Methods
General Chemistry Methods. All reactions were carried out using commercial grade reagents and solvents. NMR chemical shifts (δ) are reported in parts per million (ppm).
The purity of the oligosaccharide components were determined by HPGPC equipped with a TSKgel G2000SW XL column (7.5 mm × 300 mm) from TOSH Bioscience. The elution conditions for this HPGPC analysis have been described previously.(Shang, Gaoet al. , 2018; Zhao, Wu et al. , 2015) The columns were eluted with carefully degassed 0.2 M sodium chloride solution at a flow rate of 0.4 mL/min. The HPLC series were equipped with a refractive index detector (RID) and diode-array detector (DAD), and the DAD was set at 234 nm.
The Mw of dHG-5 and its distribution were determined by HPGPC with the TSK G2000SWXL column. The five homogeneous oligosaccharides (oHG-5, oHG-8, oHG-11, oHG-14 and oHG-17) were used as standard samples, their peak molecular weights were obtained from the MS. Oligosaccharide retention times - peak molecular weights curve was fitted by third-order polynomial using GPC software, and the Mw of dHG-5 was calculated using the same GPC software.
The oligosaccharide structures were characterized by1H /13C NMR and 2D NMR spectroscopy, which was performed in D2O at 298 K on a Bruker 800 MHz spectrometer with Topspin 3.2 software, as previously described.(Shang, Gao et al. , 2018) All spectra were recorded with HOD suppression by presaturation. The purified oligosaccharide samples (15-25 mg) were dissolved in 1.0 mL 99.9% D2O (Sigma) and lyophilized three times to remove the exchangeable protons. Finally, the samples were re-dissolved in 0.5 mL D2O and transferred to NMR microtubes (outside diameter, 5 mm, Norrell).
The 1D (1H /13C) and 2D (1H-1H COSY, TOCSY, NOESY, and1H-13C HSQC, HMBC) NMR spectra were recorded under conditions described earlier (Cai, Yang et al. , 2019) with minor modifications and processed using a trial MestReNova v9.0.1-13254 software.
The MS spectra analysis was conducted on a micrOTOF-QII mass spectrometer (Bruker Daltonic, Germany) equipped with an electrospray ion source (ESI). The fractionated and desalted samples were dissolved in 100% pure water and introduced into an ion source at a flow rate of 5 μL/min. The MS spectrometric conditions were as follows: ESI in negative ionization mode, capillary voltage of 2500 V, nebulizer pressure of 0.8 bar, drying gas flow rate of 5.0 L/min, and drying gas temperature of +180 °C. The mass spectra of the oligosaccharides were acquired in scan mode (m/z scan range 400-3000). Data analysis was performed using Bruker Compass Data-Analysis 4.0 software (Bruker Daltonic, Germany).
Preparation of dHG-5. The native FG (HPLC purity 99.9%; average molecular mass 45 kDa) was isolated and purified from the sea cucumberHolothuria fuscopunctata as previously described.(X. M. Li, Luoet al. , 2017) Briefly, the 30 kg dried body wall of the sea cucumber was digested by papain (EC 3.4.22.2) and the polysaccharide components were released by 0.25 M sodium hydroxide. The crude polysaccharide was obtained by repeated salting-out with KOAc and precipitation by ethanol, which was further purified by strong anion-exchange chromatography using FPA98 resin. Finally, about 246 g of native FG was obtained with 0.8 % yield and further used for chemical cleavage to prepare its low molecular weight fragments.
