title: 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
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Experimental Haematology and Transfusion Medicine Clinic for Orthopedic Surgery, University Clinic Bonn Life and Medical Sciences Institute, University of Bonn, Bonn, Germany. jens.mueller@ukb.uni-bonn.de
authors.forename: J
authors.initials: J
authors.lastname: Müller
authors.forename: M
authors.initials: M
authors.lastname: Friedrich
authors.forename: T
authors.initials: T
authors.lastname: Becher
authors.forename: J
authors.initials: J
authors.lastname: Braunstein
authors.forename: T
authors.initials: T
authors.lastname: Kupper
authors.forename: P
authors.initials: P
authors.lastname: Berdel
authors.forename: S
authors.initials: S
authors.lastname: Gravius
authors.forename: F
authors.initials: F
authors.lastname: Rohrbach
authors.forename: J
authors.initials: J
authors.lastname: Oldenburg
authors.forename: G
authors.initials: G
authors.lastname: Mayer
authors.forename: B
authors.initials: B
authors.lastname: Pötzsch
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D052157:Aptamers, Nucleotide; D019644:Arthroplasty, Replacement, Hip; D015415:Biomarkers / Q000097:blood; D001780:Blood Coagulation Tests; D002863:Chromogenic Compounds; D004796:Clinical Enzyme Tests; D005260:Female; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D016343:Monitoring, Intraoperative / Q000379:methods*; D011237:Predictive Value of Tests; D011486:Protein C / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D052157:Aptamers, Nucleotide; D015415:Biomarkers; D002863:Chromogenic Compounds; D011486:Protein C; D013917:Thrombin