A Polymer Therapeutic Having Universal Heparin Reversal Activity: Molecular Design and Functional Mechanism

Abstract

Heparins are widely used to prevent blood clotting during surgeries and for the treatment of thrombosis. However, bleeding associated with heparin therapy is a concern. Protamine, the only approved antidote for unfractionated heparin (UFH) could cause adverse cardiovascular events. Here, we describe a unique molecular design used in the development of a synthetic dendritic polycation named as universal heparin reversal agent (UHRA), an antidote for all clinically used heparin anticoagulants. We elucidate the mechanistic basis for the selectivity of UHRA to heparins and its nontoxic nature. Isothermal titration calorimetry based binding studies of UHRAs having different methoxypolyethylene glycol (mPEG) brush structures with UFH as a function of solution conditions, including ionic strength, revealed that mPEG chains impose entropic penalty to the electrostatic binding. Binding studies confirm that, unlike protamine or N-UHRA (a truncated analogue of UHRA with no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity toward heparins through a combined steric repulsion and Donnan shielding effect (a balance of F and F). Clotting assays reveal that UHRA with mPEG chains did not adversely affect clotting, and neutralized UFH over a wide range of concentrations. Conversely, N-UHRA and protamine display intrinsic anticoagulant activity and showed a narrow concentration window for UFH neutralization. In addition, we found that mPEG chains regulate the size of antidote-UFH complexes, as revealed by atomic force microscopy and dynamic light scattering studies. UHRA molecules with mPEG chains formed smaller complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes to neutralize heparin's activity.

The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways

“…With respect to safety aspects, the antidote situation for heparin therapy using protamine sulfate as the sole approved compound is not satisfactory with respect to inactivity towards LMWH and fondaparinux and the potential to cause severe adverse reactions in patients. However, recent efforts were reported to potentially replace protamine by other more effective heparin-binding copolymers [ 35 , 36 , 37 ], but these approaches are mainly at a preclinical level.…”

A supramolecular host–guest complex for heparin binding and sensing

“…Similar effect of concentration and number of charges on neutralization efficiency has been detected also previously with polymeric heparin binders. [48][49][50] In short, the anti-Xa assay confirms that heparin neutralization is efficient and linearly dose-dependent with hosts P10+ and R16+. Moreover, at higher host concentrations, full neutralization of heparin is essentially achieved with P10+.…”

Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups

“…To validate the key finding in the simulation studies that binding affinity was not influenced by the length of the PEG chain, we synthesized two of the cationic PEG-based molecules studied by simulation. m -PEG epoxides-350 and 550 were synthesized as reported in the literature 21 Those epoxides were independently refluxed with tris(2-aminoethyl)amine (TREN) and purified by precipitation from DCM and cold pentane. The aminated m -PEGs were methylated by Eschweiler–Clarke reaction (Scheme 1) to generate compounds II (PEG 8 -R 1 (studied in simulation system 3)) and III (PEG 12 -R 1 (simulation system 2)), and the purity of the compounds was confirmed by 1 H NMR.…”

“…Synthesis of m -PEG epoxide-350 21 and 550 21 was followed as reported in literature. 21 See supporting information for NMR spectra (Figures S23 to S26).…”

“…19–21 The basic chemistry used to synthesize these molecules is highly flexible, offering the ability to change molecule size, ligand type and density, and PEG length and grafting density. While the cationic ligands, with their spacing and the properties of the PEG brush layer used within UHRA, make it highly effective in binding and neutralizing the polyanion heparin 21 , they are not designed to specifically bind polyP, as both the rigidity and charge spacing of the polyP polyanion differ from those of heparin. Changes to both the ligand used and the grafted PEG chain length employed are required to strengthen the binding and specificity to polyP.…”