The reaction principle was based on a β-eliminative cleavage of FG through its activated benzyl ester derivative according to previously research.(Na Gao, Lu et al. , 2015) Approximately 130 g of sodium FG was dissolved in 1.9 L of distilled water and completed transalification with benzethonium salts. FG benzethonium salts (325 g) were obtained by precipitation and centrifugation and dried under vacumm condition at room temperature. The FG benzethonium salts were dissolved in 1.6 L N, N-dimethyl formamide (DMF) and esterified by 32 mL benzyl chloride (~2 equals of carboxyl groups in native FG) under continually stirring at 35 °C for 24 h. Then the FG in the DMF solution was cooled to 25°C and depolymerized by adding 550 mL freshly prepared 0.08 M EtONa in ethanol. About 2.2 L saturated sodium chloride solution and 24 L of ethanol were added to the reaction solution successively to complete the transalification of benzethonium salt to sodium salts. Particularly, the saponification procedure in alkaline solution was necessary to hydrolyze the residual benzyl esters, and the reducing ends were reduced to its alcoholic hydroxyl by NaBH4. The depolymerized product was isolated using tangential flow filtration on a Pellicon Mini device equipped with a 0.1 m2 PLCTK membrane with a molecular weight cutoff (MWCO) of 3 kDa (Millipore) or 10 kDa. The fraction with Mw higher than 3 kDa and lower than 10 kDa were collected and freeze-dried, finally ~30 g dHG-5 was obtained.
Purification of oligosaccharides from dHG-5. The oligosaccharides contained in dHG-5 were fractionated by GPC on a column packed with Bio-gel P6 (fine, 2×150 cm, Bio-Rad) or Bio-gel P10 (medium, 2×200 cm, Bio-Rad). dHG-5 (0.8 g) was dissolved in 10 mL of deionized water, subjected to the Bio-Gel P10 column equilibrated well by 0.2 M NaCl and then eluted with the same solution. The flow rate was approximately 12 mL/h and 100 fractions (2 mL/tube) were collected. Absorbance of each fraction were measured at 234 nm. The oligosaccharide fractions were collected and then desalted by a column packed with Sephadex G-10 (1.5×100 cm). The elution volume - absorbance was plotted to show the oligosaccharide distribution in eluted fractions, those containing the uniform oligosaccharide were pooled (as detected by HPGPC) and lyophilized after repeated fractionating. Five sized homogeneous oligosaccharides from dHG-5 were desalted on a Bio-Gel P-2 column (fine, 1.5 ×100 cm, Bio-Rad) and were freeze-dried, the obtained white powders were compound oHG-5 (pentasaccharide), oHG-8 (octasaccharide), oHG-11 (hendecasaccharide), oHG-14 (tetradecasaccharide) and oHG-17 (heptadecasaccharide). Similarly, another four larger components were obtained and identified as oHG-20 (eicosasaccharide), oHG-23 (tricosasaccharide), oHG-26 (hexacosasaccharide) and oHG-29 (nonacosasaccharide).
