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. 2019 Jan 23;9(6):3020-3029.
doi: 10.1039/c8ra09724k. eCollection 2019 Jan 22.

The neutralization of heparan sulfate by heparin-binding copolymer as a potential therapeutic target

Bartlomiej Kalaska  1 , Joanna Miklosz  1 , Kamil Kamiński  2 , Bogdan Musielak  2 , Shin-Ichi Yusa  3 , Dariusz Pawlak  1 , Maria Nowakowska  2 , Krzysztof Szczubiałka  2 , Andrzej Mogielnicki  1
Affiliations

The neutralization of heparan sulfate by heparin-binding copolymer as a potential therapeutic target

Bartlomiej Kalaska et al. RSC Adv. .

Abstract

Besides regulating ligand-receptor and cell-cell interactions, heparan sulfate (HS) may participate in the development of many diseases, such as cancer, bacterial or viral infections, and their complications, like bleeding or inflammation. In these cases, the neutralization of HS could be a potential therapeutic target. The heparin-binding copolymer (HBC, PEG41-PMAPTAC53) was previously reported by us as a fully synthetic compound for efficient and safe neutralization of heparins and synthetic anticoagulants. In a search for molecular antagonists of HS, we examined the activity of HBC as an HS inhibitor both in vitro and in vivo and characterized HBC/HS complexes. Using a colorimetric Azure A method, isothermal titration calorimetry and dynamic light scattering techniques we found that HBC binds HS by forming complexes below 200 nm with less than 1 : 1 stoichiometry. We confirmed the HBC inhibitory effect in rats by measuring activated partial thromboplastin time, prothrombin time, anti-factor Xa activity, anti-factor IIa activity, and platelet aggregation. HBC reversed the enhancement of all tested parameters caused by HS demonstrating that cationic synthetic block copolymers may have a therapeutic value in various disorders involving overproduction of HS.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Anticoagulant properties of heparan sulfate (HS) in rat plasma. Effects of HS were monitored by measuring activated partial thromboplastin time (aPTT, A), prothrombin time (PT, B), anti-factor Xa (anti-fXa, C) and anti-factor IIa (anti-fIIa) activity (D). Results are shown as median values with lower and upper limits.
Fig. 2
Fig. 2. The chemical structure of heparin-binding copolymer (HBC).
Fig. 3
Fig. 3. The binding of heparan sulfate (HS) by heparin-binding copolymer (HBC) in phosphate buffer solution (PBS).
Fig. 4
Fig. 4. Neutralization of heparan sulfate (HS) by heparin-binding copolymer (HBC) in rat plasma. Neutralization was monitored in vitro by measuring activated partial thromboplastin time (aPTT, A), prothrombin time (PT, B), anti-factor Xa (anti-fXa) activity (C), and anti-factor IIa (anti-fXa) activity (D). Results are shown as median values with lower and upper limits. Dotted lines indicate normal values for coagulation parameters of rat plasma.
Fig. 5
Fig. 5. Neutralization of heparan sulfate (HS) (6 mg kg−1, 1 mL kg−1) by heparin-binding copolymer (HBC) (4.5 mg kg−1, 1 mL kg−1 by weight) in Wistar rats. Neutralization was assessed by measuring activated partial thromboplastin time (aPTT, A), prothrombin time (PT, B), anti-factor Xa activity (anti-fXa, C), and anti-factor IIa activity (anti-fXa, D). ***p < 0.001, unpaired Student's t-test. Results are shown as mean ± SD.
Fig. 6
Fig. 6. Neutralization of heparan sulfate (HS) (6 mg kg−1, 1 mL kg−1) by heparin-binding copolymer (HBC) (4.5 mg kg−1, 1 mL kg−1) in Wistar rats. Neutralization was assessed by measuring platelet aggregation expressed as the maximal extension (MaxA, A), the slope of platelet aggregation (Slp, B), lag time (Lag, C), and area under the curve (AUC, D) in rats. *p < 0.05, unpaired Student's t-test. Results are shown as mean ± SD.
Fig. 7
Fig. 7. The size distribution of heparin-binding copolymer-heparan sulfate (HBC–HS) complexes measured by dynamic light scattering (DLS) analysis. The concentrations of HS and HBC were 0.4 mg mL−1 and 12 mg mL−1, respectively.
Fig. 8
Fig. 8. The estimated heat of binding for the titration of heparan sulfate (HS) with heparin-binding copolymer (HBC) measured by isothermal titration calorimetry (ITC) method.
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