{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T08:43:52Z","timestamp":1766738632990,"version":"3.41.2"},"reference-count":26,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T00:00:00Z","timestamp":1633910400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cardiovasc. Med."],"abstract":"<jats:p>Protein C (PC) activity tests are routinely performed in a thrombophilia workup to screen for PC deficiency. Currently used tests combine conversion of PC to activated PC (APC) by the snake venom Protac with subsequent APC detection through hydrolysis of a chromogenic peptide substrate or prolongation of a clotting time. In this prospective cohort study, we analyzed how different modes of PC activation and subsequent APC determination influence the diagnostic accuracy of PC activity testing in a cohort of 31 patients with genetically confirmed PC deficiency. In addition to chromogenic and clot-based measurement, an oligonucleotide-based enzyme capture assay utilizing a basic exosite-targeting aptamer was used for APC detection. To study the influence of the PC activation step on diagnostic sensitivity, PC activation through Protac and through the thrombin-thrombomodulin (TM) complex were compared. Twenty-six (84%) and 24 (77%) PC deficient patients were identified as true-positive using the chromogenic and the clot-based PC activity assay, respectively. True-positive results increased to 27 (87%) when the basic exosite-targeting aptamer approach was used for APC measurement. Additional replacement of the PC activator Protac by thrombin-TM gave true-positive results in all patients. These data indicate that the mode of PC activation is crucial in determining the accuracy of PC activity testing and that diagnostic sensitivity can be significantly improved by replacing the PC activator Protac with thrombin-TM. APC detection using a basic exosite-targeting aptamer achieves high sensitivity toward mutations outside the active center while being less subject to interfering factors than clot-based PC activity assays.<\/jats:p>","DOI":"10.3389\/fcvm.2021.755281","type":"journal-article","created":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T11:24:08Z","timestamp":1633951448000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":6,"title":["PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy"],"prefix":"10.3389","volume":"8","author":[{"given":"Sara","family":"Reda","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Heiko","family":"R\u00fchl","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jana","family":"Witkowski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jens","family":"M\u00fcller","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Pavlova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johannes","family":"Oldenburg","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bernd","family":"P\u00f6tzsch","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,10,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1111\/j.1538-7836.2007.02491.x","article-title":"Activated protein C","volume":"5","author":"Griffin","year":"2007","journal-title":"J Thromb Haemost."},{"key":"B2","doi-asserted-by":"publisher","first-page":"1034","DOI":"10.1160\/TH11-08-0522","article-title":"The structure-function relationship of activated protein C. Lessons from natural and engineered mutations","volume":"106","author":"Wildhagen","year":"2011","journal-title":"Thromb Haemost."},{"key":"B3","doi-asserted-by":"publisher","first-page":"1311","DOI":"10.1161\/01.ATV.0000168421.13467.82","article-title":"Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition","volume":"25","author":"Dahlb\u00e4ck","year":"2005","journal-title":"Arterioscler Thromb Vasc Biol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"2059","DOI":"10.2174\/092986710791233706","article-title":"Regulation of the protein C anticoagulant and antiinflammatory pathways","volume":"17","author":"Rezaie","year":"2010","journal-title":"Curr Med Chem."},{"key":"B5","doi-asserted-by":"publisher","first-page":"1880","DOI":"10.1126\/science.1071699","article-title":"Activation of endothelial cell protease activated receptor 1 by the protein C pathway","volume":"296","author":"Riewald","year":"2002","journal-title":"Science."},{"key":"B6","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1136\/adc.2010.199919","article-title":"Purpura fulminans: recognition, diagnosis and management","volume":"96","author":"Chalmers","year":"2011","journal-title":"Arch Dis Child."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1055\/s-0038-1660995","article-title":"The use of a functional and immunologic assay for plasma protein C in the study of the heterogeneity of congenital protein C deficiency","volume":"51","author":"Bertina","year":"1984","journal-title":"Thromb Haemost."},{"key":"B8","doi-asserted-by":"publisher","first-page":"876","DOI":"10.1055\/s-0038-1653885","article-title":"Protein C deficiency: a database of mutations, 1995 update. On behalf of the Subcommittee on Plasma Coagulation Inhibitors of the Scientific and Standardization Committee of the ISTH","volume":"73","author":"Reitsma","year":"1995","journal-title":"Thromb Haemost."},{"key":"B9","doi-asserted-by":"publisher","first-page":"253","DOI":"10.1016\/0049-3848(86)90145-3","article-title":"Fast functional protein C assay using Protac, a novel protein C activator","volume":"43","author":"Martinoli","year":"1986","journal-title":"Thromb Res."