oHG-5, pentasaccharide,L-Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-α(1,] 3)-D-GlcA-ol:1H NMR (800 MHz, D2O) δ 5.693 (1H, d, H∆U-4, J (3,4) = 2.40 Hz), 5.201 (1H, d, HdF-1, J (1,2) = 3.78 Hz), 5.039 (1H, d, HrF-1,J (1,2) = 3.96 Hz), 4.918 (1H, m, HA-4, J (3,4) = 2.16 Hz), 4.859 (1H, d, H∆U-1, J (1,2) = 8.64 Hz), 4.831 (1H, m, HrF-4, J (3,4) = 2.76 Hz), 4.831 (1H, m, HdF-4,J (3,4) = 2.82 Hz), 4.639 (1H, d, HA-1, J (1,2) = 8.52 Hz), 4.546 (1H, dd, HrF-3, J (3,4) = 2.76 Hz), 4.529 (1H, dd, HdF-3, J (2,3)= 10.38 Hz, J (3,4) = 2.82 Hz), 4.418 (1H, dd, H∆U-3, J (2,3) = 7.56 Hz,J (3,4) = 2.82 Hz), 4.363 (1H, m, HrF-5, J (5,6) = 6.84 Hz), 4.273 (1H, m, HdF-5, J (5,6) = 6.48 Hz), 4.268 (1H, m, HA-6, J (6,6’) = 10.68 Hz), 4.256 (1H, d, HrU-5,J (4,5) = 7.44 Hz), 4.167 (1H, dd, HA-3, J (2,3) = 8.70 Hz,J (3,4) = 2.16 Hz), 4.146 (1H, m, HA-6’, J (5,6’) = 3.52 Hz), 4.071 (1H, dd, HrU-4, J (4,5) = 7.44 Hz), 4.060 (1H, dd, HrU-2, J (1,2)= 3.96 Hz, J (1’,2) = 6.78 Hz), 4.043 (1H, dd, HA-2, J (1,2) = 8.52 Hz,J (2,3) = 8.70 Hz), 4.019 (1H, m, HA-5, J (5,6/6’) = 8.24 Hz, 3.52 Hz), 4.019 (1H, dd, HrU-3), 3.886 (1H, dd, HdF-2, J (1,2) = 3.78 Hz,J (2,3) = 10.34 Hz), 3.886 (1H, dd, HrF-2, J (2,3) = 10.38 Hz), 3.822 (1H, dd, H∆U-2, J (1,2) = 8.64 Hz,J (2,3) = 7.56 Hz), 3.743 (1H, d, HrU-1, J (1,2) = 3.96 Hz,J (1,1’) = 11.82 Hz), 3.700 (1H, d, HrU-1’, J (1’,2) = 6.78 Hz), 1.986 (3H, s, HA-8), 1.240 (3H, d, HrF-6,J (5,6) = 6.84 Hz), 1.231 (3H, d, HdF-6, J (5,6) = 6.32 Hz);13C NMR (200 MHz, D2O) δ 180.08 (CrU-6), 177.65 (CA-7), 171.65 (C∆U-6), 149.51 (C∆U-5), 109.52 (C∆U-4), 105.82 (C∆U-1), 104.31 (CA-1), 104.26 (CrF-1), 101.04 (CdF-1), 84.36 (CrU-4), 82.44 (CrU-3), 81.70 (CrF-4), 81.72 (CdF-4), 79.12 (C∆U-3),78.94 (CA-4), 78.56 (CA-3), 75.17 (CrU-5), 74.64 (CA-5), 72.90 (C∆U-2), 72.59 (CrU-2), 70.64 (CA-6), 69.55 (CrF-5), 69.39 (CrF-2), 69.23 (CdF-5), 69.02 (CdF-2), 65.28 (CrU-1), 54.35 (CA-2), 25.19 (CA-8), 18.85 (CrF-6), 18.56 (CdF-6); HRMS (ESI-Q-TOF) m/z: [M-2Na]2- calculated for C32H43O43NS6Na6729.4465; Found for 729.4412.
