Abstract: In a method of determining heparin content, a known amount of thrombin (FIIa) or activated coagulation factor X (FXa) is added to a mixture which comprises a known concentration of chromogenic substrate (S), a known concentration of antithrombin (AT) and a sample having an unknown heparin concentration. The amount of FIIa or FXa is chosen such that at most 20%, of the S present is reacted during the period which the AT needs to deactivate all the FIIa or FXa present. The final concentration of the reacted chromogenic substrate p-nitroanilide ([pNA].sub.final) is determined after completion of the reactions, and the [pNA].sub.final is used to determine the rate constant (k.sub.dec) of the reaction of FIIa or FXa with AT using the relationship: ##EQU1## The heparin concentration in the sample can then be determined using the value of k.sub.dec from a predetermined calibration curve of k.sub.dec against heparin concentration.

Abstract: The present invention relates to a method to determine the concentration of anticoagulants as a function of the inhibition of thrombin formation in an assay mixture. The assay mixture is composed of two reagents and the diluted plasma sample. The diluted plasma sample containing an anticoagulatory substance is mixed with a clotting factor reagent, which is a combination of an excess amount of purified coagulation factors of the endogenous system, phospholipids and a weak thrombin inhibitor, which can be potentiated by an anticoagulatory substance. Then a coagulation cascade activator and calcium chloride, an activator reagent, is added to the mixture and formed thrombin is measured after a chosen incubation time. A proportionality between the inhibition of the thrombin formation and the anticoagulatory active components allows for a determination of the concentration of anticoagulatory active components in the sample.