Prevention of the influence of fibrin and α2-macroglobulin in the continuous measurement of the thrombin potential: Implications for an endpoint determination of the optical density

We proposed the endogenous thrombin potential (ETP) as an overall function test of the coagulation system. We recently introduced a routine test which requires defibrinated plasma. In order to develop an assay in which the ETP-value can be directly obtained by measuring the optical density, we investigated two methods to inhibit fibrinogen clottability and to inactivate α₂-macroglobulin. The first method makes use of hydroxylamine to inactivate α₂-macroglobulin and H-Gly-Pro-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incubated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37°C resulted in a reduced endlevel of the amidolytic activity on small chromogenic substrates. The second method uses a metalloprotease purified from Crotalus basiliscus to remove α₂-macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro- OH. Herein plasma is incubated with 3.5 μM protease during 15 minutes at 37°C in the presence of 1 mg/mL polymerization inhibitor. The enzymatic method results in a zero endlevel of the amidolytic activity and this would imply that measurement of the ETP is reduced to an endpoint determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculated ETP in plasmas with different degrees of anticoagulation.