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 ± 0.005 ng mL(-1) (0.39 ± 0.10 pmol L(-1)) and a limit of quantification of 0.116 ± 0.055 ng mL(-1) (2.06 ± 0.98 pmol L(-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 12 ng mL(-1) (214 pmol L(-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 < 7 h, the APC-OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.