PREVENTION OF THE INFLUENCE OF FIBRIN AND ALPHA(2)-MACROGLOBULIN IN THE CONTINUOUS MEASUREMENT OF THE THROMBIN POTENTIAL - IMPLICATIONS FORAN END-POINT DETERMINATION OF THE OPTICAL-DENSITY

We proposed the endogenous thrombin potential (ETP) as an overall func tion 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 optic al density, we investigated two methods to inhibit fibrinogen clottabi lity and to inactivate alpha(2)-macroglobulin. The first method makes use of hydroxylamine to inactivate alpha(2)-macroglobulin and H-Gly-Pr o-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incu bated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37 degrees C resulted in a reduced endlevel of the amidol ytic activity on small chromogenic substrates, The second method uses a metalloprotease purified from Crotalus basiliscus to remove alpha(2) -macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro-OH. H erein plasma is incubared with 3.5 mu M protease during 15 minutes at 37 degrees 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 end point determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculat ed ETP in plasmas with different degrees of anticoagulation. (C) 1998 Elsevier Science Ltd.