},{"key":"B10","doi-asserted-by":"publisher","first-page":"39309","DOI":"10.1074\/jbc.M508502200","article-title":"Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases: crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator","volume":"280","author":"Murakami","year":"2005","journal-title":"J Biol Chem."},{"key":"B11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/ijlh.12004","article-title":"Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis","volume":"35","author":"Mackie","year":"2013","journal-title":"Int J Lab Hematol."},{"key":"B12","doi-asserted-by":"publisher","first-page":"440","DOI":"10.1002\/ajh.21679","article-title":"Laboratory tests for protein C deficiency","volume":"85","author":"Khor","year":"2010","journal-title":"Am J Hematol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"748","DOI":"10.1046\/j.1538-7836.2003.00141.x","article-title":"The long-term within- and between-laboratory variability for assay of antithrombin, and proteins C and S: results derived from the external quality assessment program for thrombophilia screening of the ECAT Foundation","volume":"1","author":"Meijer","year":"2003","journal-title":"J Thromb Haemost."},{"key":"B14","doi-asserted-by":"publisher","first-page":"909","DOI":"10.1309\/AJCP8MWU4QSTCLPU","article-title":"Protein C assay performance: an analysis of North American specialized coagulation laboratory association proficiency testing results","volume":"137","author":"Baron","year":"2012","journal-title":"Am J Clin Pathol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"336","DOI":"10.1111\/j.1751-553X.2012.01401.x","article-title":"The phenotypic and genetic assessment of protein C deficiency","volume":"34","author":"Cooper","year":"2012","journal-title":"Int J Lab Hematol."},{"key":"B16","doi-asserted-by":"publisher","first-page":"646","DOI":"10.1007\/s004390000315","article-title":"Molecular genetic analysis of severe protein C deficiency","volume":"106","author":"Millar","year":"2000","journal-title":"Hum Genet."},{"key":"B17","doi-asserted-by":"publisher","first-page":"e44378","DOI":"10.1371\/journal.pone.0044378","article-title":"The prothrombotic phenotypes in familial protein C deficiency are differentiated by computational modeling of thrombin generation","volume":"7","author":"Brummel-Ziedins","year":"2012","journal-title":"PLoS ONE."},{"key":"B18","doi-asserted-by":"publisher","first-page":"442","DOI":"10.1016\/j.chembiol.2009.03.007","article-title":"An exosite-specific ssDNA aptamer inhibits the anticoagulant functions of activated protein C and enhances inhibition by protein C inhibitor","volume":"16","author":"M\u00fcller","year":"2009","journal-title":"Chem Biol."},{"key":"B19","doi-asserted-by":"publisher","first-page":"390","DOI":"10.1111\/j.1538-7836.2012.04623.x","article-title":"Monitoring of plasma levels of activated protein C using a clinically applicable oligonucleotide-based enzyme capture assay","volume":"10","author":"M\u00fcller","year":"2012","journal-title":"J Thromb Haemost."},{"key":"B20","doi-asserted-by":"publisher","first-page":"1215","DOI":"10.1093\/nar\/16.3.1215","article-title":"A simple salting out procedure for extracting DNA from human nucleated cells","volume":"16","author":"Miller","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"B21","doi-asserted-by":"publisher","first-page":"273","DOI":"10.1097\/00001721-199304000-00009","article-title":"Twelve novel and two recurrent mutations in 14 Austrian families with hereditary protein C deficiency","volume":"4","author":"Poort","year":"1993","journal-title":"Blood Coagul Fibrinolysis."},{"key":"B22","doi-asserted-by":"publisher","first-page":"S53","DOI":"10.1515\/CCLM.2010.369","article-title":"Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game","volume":"48","author":"Bereczky","year":"2010","journal-title":"Clin Chem Lab Med."},{"key":"B23","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1097\/MBC.0000000000000806","article-title":"Comparison of clot-based and chromogenic assay for the determination of protein c activity","volume":"30","author":"Roshan","year":"2019","journal-title":"Blood Coagul Fibrinolysis."},{"key":"B24","doi-asserted-by":"publisher","first-page":"8","DOI":"10.1016\/S1050-1738(02)00191-3","article-title":"Exosite-dependent regulation of the protein C anticoagulant pathway","volume":"13","author":"Rezaie","year":"2003","journal-title":"Trends Cardiovasc Med."},{"key":"B25","doi-asserted-by":"publisher","first-page":"44596","DOI":"10.1038\/srep44596","article-title":"Rational Design of Protein C Activators","volume":"7","author":"Barranco-Medina","year":"2017","journal-title":"Sci Rep."},{"key":"B26","doi-asserted-by":"publisher","first-page":"26104","DOI":"10.1016\/S0021-9258(18)35723-5","article-title":"The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives","volume":"267","author":"Rezaie","year":"1992","journal-title":"J Biol Chem."}],"container-title":["Frontiers in Cardiovascular Medicine"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcvm.2021.755281\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T11:24:10Z","timestamp":1633951450000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcvm.2021.755281\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,11]]},"references-count":26,"alternative-id":["10.3389\/fcvm.2021.755281"],"URL":"https:\/\/doi.org\/10.3389\/fcvm.2021.755281","relation":{},"ISSN":["2297-055X"],"issn-type":[{"type":"electronic","value":"2297-055X"}],"subject":[],"published":{"date-parts":[[2021,10,11]]},"article-number":"755281"}}