Abstract 30: Tissue Factor Pathway Inhibitor α Inhibits Prothrombinase via a Three-Step Mechanism

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TFPIα inhibits early forms of the prothrombinase complex (factor Xa (FXa), factor Va (FVa)), though the inhibitory mechanism is not entirely understood. One step of inhibition is a high affinity interaction between a TFPIα C-terminal basic region (BR) (252-LIKTKRKRKK-261) and an acidic region (AR) present in FXa-activated and platelet-released forms of FVa. We investigated two additional potential mechanistic steps: (1) binding of the second Kunitz-type inhibitory domain (K2) of TFPIα to the FXa active site; and (2) the function of uncharged residues L252, I253, and T255 within the BR, which are evolutionarily conserved, suggesting they have activity. Direct inhibition of FXa was investigated using TFPIα with an altered K2 (TFPI-R107A) incapable of binding FXa. TFPI-R107A inhibited purified prothrombinase 17-fold weaker than TFPIα (IC50 = 30.6nM vs. 1.8nM) and did not inhibit FXa-initiated thrombin generation in platelet-rich plasma (PRP). Therefore, direct binding of FXa and K2 is required for efficient inhibition of prothrombinase under physiological conditions. Similarly, the role of L252, I253, and T255 was investigated by substituting them with alanine (TFPI-AAKA). The IC50 for prothrombinase inhibition by TFPI-AAKA was 10.4nM, and it had reduced inhibitory activity in PRP, revealing that these residues are also required for efficient prothrombinase inhibition. The role of L252, I253, and T255 was further probed using the peptide LIKTKRKRKK, which inhibited purified prothrombinase (IC50 = 1.0μM) and thrombin generation in PRP at 1μM. AAKAKRKRKK had very little activity in either assay (~20% prothrombinase inhibition with 225μM peptide), but bound the FVa AR equivalently to LIKTKRKRKK (K d = 5.9nM and 6.0nM, respectively). Thus, the basic residues are responsible for AR binding, while a second step, mediated by L252-T255, is necessary for inhibitory activity. These residues may be necessary for displacement of FXa from FVa, as proposed by Bunce et al. We propose that prothrombinase inhibition by TFPIα involves three steps: (1) the TFPIα BR basic residues bind the FVa AR; (2) residues L252-T255 block prothrombinase assembly; and (3) K2 binds the FXa active site. All three steps are required for physiologic inhibition of prothrombinase by TFPIα.