Monitoring of plasma levels of activated protein C using a clinically applicable oligonucleotide-based enzyme capture assay

Abstract

Background: Human-activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular-inflammatory biomarker.Objective: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions.Methods/patients:Based on the APC-binding ssDNA aptamer HS02-52G we developed an oligonucleotide-based enzyme capture assay (OECA) that quantifies aptamer-captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre-analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery.Results and conclusion: A combination of APC-OECA with an aprotinin-based quenching strategy allowed APC analysis with a limit of detection as low as 0.022±0.005ngmL-1 (0.39±0.10pmolL-1) and a limit of quantification of 0.116±0.055ngmL-1 (2.06± 0.98 pmolL-1). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC-OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12ngmL-1 (214pmolL-1). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn-around time from blood sampling to generation of test results of <7h, the APC-OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions. © 2012 International Society on Thrombosis and Haemostasis.