Development of Methods for Measuring Protein C Inhibitor and Antithrombin: Use of Monoclonal Antibodies against the Reactive Center Loop-Inserted Forms of the Serpins
Abstract: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that are involved in the regulation of coagulation. Like other inhibitory serpins, PCI and AT adopt different structural conformations that are related to their functions. The cleaved, inactive form is a result of cleavage by a protease, and the latent form, which is also inactive, can arise due to a mutation in the serpin. Methods that quantify the different forms can be useful as diagnostic tools. The aim of this work was to develop such methods for measuring AT and for measuring cleaved PCI in complex with APC (APCI-PCI). The high-affinity monoclonal antibody M36 (KD ~50 pM) was produced against cleaved PCI. M36 is specific enough to discriminate between cleaved and native PCI and thus it was used as the catcher antibody, along with a monoclonal antibody against protein C as detector, in an immunoassay developed to measure the APC-PCI complex in blood plasma. The mean concentration of APC-PCI was found to be 0.30 µg/L in healthy individuals, and the levels of this complex were elevated in patients who had undergone hip surgery and were lowered in patients treated with warfarin. A crystal of cleaved PCI was obtained, which revealed a short helix A, and this is similar to the two hormone-binding serpins called corticosteroid-binding globulin and thyroxine-binding globulin. A potential binding site for retinoic acid was found in PCI, and moreover, possible novel binding sites for heparin were exposed, which suggested an alternative model for heparin-activated inhibition of APC by PCI. The monoclonal antibody M9 against reactive center loop-inserted antithrombin (AT) was produced and was found to have affinities (KD values) of 3.07 and 2.27 µM for latent and cleaved AT, respectively. An immunoassay for measuring cleaved AT was developed using M9 as catcher antibody and the monoclonal antibody M27 (specific for cleaved and native AT) as detector. The median level of cleaved AT was 1.3 mg/L in healthy individuals. No correlation was found between cleaved AT and the thrombin-antithrombin (TAT) complex in patients with deep venous thrombosis. The monoclonal antibody 8C8 was produced against latent AT, and it showed affinities (KD values) of 0.92 nM, 0.57 µM, and 5.65 µM for latent, native and cleaved AT, respectively. An immunoassay was developed for measuring latent AT, using M9 (specific for cleaved and latent AT) as catcher antibody and 8C8 as detector. The assay showed that the median level of latent AT was 4.7 mg/L in healthy individuals. Commercial preparations of native AT were found to contain ?10% latent AT. In addition, a weak interaction (KD = 51 µM) was observed between latent and native AT, which contradicts the formation of a stable dimer in vivo.
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