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Heparin microarray-based specific interaction assay of marine sulfated polysaccharides with antithrombin Ⅲ.
Wang F, Li Q, Zhou H, Liu C, Yu G, Li G.
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Marine sulfated polysaccharides (SPs) are promising candidates for new anticoagulant drug development, necessitating efficient method to evaluate their anticoagulant activity at the molecular level. In this work, a heparin microarray-based competitive strategy was developed to investigate the specific interactions between SPs and antithrombin Ⅲ (AT). The strategy is based on the principle that SPs competitively bind to the active domain of AT, reducing the signal of AT binding to immobilized heparin on microarrays. Validation was performed using established anticoagulants heparin and enoxaparin as model analytes. After optimizing key experimental conditions, the method successfully determined the IC50 values for three SPs: fucoidan derived from Ascophyllum nodosum (AnF, 50.55 ± 2.79 μg‧mL-1), fucosylated chondroitin sulfate derived from Holothuria tubulosa (FCSht, 44.18 ± 4.05 μg‧mL-1), and its selectively degraded sulfation product (S-dFCSht, 16.99 ± 6.56 μg‧mL-1). The reliability of these results was confirmed by surface plasmon resonance assay. The strategy's versatility was further demonstrated by assessing SP interactions with side effect-related proteins, providing valuable insights into both efficacy and safety profiles. Although currently applicable primarily to heparin-interacting anticoagulant targets, this strategy can be extended to other targets by fabricating specialized glycan microarrays, enabling comprehensive evaluation of SP interactions with various anticoagulant targets.
31900921;32371338 National Natural Science Foundation of China (National Science Foundation of China), ZR2018BH042 Natural Science Foundation of Shandong Province (Shandong Provincial Natural Science Foundation), TSQN20210304,TSPD202408073 Taishan Scholar Project of Shandong Province
Scheme 1. The heparin microarray-based competitive strategy for investigating the interactions between SPs and AT.
Fig. 1. Heparin microarray-based competitive strategy for investigating SPs and AT specific interactions was feasible.Fluorescence images of carbohydrate microarray containing heparin, dextran, and water spots before (a1, b1, and c1) and after recognition with AT-His tag/Ab-His tag-FITC (a2), Ab-His tag-FITC (b2), or NA-His tag/Ab-His tag-FITC (c2). The images were in pseudocolor, and the default color was set according to the wavelength of excitation laser. d AT binding ratio on heparin microarrays after AT preincubating with heparin, enoxaparin, or dextran solutions at different concentrations. e IC50 values of heparin or enoxaparin on AT-immobilized heparin binding events. The data were expressed as means ± standard deviation (SD) and determined by t-test. NS not significant; **P < 0.01.
Fig. 2. Key conditions for heparin microarray-based competitive strategy for investigating SP-AT interactions.a Fluorescence images (inset) and fluorescence intensity of heparin at different spotting concentrations binding with AT-His tag (50 μg‧mL−1) then labelled with Ab-His tag-FITC (100 μg‧mL−1). b Fluorescence images (inset) and fluorescence intensity of immobilized heparin binding with AT-His tag at different concentrations then labelled with Ab-His tag-FITC (100 μg‧mL−1). c Fluorescence images (inset) and fluorescence intensity of immobilized heparin binding with AT (50 μg‧mL−1) then labelled with Ab-His tag-FITC at different concentrations. d Fluorescence images (inset) and fluorescence intensity of immobilized heparin binding with remaining AT-His tag then labelled with Ab-His tag-FITC after AT-His tag preincubating with heparin solution (50 μg‧mL−1) for different time.
Fig. 3. The investigation of binding potency between marine SPs and AT by adopting heparin microarray-based competitive strategy.a Relationship of AT binding ratio on heparin microarrays with the concentrations of marine SP (AnF, FCSht, and S-dFCSht) that preincubated with AT. b IC50 values for heparin, AnF, FCSht, and S-dFCSht on binding events of AT and immobilized heparin on microarrays. c IC50 values for enoxaparin, AnF, FCSht, and S-dFCSht on binding events of AT and immobilized heparin on microarrays. The data were expressed as means ± SD and determined by t-test. NS not significant; **P < 0.01.
Fig. 4. SPR analysis of the affinity between SPs and AT.The typical SPR sensing curves (solid lines) and fitting curves (dotted lines) of heparin (a), enoxaparin (b), AnF (c), FCSht (d), and S-dFCSht (e) at different concentrations binding with AT. KD values for heparin-AT, enoxaparin-AT, AnF-AT, FCSht-AT, and S-dFCSht-AT specific interaction (f, g). The data were expressed as means ± SD and determined by t-test. NS not significant; **P < 0.01, ***P < 0.001.
Fig. 5. The heparin microarray-based competitive strategy used for evaluating SP-induced side effects.Fluorescence images of carbohydrate microarray containing heparin, dextran, and water spots before (a1, b1) and after recognition with PF4-His tag/Ab-His tag-FITC (a2), and Ab-His tag-FITC (b2). The images were in pseudocolor, and the default color was set according to the wavelength of excitation laser. c PF4 binding ratio on heparin microarrays after PF4 preincubating with heparin, enoxaparin, and dextran solutions at different concentrations. d IC50 for heparin, enoxaparin, AnF, FCSht, and S-dFCSht on binding event of PF4 and immobilized heparin. The data were expressed as means ± SD and determined by t-test., **P < 0.01, ***P < 0.001, and ****P < 0.0001.
