Abstract Number: PB0414
Meeting: ISTH 2020 Congress
Background: The membrane-adjacent tissue factor (TF) residues S160-S161-S162-S163 form a flexible, solvent-exposed loop that is poorly resolved in almost all crystal structures. Mutating some of these four serines to alanine decreases activation of factor X (FX) and factor IX (FIX). However, the exact role of this serine loop in facilitating substrate activation and selection by TF-FVIIa is still unknown.
Aims: Investigate if the TF serine loop facilitates selective activation of TF-FVIIa substrates.
Methods: We generated thirteen different TF serine loop mutants. In the first set of mutants, each serine was substituted to threonine. In the second set, the importance of loop length was tested by inserting one or more serines or threonines, or deleting one or more serines. Membrane-anchored TF was expressed in E. coli and affinity-purified. TF mutants were incorporated into liposomes and characterized using substrate activation assays as well as thrombin generation assays.
Results: Serine-to-threonine substitutions had little effect on rates of FX activation by TF-FVIIa. Surprisingly, however, increasing or decreasing the length of the serine loop by even one amino acid decreased rates of FX activation 20- to 200-fold, while having little to no effect on FIX activation. Thrombin generation assays with FVIII- and FIX-deficient plasma showed that the thrombin concentration was reduced two- to fourfold and time-to-peak was increased twofold for these TF serine loop mutants compared to wildtype TF.
Conclusions: The exact length of the serine loop on TF is essential for FX activation by TF-FVIIa, while altering its length has negligible effects on FIX activation. Thus, the precise length of this apparently unstructured TF loop plays a selective role in facilitating FX activation, which we believe is the most extreme example to date of substrate selectivity mediated by a portion of the TF structure.
To cite this abstract in AMA style:Birkle F, Morrissey JH. A Membrane-Adjacent Region in Tissue Factor Provides Substrate Selectivity [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-membrane-adjacent-region-in-tissue-factor-provides-substrate-selectivity/. Accessed May 16, 2021.
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