oHG-8, octasaccharide ,L-Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-β(1,3)-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-(α1,] 3)-D-GlcA-ol: 1H NMR (800 MHz, D2O) δ5.686 (1H, d, H∆U-4), 5.286 (1H, d, HF-1,J (1,2) = 3.72 Hz), 5.204 (1H, d, HdF-1, J (1,2) = 3.78 Hz), 5.033 (1H, d, HrF-1, J (1,2) = 3.84 Hz), 4.973 (1H, d, HF-4, J (3,4) = 2.46 Hz), 4.912 (1H, m, HdA-4), 4.846 (1H, d, H∆U-1, J (1,2) = 8.28 Hz), 4.831 (1H, d, HdF-4, J (3,4) = 2.82 Hz), 4.837 (1H, d, HrF-4, J (3,4) =3.06 Hz), 4.799 (1H, m, HF-5, J (5,6) = 6.36 Hz), 4.622 (1H, d, HrA-1), 4.545 (1H, d, HrF-3, J (3,4) = 3.06 Hz), 4.530 (1H, d, HdA-1, J (1,2) = 8.52 Hz), 4.530 (1H, d, HdF-3, J (3,4) = 2.82 Hz), 4.458 (1H, d, HF-3,J (3,4) = 2.46 Hz), 4.426 (1H, dd, H∆U-3, J (2,3) = 8.22 Hz,J (3,4) = 2.40 Hz), 4.426 (1H, d, HU-1, J (1,2) = 8.16 Hz), 4.370 (1H, m, HrF-5, J (5,6) = 6.84 Hz), 4.532 (1H, d, HdA-6,J (1,2) = 8.16 Hz), 4.418 (1H, dd, HdF-5, J (1,2) = 3.92 Hz,J (2,3) = 10.60 Hz), 4.321 (1H, m, HrA-6, J (6,6’) = 12.06 Hz), 4.129 (1H, m, HrA-6’, J (6,6’) = 10.68 Hz), 4.090 (1H, dd, HdA-2,J (1,2) = 8.52 Hz), 4.087 (1H, dd, HdA-3), 4.066 (1H, m, HrU-2, J (1,2) = 3.96 Hz), 4.012 (1H, m, HrU-3), 4.012 (1H, m, HrU-4), 4.022 (1H, m, HrA-5, J (5,6) = 8.24 Hz), 4.019 (1H, m, HdA-5), 3.976 (1H, m, HrA-2, J (1,2) = 7.26 Hz), 3.981 (1H, m, HrA-3), 3.954 (1H, d, HU-4,J (4,5) = 9.42 Hz), 3.886 (1H, dd, HrF-2, J (1,2) = 3.84 Hz,J (2,3) = 10.38 Hz), 3.886 (1H, dd, HdF-2,J (1,2) = 3.78 Hz, J (2,3) = 10.38 Hz), 3.871 (1H, dd, HF-2, J (1,2) = 3.72 Hz, J (2,3) = 10.50 Hz), 3.830 (1H, dd, H∆U-2, J (1,2) = 8.28 Hz,J (2,3) = 8.22 Hz), 3.733 (1H, d, HrU-1,J (1,1’) = 11.88 Hz, J (1,2)= 3.96 Hz), 3.691 (1H, d, HrU-1’,J (1’,2) = 6.84 Hz),3.632 (1H, dd, HU-3, J (2,3) = 9.06 Hz,J (3,4) = 8.61 Hz), 3.641 (1H, d, HU-5, J (4,5) = 9.42 Hz), 3.546 (1H, dd, HU-2, J (1,2) = 8.16 Hz,J (2,3) =9.06 Hz), 1.997 (3H, s, HdA-8), 1.992 (3H, s, HrA-8), 1.343 (3H, d, HF-6, J (5,6) = 6.36 Hz),1.241 (3H, d, HrF-6, J (5,6) = 6.84 Hz), 1.231 (3H, d, HdF-6, J (5,6) = 6.48 Hz); 13C NMR (200 MHz, D2O)δ 180.08 (CrU-6), 177.88 (CU-6), 177.74 (CrA-7, CdA-7), 171.65 (C∆U-6), 149.61 (C∆U-5), 109.44 (C∆U-4), 106.44 (CU-1),105.81 (C∆U-1), 104.17 (CrA-1), 104.17 (CrF-1), 102.39 (CdA-1), 101.97 (CF-1), 100.94 (CdF-1), 84.03 (CrU-4), 82.33 (CrU-3), 82.09 (CF-4), 81.88 (CU-3), 81.68 (CrF-4), 81.69 (CdF-4), 79.86 (CU-5), 79.10 (C∆U-3), 78.98 (CdA-4, CrA-4), 78.55 (CdA-3), 78.07 (CrA-3), 78.07 (CF-3), 77.91 (CrF-3), 77.96 (CdF-3), 77.91 (CU-4), 76.24 (CU-2), 75.03 (CrA-5), 74.62 (CrU-5, CdA-5), 72.96 (C∆U-2), 72.59 (CrU-2), 70.52 (CrA-6), 69.99 (CdA-6), 69.52 (CrF-5), 69.36 (CdF-5), 69.24 (CrF-2), 69.10 (CdF-2), 69.10 (CF-2), 69.00 (CF-5), 65.21 (CrU-1), 54.26 (CrA-2), 54.26 (CdA-2), 25.28 (CrA-8), 25.26 (CdA-8), 18.84 (CrF-6), 18.78 (CF-6), 18.56 (CdF-6); HRMS (ESI-Q-TOF) m/z: [M-3Na]3- calculated for C52H69N2Na10O70S103-796.9367; Found for 796.9317.
