PB0543 - Like heparin, polyphosphate promotes the formation of a ternary complex with thrombin and fibrin

Background: Heparin binds thrombin and fibrin and promotes the formation of a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin. Polyphosphate (polyP) is a procoagulant polyanion released from activated platelets that, like heparin, binds thrombin and fibrin(ogen).

Aims: To determine whether polyP promotes thrombin binding to fibrin, thereby forming a ternary complex that protects thrombin from inhibition by antithrombin.

Methods: The polyP-fibrinogen interaction was quantified using surface plasmon resonance, whereas the binding of thrombin to fibrin in the absence or presence of polyP or heparin was quantified by measuring thrombin activity in clot supernatants. The protection of bound thrombin from inhibition by the antithrombin-heparin complex was evaluated by monitoring thrombin activity, while the procoagulant activity of fibrin-bound thrombin was quantified by incubation with FITC-fibrinogen and monitoring fibrin accretion.

Results: PolyP binds fibrinogen with a Kd of 108 nM. PolyP and heparin promote thrombin binding to fibrin clots by 48% and 30%, respectively (Figure). Thrombin binds fibrin with a Kd of 5.6 µM in the absence of a polyanion; the affinity is 3- and 14-fold higher in the presence of polyP or heparin, respectively (Table). The heparin-catalyzed rate of thrombin inhibition by antithrombin is 14-fold lower in the presence of fibrin than in its absence. In contrast, with polyP, the rate of inhibition is 3-fold faster with fibrin than without it. Whereas fibrin-bound thrombin promotes fibrin accretion in the presence of heparin, there is no promotion of fibrin accretion in the presence of polyP.

Conclusion(s): Like heparin, polyP binds thrombin and fibrin(ogen) and promotes thrombin binding to fibrin. Whereas thrombin assembled within the heparin-thrombin-fibrin complex is protected from inhibition by antithrombin and is procoagulant, thrombin within the polyP-thrombin-fibrin is not. These findings identify important differences in the mechanisms and consequences of polyanion interactions with thrombin and fibrin.