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.
Document Type
Article
Publication Date
12-26-2017
Digital Object Identifier (DOI)
https://doi.org/10.1182/bloodadvances.2017011098
Funding Information
This work was supported by National Institutes of Health, National Heart, Lung, and Blood Institute grants HL068835 (A.E.M.) and HL129193 (J.P.W.).
Related Content
The full-text version of this article contains a data supplement.
Repository Citation
Wood, Jeremy P.; Petersen, Helle H.; Yu, Bingke; Wu, Xiaoai; Hilden, Ida; and Mast, Alan E., "TFPIα Interacts with FVa and FXa to Inhibit Prothrombinase During the Initiation of Coagulation" (2017). Gill Heart & Vascular Institute Faculty Publications. 17.
https://uknowledge.uky.edu/heart_facpub/17
Data supplement: Document 1. Supplemental Figures
Included in
Amino Acids, Peptides, and Proteins Commons, Biochemistry, Biophysics, and Structural Biology Commons, Cardiology Commons
Notes/Citation Information
Published in Blood Advances, v. 1, no. 27, p. 2692-2702.
This research was originally published in Blood Advances. Jeremy P. Wood, Helle H. Petersen, Bingke Yu, Xiaoai Wu, Ida Hilden and Alan E. Mast. TFPIα interacts with FVa and FXa to inhibit prothrombinase during the initiation of coagulation. Blood Adv. 2017;1:2692-2702. © 2017 by The American Society of Hematology
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