TFPIα interacts with FVa and FXa to inhibit prothrombinase during the initiation of coagulation

Abstract

Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase, the thrombin-generating complex of factor Xa (FXa) and factor Va (FVa), during the initiation of coagulation. This inhibition requires binding of a conserved basic region within TFPIα to a conserved acidic region in FXa-activated and platelet-released FVa. In this study, the contribution of interactions between TFPIα and the FXa active site and FVa heavy chain to prothrombinase inhibition were examined to further define the inhibitory biochemistry. Removal of FXa active site binding by mutation or by deletion of the second Kunitz domain (K2) of TFPIα produced 17- or 34-fold weaker prothrombinase inhibition, respectively, establishing that K2 binding to the FXa active site is required for efficient inhibition. Substitution of the TFPIα basic region uncharged residues (Leu252, Ile253, Thr255) with Ala (TFPI-AAKA) produced 5.8-fold decreased inhibition. This finding was confirmed using a basic region peptide (Leu252-Lys261) and Ala substitution peptides, which established that the uncharged residues are required for prothrombinase inhibitory activity but not for binding the FVa acidic region. This suggests that the uncharged residues mediate a secondary interaction with FVa subsequent to acidic region binding. This secondary interaction seems to be with the FVa heavy chain, because the FV Leiden mutation weakened prothrombinase inhibition by TFPIα but did not alter TFPI-AAKA inhibitory activity. Thus, efficient inhibition of prothrombinase by TFPIα requires at least 3 intermolecular interactions: (1) the TFPIα basic region binds the FVa acidic region, (2) K2 binds the FXa active site, and (3) Leu252-Thr255 binds the FVa heavy chain.

Graphical abstract

Figure 1.

TFPIα variant proteins used in these studies. (A) Shown are the domain structures of TFPIα, K1K2 (amino acids 1-160), TFPIΔK2 (R107A mutation), TFPI-AAKA (L252A, I253A, T255A mutations), and K3C (amino acids 185-271). The 3 Kunitz domains are shown in gray. The C-terminal basic region is blue. (B) Western blot of the TFPI variant proteins, probed with antibodies against the K2 domain (red) and the last 12 amino acids of the C-terminus (green), with overlay indicated in yellow. The K1K2 sample contains some dimerized protein.

Figure 2.

The K2 domain of TFPIα enhances prothrombinase inhibition. (A) FVa_Xa (0.5 nM) was incubated with phospholipid vesicles (20 μM), the thrombin inhibitor DAPA (3 μM), and TFPIα (open circle), TFPIΔK2 (open square), or K3C (filled square). Reactions were initiated by addition of prothrombin (1.4 μM) and FXa (5 nM). After dilution to reduce the effect of DAPA, thrombin was quantified by the rate of cleavage of a chromogenic substrate (0.32 mM). The initial rate of thrombin generation is shown as a percentage of control (mean ± standard deviation; n ≥ 3). Lines represent best-fit inhibition curves. (B-F) Thrombin generation was measured in TFPI-depleted plasma (B-C) or PRP (D-F). Reactions were initiated with a mixture of FXa (0.1 nM) and phospholipid vesicles (4 μM) (B-C) or FXa and collagen (15 µg/mL) (D-F), in the presence of the indicated concentrations of TFPIα (B,D), TFPIΔK2 (C,E), or K3C (F). Shown are the average thrombin generation curves from experiments performed in triplicate using TFPI-depleted plasma (B-C) or using PRP from 4 different donors (D-F).

Figure 3.

The basic region uncharged residues enhance prothrombinase inhibition. (A) Prothrombinase activity was measured as in Figure 2A, in the presence of the indicated concentrations of TFPI-AAKA (filled square). The initial rate of thrombin generation is shown as a percentage of control (mean ± standard deviation; n ≥ 3). Lines represent best-fit inhibition curves. Inhibition of prothrombinase by TFPIα (open circle) and TFPIΔK2 (open square) is reproduced from Figure 2 for reference. (B-C) Thrombin generation was measured in TFPI-depleted plasma (B) or PRP (C) as in Figure 2, in the presence of the indicated concentrations of TFPI-AAKA. Shown are average thrombin generation curves from experiments performed in triplicate using TFPI-depleted plasma (B) or PRP from 4 donors (C).

Figure 4.

The uncharged residues are required for prothrombinase inhibition by a peptide mimicking the TFPIα basic region. (A) Prothrombinase activity assays were performed as in Figure 2A using either FVa_Xa (filled square) or FVa_IIa (open circle) and the indicated concentrations of LIKTKRKRKK (LIKT). The initial rate of thrombin generation is shown as a percentage of control (mean ± standard deviation; n ≥ 3). Lines represent best-fit inhibition curves. (B,D) Thrombin generation was measured in platelet-rich plasma as in Figure 2D, in the presence of the indicated concentrations of LIKT (B) or AAKAKRKRKK (AAKA) (D). Shown are average thrombin generation curves from experiments using at least 3 donors. (C) Prothrombinase activity assays were performed using FVa_Xa and the indicated concentrations of AAKA (open circle). Inhibition of FVa_Xa prothrombinase by LIKT (filled square) is reproduced from panel A for reference.

Figure 5.

Leu252, Ile253, and Thr255 each contribute to prothrombinase inhibition. (A) Prothrombinase activity was measured as in Figure 2A, in the presence of the indicated concentrations of AIKTKRKRKK (AIKT; open diamond), LAKTKRKRKK (LAKT; open circle), or LIKAKRKRKK (LIKA; filled triangle). The initial rate of thrombin generation is shown as a percentage of control (no peptide; mean ± standard deviation; n ≥ 3). Lines represent best-fit inhibition curves. Inhibition of prothrombinase by LIKTKRKRKK (LIKT; filled square) is reproduced from Figure 3 for reference. (B-D) Thrombin generation was measured in PRP as in Figure 2D, using the indicated concentrations of AIKT (B), LAKT (C), or LIKA (D). Shown are the average thrombin generation curves from experiments using 3 donors.

Figure 6.

The basic amino acids, but not the uncharged amino acids, mediate binding of the TFPIα basic region to the FVa acidic region. (A) Rhod-LIKTKRKRKK (30 nM) was incubated with FV810 (30 nM) and the indicated concentrations of LIKTKRKRKK (LIKT; filled square), AAKAKRKRKK (AAKA; open square), AIKTKRKRKK (AIKT; filled circle), LAKTKRKRKK (LAKT; open circle), or LIKAKRKRKK (LIKA; filled triangle). Fluorescence anisotropy was measured and curve fits generated as described in Materials and methods. (B) FV810 (0.5 nM), phospholipid vesicles (20 µM), and the thrombin inhibitor DAPA (3 µM) were incubated with LIKTKRKRKK (LIKT; 3 µM) and varying concentrations of AAKA, and thrombin generation was measured as in Figure 2A. The initial rate of thrombin generation is shown as a percentage of control (no peptide; mean ± standard deviation; n = 3).

Figure 7.

The Leu252-Thr254 region is responsible for the effect of FVL on prothrombinase inhibition by TFPIα. FV810 (filled circle) or FVL810 (open circle; 0.5 nM) was incubated with phospholipid vesicles (20 μM), the thrombin inhibitor DAPA (3 μM), and the indicated concentrations of TFPI-AAKA (A) or TFPIΔK2 (B), and thrombin generation was measured as in Figure 2A. The initial rate of thrombin generation is shown as a percentage of control (mean ± standard deviation; n ≥ 3). Lines represent best-fit inhibition curves.