Formation of a ternary polyphosphate-thrombin-fibrin complex attenuates thrombin activity

Polyphosphate (polyP) released from activated platelets acts as a procoagulant, promoting thrombin generation and fibrin formation. Like heparin, polyP binds to thrombin and fibrin(ogen).

To determine whether, like heparin, polyP promotes thrombin binding to fibrin, thereby forming a ternary polyP-thrombin-fibrin complex that modulates thrombin activity.

The affinity of fluorescein isothiocyanate-labeled polyP or heparin for fibrin was determined by measuring unbound polyanion in the supernatants of fibrin clots. Likewise, the affinity of thrombin for fibrin in the absence or presence of polyanion was determined by quantifying unbound thrombin by chromogenic assay. Rate constants of thrombin inhibition by antithrombin-heparin were determined in the absence or presence of polyanion and fibrin. Clots formed with polyanions were incubated with labeled fibrinogen to monitor clot accretion.

PolyP and heparin bind fibrin with Kd values of 1.7 ± 0.1 μM and 1.1 ± 0.1 μM, respectively. In the presence of polyP or heparin, thrombin binds to fibrin with Kd values of 1.2 ± 0.2 μM or 0.9 ± 0.1 μM, respectively, whereas the Kd is 5.6 ± 0.4 μM in the absence of a polyanion. Thrombin within the ternary polyP-thrombin-fibrin complex is protected from inhibition by the antithrombin-heparin complex but has reduced capacity to induce fibrin accretion.

Like heparin, polyP promotes the formation of a ternary polyP-thrombin-fibrin complex, which protects thrombin from inhibition by antithrombin-heparin. However, the ternary complex reduces the ability of clot-bound thrombin to generate fibrin. These findings highlight the interplay among polyP, thrombin, and fibrin in regulating coagulation.