Anticoagulant therapy associated bleeding and pathological thrombosis, pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit newly identified activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids (ecNA). The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described inhibitors of polyP and ecNAs, exhibit both non-specific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation, protamine used to counteract heparin-associated bleeding exhibit adverse side-effects. To address unmet clinical need, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. To understand the contribution of UHRA design towards activity and nontoxicity, we synthesized UHRAs having different methoxy polyethylene glycol (mPEG) brush structures. Isothermal titration calorimetry based binding of UHRAs with heparin at different conditions revealed unique contributions of mPEG chains on the molecule. Binding studies confirm that unlike protamine or N-UHRA (UHRA analog with no mPEG chains) which follows classic polyelectrolyte theory, the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity towards heparins through a combined steric repulsion and Donnan shielding effect. In support to this observation, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization or abrogate plasma clotting. Using electron microscopy, confocal microscopy and clot lysis assays, we confirm that UHRA does not incorporate into clots, and clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury as seen with protamine.

Molecular design of non-toxic polymeric inhibitors as novel anti-thrombotics and antidotes for anticoagulants

254th National Meeting (2017)

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