ALBERT

Description: Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-▵19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-▵19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-▵19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-▵19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-▵19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-▵19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-▵19 bound to kaolin, which binding was equally well inhibited by either, rFXII-▵19 or full-length FXII (IC50 = 7.2 ± 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-▵19 generated a similar amount of FXIIa- and kallikrein-C1–inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1–inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-▵19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.

Description: 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 Lewisx (SLeX), 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 SLeX - selectin interactions is not clear. METHOD: We investigated the presence of SLeX -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 SLeX -selectin interaction was investigated in an a model system where SLeX -beads were rolled on immobilized E-selectin molecules. RESULT: We do not find any evidence of SLeX on C1-inh using either western blotting with anti-SLeX antibodies or by mass spectrometric analysis of C1-inh N- glycans. C1-inh products show modest but significant interference in SLeX -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 SLeX -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 SLeX -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 SLeX 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. Disclosures Engel: ViroPharma Inc.: Research Funding. Nunez:ViroPharma Inc.: Research Funding. Roem:ViroPharma Inc.: Research Funding. van Mierlo:ViroPharma Inc.: Research Funding. Wouters:ViroPharma: Research Funding. Zeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Description: Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-▵19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-▵19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-▵19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-▵19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-▵19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-▵19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-▵19 bound to kaolin, which binding was equally well inhibited by either, rFXII-▵19 or full-length FXII (IC50 = 7.2 ± 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-▵19 generated a similar amount of FXIIa- and kallikrein-C1–inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1–inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-▵19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.