A Biomarker Assay For The Detection Of Contact System Activation
Authors: Faucette Ryan R , Cicardi Marco , Suffritti Chiara , Kenniston Jon A , Sexton Daniel J , Adelman Burt 
Published: October 2019

The contact system is composed of circulating plasma proteins that can both initiate coagulation through the intrinsic pathway and promote inflammation through the generation of bradykinin. Activation of the contact system is mediated by the assembly of factor XII (FXII), high molecular weight kininogen (HMWK), and prekallikrein (inactive form of the serine protease plasma kallikrein [pKal]) on a negatively charged surface. Bradykinin is produced from contact system activation by the proteolytic action of active pKal on its substrate HMWK. In vivo, vascular tissue injury or other unknown precipitants may expose negatively charged surfaces capable of supporting contact system activation. In vitro, the contact system is activated following exposure of plasma to substances such as kaolin, dextran sulfate, phospholipids, or surfaces such as glass. We developed a Western blot assay to detect the presence of both intact and cleaved HMWK in human and cynomolgus monkey plasma samples. Using this assay we showed that endogenous HMWK in normal human plasma was converted to cleaved kininogen following the addition of contact system activators, such as kaolin, dextran sulfate, or ellagic acid. HMWK was also shown to be cleaved by the addition of trace amounts of either FXIIa or pKal to normal human plasma. The utility of the Western blot assay was further demonstrated using plasma samples from patients with hereditary angioedema with C1 inhibitor (C1-INH) deficiency (HAE-C1-INH). HAE-C1-INH is a rare, autosomal dominant disease caused by mutations in one of the two alleles of the C1-INH gene (SERPING1). C1-INH is a serine protease inhibitor that blocks the activity of FXIIa and pKal in the contact system. HAE-C1-INH patients suffer from recurrent, localised angioedema attacks due to episodic activation of the contact system arising from unregulated pKal activity, which cleaves HMWK to release bradykinin, a potent vasoactive peptide mediating edema formation. Our results demonstrate that the method could detect significant contact system activation in HAE patient samples collected between attacks (i.e. quiescent disease samples) as compared to normal human plasma. Plasma samples collected during an HAE-C1-INH attack exhibited further elevations in cleaved kininogen. We also measured contact activation in plasma samples from cynomolgus monkeys that were dosed subcutaneously (SC) with DX-2930, a potent and specific monoclonal antibody inhibitor of plasma kallikrein being developed for HAE-C1-INH. DX-2930 blocked the cleavage of HMWK induced by kaolin-mediated contact system activation in a dose-dependent manner (5, 25, or 50 mg/Kg DX-2930, SC). In summary, this study presents a simple method that can be used both to identify disease states associated with contact system activation, and to serve as a pharmacodynamic biomarker for the development of DX-2930 as a treatment for HAE-C1-INH.

Disclosures:

Faucette: Dyax Corp: Employment. Cicardi:Dyax Corp: Consultancy. Kenniston:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.