Thrombin generation assay in untreated whole human blood

Augusta Modestino, Matthew Tyndall, Johnson Yu, Roy B. Lefkowitz, Geert W. Schmid-Schönbein, Michael (Mike) Heller

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2248-2256
Number of pages9
JournalELECTROPHORESIS
Volume37
Issue number15-16
DOIs
StatePublished - Aug 1 2016
Externally publishedYes

Fingerprint

Thrombin
Assays
Blood
Coagulation
Aspirin
Volunteers
Substrates
Limit of Detection
Healthy Volunteers
Buffers
Peptide Hydrolases
Phosphates
Personnel
Peptides

Keywords

  • Charge-changing substrates
  • Electrophoresis
  • Protease
  • Thrombin
  • Whole-blood assay

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry

Cite this

Modestino, A., Tyndall, M., Yu, J., Lefkowitz, R. B., Schmid-Schönbein, G. W., & Heller, M. M. (2016). Thrombin generation assay in untreated whole human blood. ELECTROPHORESIS, 37(15-16), 2248-2256. https://doi.org/10.1002/elps.201600061

Thrombin generation assay in untreated whole human blood. / Modestino, Augusta; Tyndall, Matthew; Yu, Johnson; Lefkowitz, Roy B.; Schmid-Schönbein, Geert W.; Heller, Michael (Mike).

In: ELECTROPHORESIS, Vol. 37, No. 15-16, 01.08.2016, p. 2248-2256.

Research output: Contribution to journalArticle

Modestino, A, Tyndall, M, Yu, J, Lefkowitz, RB, Schmid-Schönbein, GW & Heller, MM 2016, 'Thrombin generation assay in untreated whole human blood', ELECTROPHORESIS, vol. 37, no. 15-16, pp. 2248-2256. https://doi.org/10.1002/elps.201600061
Modestino A, Tyndall M, Yu J, Lefkowitz RB, Schmid-Schönbein GW, Heller MM. Thrombin generation assay in untreated whole human blood. ELECTROPHORESIS. 2016 Aug 1;37(15-16):2248-2256. https://doi.org/10.1002/elps.201600061
Modestino, Augusta ; Tyndall, Matthew ; Yu, Johnson ; Lefkowitz, Roy B. ; Schmid-Schönbein, Geert W. ; Heller, Michael (Mike). / Thrombin generation assay in untreated whole human blood. In: ELECTROPHORESIS. 2016 ; Vol. 37, No. 15-16. pp. 2248-2256.
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