Abstract Number: PB0241
Meeting: ISTH 2020 Congress
Background: There is mounting evidence that feedback activation of factor (F) XI by thrombin is important for thrombus stabilization and growth. Polyphosphate (polyP) released from activated platelets enhances this reaction by binding FXI and thrombin and bridging them together.
Aims: To determine the contribution of thrombin exosites 1 and 2 to the potentiation of thrombin activation of FXI by polyP.
Methods: FXI was incubated with α-thrombin or a recombinant thrombin exosite 1 variant (K109E/K110E) in the absence or presence of polyP (6-mer, 20-mer, or 70-mer) and FXIa generation was quantified by measuring S2366 hydrolysis. The effect of HD1 and HD22, DNA aptamers that bind specifically to exosite 1 and 2 on thrombin, respectively, on these reactions was then determined.
Results: Whereas 70-mer polyP accelerated the rate of FXI activation by thrombin up to 1500-fold in a bell-shaped and concentration-dependent manner, shorter polyP polymers did not, suggesting that the template effect of polyP requires more than 20 phosphate units. In the presence of 70-mer polyP, HD1 and HD22 saturably attenuated polyP catalyzed FXI activation by thrombin by 50% and 70%, respectively. In contrast, in the absence of polyP, HD1 had no effect on FXIa activation, suggesting that HD1 is unlikely to sterically inhibit FXI binding to thrombin. With 70-mer polyP, the maximal activation of FXI by K109E/K110E thrombin was 30% less than that with α-thrombin.
Conclusions: When composed of at least 70 phosphate units, polyP serves as a template and binds FXI and thrombin and promotes their interaction. HD22 attenuates the reaction because exosite 2 mediates the interaction of thrombin with polyP. The attenuating effect of HD1 likely reflects allosteric modulation of exosite 2 and/or the active site. These findings suggest that allosteric modulation of thrombin’s exosites may represent a novel strategy for downregulating FXI activation.
To cite this abstract in AMA style:Yin R, Patel V, Ajewole E, Fredenburgh J, Weitz J. Allosteric Modulation of Exosite 1 Attenuates the Enhancing Effect of Polyphosphate on FXI Activation by Thrombin [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/allosteric-modulation-of-exosite-1-attenuates-the-enhancing-effect-of-polyphosphate-on-fxi-activation-by-thrombin/. Accessed May 6, 2021.
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