Thrombin Generation Assay

Background

Thrombin generation (TG) reflects the end product of the coagulation system and hence could be used to assess the coagulation system in sick patients, or to monitor any anticoagulant drug.  TG assay is the most sensitive and reliable method for determining the hemostatic status of a patient.

Problem

Current TG assays are not accurate as they measure free and bound Thrombin (α2M-thrombin complexes), and depend on calculations to estimate free thrombin.

There is a need for better assays to predict bleeding or clotting risk, and coagulation disorders. Ideally, this should come in the form of a single assay that can monitor a wide range of common anticoagulants or that can be used during cardiopulmonary bypass, extracorporeal circulation or hemodialysis.

The Technology

Thrombin Generation Assay

University of Melbourne and McMaster University scientists have developed a simple assay that incorporates a novel substrate that only detects free thrombin and is insensitive to α2M-bound thrombin. This is achieved with a macromolecule and a spacer that sterically hinders interaction with α2M-Thrombin complexes.

Advantages of this assay

  • It measures directly and accurately free thrombin.
  • A single assay to monitor the effect of any anticoagulant with a single assay.
  • It works with plasma and whole blood
  • The assay can be modified to work with fluorogenic or electrogenic substrates
  • It is intended for use with plate readers, but could be adapted into a point of care device

Free thrombin vs. a2M-bound Thrombin detection assay. Our modified-substrate only reacts with free Thrombin:

Reactions of standard soluble substrate S2238 (0.3192 mg/mL) mixed with either Thrombin or Thrombin-α2M (137nM); and our modified substrate (1.389 mg/mL; insoluble attached to bottom of wells) mixed with Thrombin or Thrombin-α2M (90nM).

Difference in kinetics are due to the fluid phase and solid phase nature of the 2 substrates.

The assay works with whole blood and can monitor different anticoagulants.

Thrombin Generation Assay (with and without anticoagulant) in Whole Blood using our substrate. Citrated blood +/- hirudin was activated with calcium + dilute lipid-containing tissue factor reagent.

Further information

Download Thrombin Generation Assay flyer.