribute to the anti-inflammatory effects observed in studies with C1-inh products.

CONCLUSION: We conclude that the ‘novel’ anti-inflammatory effects of C1-inh are unlikely due to SLe^X and can in fact be partly due to ACT. This fresh evidence challenges a long held assumption and paves the way for development of ACT, alone or in combination with C1-inh, as a new anti-inflammatory therapeutic.