Polymeric Polyphosphate Inhibitors as Novel Antithrombotics

Chanel C. La (Centre for Blood Research and Department of Chemistry, University of British Columbia)
Srinivas Abbina (Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British)
Sreeparna Vappala (Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British)
Manu Thomas Kalathottukaren (Centre for Blood Research and Department of Pathology and Laboratory Medicine)
James H. Morrissey (Departments of Biological Chemistry )
Charles A. Haynes (Michael Smith Laboratories and Department of Chemical and Biological Engineering)
Jayachandran N Kizhakkedathu (Centre for Blood Research, Department of Chemistry and Department of Pathology and Laboratory Medici)

Introduction

Inorganic polyphosphates (polyP) are highly anionic macromolecules secreted by activated human platelets. In recent years, polyP have been shown to play an important role in the blood clotting system as a procoagulant. Although the mechanisms behind the ability of polyP to enhance clotting are still being investigated and understood, it is clear that the polyP contributes heavily to the acceleration of blood coagulation via multiple pathways, serving as an appealing target in the design of a novel antithrombotic agent. By targeting polyP instead of a key component of the coagulation system, this could lower the risk of bleeding associated with anticoagulant therapy unlike current anticoagulants. This presents a polyP specific neutralizing agent as a promising antithrombotic therapeutic.

We have designed and synthesized a library of cationic polymer based polyphosphate inhibitors, with the objective of developing a biocompatible and effective antithrombotic agent.

Materials and Methods

Polymeric structures were synthesized and characterized by gel permeation chromatography (GPC) for polymer size and distribution, nuclear magnetic resonance (NMR) for structural determination, and conductometric titrations for the determination of charge density. In our initial studies, a serine protease assay was used to demonstrate the inhibition of procoagulant activity of polyphosphate (long chain polyP, 650 monomer units) by the newly synthesized polyP inhibitors (n = 3 biological replicates, human pooled plasma, 20 donors). Protonation of polyP inhibitors at physiological pH (7.4) were observed using potentiometry and thermodynamic binding properties of the polyP inhibitor and polyP (short chain polyP, 75 monomer units) were studied using isothermal calorimetry (ITC).

Results

Three main parameters were compared in the identification of an optimal binding partner for polyP: (1) polymer size, (2) charge separation and (3) total charge. Using the serine protease assay as a preliminary screening tool, polyP inhibition activity was compared for inhibitors with different molecular weight and charge density. Next, a varying amount of separation between charges (ethylene vs. propylene spacer) was compared on the same size polymer with similar total charge. Finally, effect of total charge content on each polymer size was identified to optimize the design.

Discussion and Conclusion

Overall, the effects of three parameters in the design of a polymeric polyP inhibitor have been assessed. As we have studied the binding affinity and activity of the polymer and now understand how these can be affected by the selective design process, further optimizations can be made to this model of polymer therapeutic design to generate novel inhibitors for polyP. Findings of this work have demonstrated an optimistic outlook on the synthesis of a safe and effective novel antithrombotic.

References

Smith, S. A.; Mutch, N. J.; Baskar, D.; Rohloff, P.; Docampo, R.; Morrissey, J. H. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (4), 903–908.

Smith, S. A.; Choi, S. H.; Collins, J. N. R.; Travers, R. J.; Cooley, B. C.; Morrissey, J. H. Blood 2012, 120 (26), 5103–5110.

Müller, F.; Mutch, N. J.; Schenk, W. A.; Smith, S. A.; Esterl, L.; Spronk, H. M.; Schmidbauer, S.; Gahl, W. A.; Morrissey, J. H.; Renné, T. Cell 2009, 139 (6), 1143–1156.

Travers, R. J.; Shenoi, R. A.; Kalathottukaren, M. T.; Kizhakkedathu, J. N.; Morrissey, J. H. Blood 2014, 124 (22), 3183–3190.

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