Inhibitory properties of human recombinant Arg24→Gln type-2 tissue factor pathway inhibitor (R24Q TFPI-2)

Abstract Human type-2 tissue factor pathway inhibitor (TFPI-2), also known as placental protein 5, is a 32-kDa serine proteinase inhibitor consisting of three tandemly arranged Kunitz-type domains homologous to tissue factor pathway inhibitor. TFPI-2 inhibits a variety of serine proteinases involved in coagulation and fibrinolysis through an arginine residue (R24) in its first Kunitz-type domain, which constitutes a putative P 1 residue for the substrate recognition sites of these proteinases. As recent studies have shown that this P 1 residue to be a glutamine in murine TFPI-2, we constructed, expressed, and purified a human TFPI-2 mutant with glutamine substituted for arginine at position 24 (R24Q TFPI-2). R24Q TFPI-2 lost ∼90% of its inhibitory activity towards bovine trypsin and virtually all inhibitory activity towards human plasmin and the factor VIIa–tissue factor complex, emphasizing the importance of the P 1 Arg 24 residue in the inhibition of these serine proteinases. However, whereas wild-type TFPI-2 is a relatively weak inhibitor of human factor Xa amidolytic activity (IC 50 ∼1 μM), R24Q TFPI-2 exhibited enhanced inhibitory activity towards the amidolytic and coagulant activities of this proteinase with a K i of 18 nM. While the molecular basis for the enhanced inhibition of human factor Xa by R24Q TFPI-2 is unknown, these data provide suggestive evidence that murine TFPI-2 may function as a serine proteinase inhibitor in spite of the absence of a P 1 Arg or Lys residue.