Allosteric modulation of exosite 1 attenuates polyphosphate-catalyzed activation of factor XI by thrombin

Background: Polyphosphate (polyP) promotes feedback activation of factor (F) XI by thrombin by serving as a template. The contribution of thrombin's exosites to these interactions is unclear.

Objectives: To determine the contribution of thrombin exosites 1 and 2 to polyP-induced potentiation of FXI activation by thrombin.

Methods: The affinities of α-thrombin; K109E/110E-thrombin, an exosite 1 variant, or R93E-thrombin, an exosite 2 variant; FXI; and FXIa for polyP-70 were quantified using surface plasmon resonance in the absence or presence of exosite ligands. FXI was activated with α-thrombin or thrombin variants in the absence or presence of polyP-70 and exosite ligands.

Results: α-Thrombin, K109/110E-thrombin, FXI, and FXIa bound polyP-70, whereas R93E-thrombin exhibited minimal binding. Exosite 1 and exosite 2 ligands attenuated thrombin binding to polyP-70. PolyP-70 accelerated the rate of FXI activation by α-thrombin and K109E/110E-thrombin but not R93E-thrombin up to 1500-fold in a bell-shaped, concentration-responsive manner. Exosite 1 and exosite 2 ligands had no impact on FXI activation by thrombin in the absence of polyP-70; however, in its presence, they attenuated activation by 40% to 65%.

Conclusion: PolyP-70 binds FXI and thrombin and promotes their interaction. Exosite 2 ligands attenuate activation because thrombin binds polyP-70 via exosite 2. Attenuation of FXI activation by exosite 1 ligands likely reflects allosteric modulation of exosite 2 and/or the active site of thrombin because exosite 1 is not directly involved in FXI activation. Therefore, allosteric modulation of thrombin's exosites may represent a novel strategy for downregulating FXI activation.