Thrombin generation assay in untreated whole human blood

• Published in: Electrophoresis Vol. 37; no. 15-16; pp. 2248 - 2256
• Main Authors: Modestino, Augusta, Tyndall, Matthew, Yu, Johnson, Lefkowitz, Roy B., Schmid-Schönbein, Geert W., Heller, Michael J.
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.08.2016
• Subjects: Aspirin; Aspirin - pharmacology; Assaying; Blood; Blood Coagulation - drug effects; Blood Coagulation Tests - methods; Charge-changing substrates; Coagulation; Electrophoresis; Electrophoresis - methods; Humans; Limit of Detection; Peptides; Protease; Sensitivity and Specificity; Substrates; Thrombin; Thrombin Time; Whole-blood assay; Electrophoresis; Thrombin; Charge-changing substrates; Protease; Whole-blood assay
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201600061

Present coagulation assays fail to detect mild coagulation disorders, while thrombin‐generation (TG) assays solve this problem. However, most of them only work with threated blood samples, which makes them labor intensive, time consuming, unreliable, and expensive. We have developed a TG electrophoretic assay that uses a thrombin specific charge‐changing fluorescent peptide substrate, electrophoretic separation, and requires a drop of blood. The limit of detection of the assay was 1.97 nM in phosphate buffer saline and 6.82 nM in citrated whole blood. The assay was used to determine the TG in whole blood from healthy volunteers (n = 6, one aspirin user), over 30 min, after the blood was drawn; the TG increased from a baseline level of 2 × 106 RFU to 1.2 × 1013 RFU. The lag time between the blood draw and initial burst of TG was 6 min for the volunteers (n = 5) and 15 min for the aspirin user. Specificity of the assay was evaluated by reacting our substrate with the heparinized blood samples and other proteases. The TG electrophoretic assay was designed and tested in the whole human blood, requiring no sample preparation, 5 μL of blood, 45 min, and it detected differences in coagulation patterns between a volunteer taking aspirin and non‐aspirin users.