The effect of heparin and its neutralisation on functional assays for factor VIIa, factor VII and TFPI.

Recently methods have become available to assay the haemostasis proteins tissue factor pathway inhibitor (TFPI) and activated factor VII (FVIIa). These assays are primarily used in research and in some studies patients may be receiving heparin therapy. We investigated the effect of heparin and its neutralisation by protamine sulphate, hexadimethrine bromide (polybrene) and triethylaminoethyl (TEAE) cellulose, on functional assays for TFPI, FVIIa and also factor VII (FVII). In the clotting assay for FVIIa using truncated recombinant tissue factor, heparin had little effect up to IU/ml, but concentrations higher than this grossly prolonged the clotting time. Protamine and polybrene neutralisation of heparin resulted in some prolongation of the clotting time despite adequate heparin neutralisation, and in addition, in the absence of heparin each of these substances themselves affected the clotting time. TEAE neutralisation of heparin appeared to be effective in the FVIIa assay, although in the absence of heparin we observed a 5% decrease in the clotting time. In the amidolytic substrate assays for TFPI and FVII, heparin with or without neutralisation resulted in only small changes in the optical density at 405nm and hence plasma levels of these factors were not significantly changed.