An Anti-Tissue Factor Pathway Inhibitor (TFPI) Monoclonal Antibody Recognized the Third Kunitz Domain (K3) of Free-Form TFPI but Not Lipoprotein-Associated Forms in Plasma1

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains, which inhibits the initial reactions of the extrinsic blood coagulation pathway through the first and second Kunitz domains. We prepared a monoclonal antibody against recombinant human TFPI (rTFPI) and determined the epitope as the third Kunitz domain, using fragments derived from rTFPI (K1-K2 fragment and K3 fragment) and synthetic peptides. We then developed an enzyme immunoassay (EIA) method using a combination of the monoclonal antibody and a polyclonal antibody. Although TFPI activity is distributed among LDL/VLDL-associated, HDL-associated, and free forms of TFPI after gel-filtration of human plasma, only the free form was detected by the EIA method. After incubation with LDL, the antigenicity of rTFPI was reduced, but that of K3 fragment was not. Gel-filtration analysis of the mixture of radiolabeled rTFPI or K3 with LDL demonstrated that rTFPI, but not K3, bound LDL. From these results, we concluded that the monoclonal antibody against TFPI recognized only a free form of TFPI in plasma, since the epitope of lipoprotein-associated TFPI had been masked by the interaction with lipoproteins.