A new sensitive chromogenic substrate assay of tissue factor pathway inhibitor type 1

The present assay is a modification of our previously published two-stage c hromogenic substrate assay of tissue factor pathway inhibitor type-1 (TFPI) activity [1]. In the first stage, the reaction mixture was made with facto r VIIa (FVIIa) molecules in excess of tissue factor (TF) binding sites and contained diluted plasma, recombinant FVIIa (10 nM), recombinant TF (1/400 vol/vol), bovine factor Xa (1,1 nM), I-2882(R) (100 mu g/ml), and CaCl2 (10 mM). The fibrin polymerisation inhibitor I-2882(R) was added to the reacti on mixture to prevent formation of cross-linked fibrin. In the second stage , residual TF/FVIIa catalytic activity was measured by the addition of a su bstrate mixture that contained bovine factor X and a chromogenic substrate (S-2222(R)). Standard curves were constructed using serial dilutions (0-1%) of pooled normal plasma. The dose-response relationship for serial dilutio ns of plasma was linear. The intra-assay coefficient of variations (CVs) fo r pre- and postheparin plasma samples (i.e., normal and high TFPI levels) w ere 1.7% and 9.9%, respectively; the inter-assay CVs were 10.0% and 19.7%, respectively. The effect of variation in antithrombin activity on the assay was approximately 5%. The present assay correlated fairly well with our pr eviously published assay (r=0.82, n=100) and with a commercial TFPI activit y assay (Actichrome(R) TFPI Activity Assay, American Diagnostica, Greenwich , CT, USA; r=0.90, n=100), as well as with an antigen assay for TFPI total antigen (Imubind(R), American Diagnostica; r=0,96, n=100). Altman and Bland plots revealed that our previous assay underestimated TFPI activity at hig h TFPI levels (i.e., postheparin TFPI samples) compared with the other meth ods. (C) 2000 Elsevier Science Ltd. All rights reserved.