Structure-Function Analysis of the Reactive Site in the First Kunitz-type Domain of Human Tissue Factor Pathway Inhibitor-2

Abstract

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P 1 residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P 1 Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P 6 ) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P 1 ) with Asp-735 in plasmin, and Arg-29 (P 5 ) with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P 2 -P 4 ) in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P 6 -P 5 region of KD1, mutation of a highly conserved Phe at the P 18 position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P 1 residue, the nature of other residues flanking the P 1 residue, particularly at P 6 and P 5 , strongly influences the inhibitory activity and specificity of human TFPI-2.

Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

“…Comparison of the Inhibitory Properties of KD1-WT, KD1-L17R, and BPTI-In agreement with previous studies (22,23), KD1-WT inhibited pKLK, FXIa, and Pm with K i ‫ء‬ values of 25, 18, and 6 nM, respectively ( Fig. 3 and Table 1).…”

“…2 and 3 are consistent with the concept that coagulation proteases prefer a nonpolar residue at P2Ј position in their substrates/inhibitors (6,17,23,42). In this context, KD1-L17R has no inhibitory activity toward pKLK, FXIa, and FVIIa/sTF.…”

“…3 and Table 1). Neither BPTI nor KD1-L17R inhibited FVIIa/sTF; however, as previously studied (22,23), KD1-WT inhibited FVIIa/TF with ϳ1 M K i ‫ء‬ . Moreover, similar to KD1-WT, the KD1-L17R did not inhibit IIa, IIa/sTM, APC, or tPA (Table 1).…”

“…trypsin, chymotrypsin, VIIa/TF, factor XIa (FXIa), plasma kallikrein (pKLK), and Pm (22,23). The other two Kunitz domains in TFPI-2 have no known inhibitory function.…”

Comparative Effects of Aprotinin and Human Recombinant R24K KD1 on Temporal Renal Function in Long-Evans Rats

“…As a result of numerous adverse renal complications associated with its use in surgery, aprotinin was voluntarily withdrawn from the market by the manufacturer in 2007 and replaced with antifibrinolytic agents such as e-aminocaproic acid and tranexamic acid in these patients. The mechanism whereby aprotinin induces renal dysfunction in these patients is not known with certainty, but presumably involves uptake of aprotinin by proximal tubule cells (Vio et al, 1998), where it has been shown to inhibit tubule kallikrein secretion, prostaglandin and renin synthesis, and bradykinin release (Seto et al, 1983).Our laboratory has previously reported that an R24K mutant of the first Kunitz-type domain of human tissue factor pathway inhibitor-2, designated R24K KD1, exhibits plasmin inhibitory activity comparable with aprotinin (Chand et al, 2004), suggesting its potential as an alternative to aprotinin in cardiac surgery. Whether R24K KD1 exhibits the same nephrotoxic effects as aprotinin in an animal model has not been investigated.…”