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

Jeremy P. Wood, Helle H. Petersen, Bingke Yu, Xiaoai Wu, Ida Hilden, Alan E. Mast

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase, the thrombingenerating 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 TFPIa 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 TFPIa 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 TFPIa 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.

Original languageEnglish
Pages (from-to)2692-2702
Number of pages11
JournalBlood advances
Volume1
Issue number27
DOIs
StatePublished - Dec 26 2017

Bibliographical note

Publisher Copyright:
© 2017 by The American Society of Hematology.

ASJC Scopus subject areas

  • Hematology

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