Two stage Coatest assay of F. VIII is reportedly insensitive to pretreatment of F. VIII with thrombin. Since thrombin is formed rapidly, during the incubation step, a one stage method was tried by incorporating S2222, containing thrombin inhibitor (1-2581) in the incubation system, thus making the assay highly specific to F. Xa-induced activation of F. VIII and allowing to monitor directly the formation of paranitroaniline in microtray plate at 2-5 min. intervals. Initial comparative analyses

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... rformed on cold activated and/or adsorbed/non-adsorbed samples (FFP, cryoprecipitate and hypo- or hypercoagulable state) revealed that in all cases the lag phase was prolonged (2-3 fold) in the one stage method. Cold activation had little effect on the lag phase/reaction rate, whereas AL(0E)3 decreased up to 50% F. VIII like activity, prolonged the lag phase and dose-response curves become non-parallel. Substituting phospholipid (pL) by tissue factor (TF) or addition of diluted TF (1/500) to reaction mixture increased synergistically the rate of F. Xa generation in both adsorbed and non-adsorbed system. In contrast washed platelets (PLT), up to 3000 x 109/1, were less effective on both TF or PL-induced F. Xa generation. The presence of 1-2581 in this system prolonged the lag phase to Ih. Substitution of the conventional O. D. reading by the time required to achieve a fixed absorbancy (O. D. =0.5) make the one stage coatest F. VIII equivalent to the APPT-type assay. Based on these results it is concluded that thrombin is involved in increasing F. VIII catalytic activity. TF and F. VII contribute to the shortening of the lag phase and increased F.a generation. The Kinetic property of cell surface-bound F. VIII is not the same in the presence or absence of thrombin. The reported insensitivity coatest F. VIII to thrombin is probably due to the fact that thrombin activated F. VIII is a good substrate for F. Xa and is cleaved by F. Xa which is produced in abundance in the two-stage chromogenic assays. A new procedure for monitoring various pathways of F. Xa generation, based on the coatest reagent is provided, which is particularly suitable for large scale screening of blood donors and blood products.