oHG-11, hendecasaccharide,L-Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-{D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-β(1,3)-}2-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-(α1,]3)-D-GlcA-ol:1H NMR (800 MHz, D2O) δ 5.686 (1H, d, H∆U-4), 5.277 (1H, d, HF-1,J (1,2) = 4.88 Hz), 5.204 (1H, d, HdF-1, J (1,2) = 3.78 Hz), 5.033 (1H, d, HrF-1, J (1,2) = 3.84 Hz), 4.957 (1H, d, HF-4, J (3,4) = 4.88 Hz), 4.905 (1H, m, HdA-4), 4.842 (1H, d, H∆U-1, J (1,2) = 8.30 Hz), 4.832 (1H, d, HrF-4,J (3,4)=3.06 Hz), 4.826 (1H, d, HdF-4,J (3,4) = 2.82 Hz), 4.783 (1H, m, HF-5, J (5,6) = 6.36 Hz), 4.752(1H, m,HrA-4), 4.750 (1H, HA-4), 4.620 (1H, d, HrA-1, J (1,2) =7.68 Hz), 4.544 (1H, d, HrF-3, J (3,4)= 3.06 Hz), 4.530 (1H, d, HdF-3, J (3,4) = 2.82 Hz), 4.515 (1H, d, HdA/A-1, J (1,2) = 8.52 Hz), 4.498 (1H, d, HA-1, J (1,2) = 8.52 Hz), 4.449 (1H, d, HF-3,J (3,4) = 2.64 Hz), 4.425 (1H, dd, H∆U-3, J (2,3) = 8.16 Hz,J (3,4) = 2.72 Hz), 4.425 (1H, d, HU-1, J (1,2) = 8.16 Hz), 4.354 (1H, m, HrF-5, J (5,6) = 6.64 Hz), 4.315 (1H, d, HdA-6, J (6,6’) =12.06 Hz), 4.283 (1H, dd, HdF-5, J (5,6) = 6.48 Hz), 4.200 (1H, m, HrA-6, J (6,6’)= 10.68 Hz), 4.199 (1H, d, HA-6,J (6,6’) = 12.06 Hz), 4.117 (1H, m, HrA-6’,J (6,6’) = 10.68 Hz), 4.105 (1H, m, HdA/A-6’, J (6,6’) = 12.06 Hz), 4.094 (1H, dd, HdA-2, J (1,2) = 8.52 Hz), 4.082 (1H, dd, HdA-3), 4.064 (1H, m, HrU-2, J (1,2) = 4.00 Hz), 4.013 (1H, m, HA-2, J (1,2) = 8.52 Hz), 4.009 (1H, m, HdA-5,J (5,6’) = 5.34 Hz), 4.006 (1H, m, HrU-3), 4.006 (1H, m, HrU-4), 4.008 (1H, m, HrA-5), 3.993 (1H, m, HrA-2,J (1,2) = 7.68 Hz), 3.980 (1H, m, HrA-3), 3.940 (1H, m, HA-5,J (5,6’) = 5.34 Hz), 3.936 (1H, d, HU-4, J (4,5) = 9.42 Hz), 3.895 (1H, m, HA-3), 3.884 (1H, dd, HrF-2,J (1,2) = 4.08 Hz, J (2,3) = 10.72 Hz), 3.884 (1H, dd, HdF-2,J (1,2) = 4.00 Hz, J (2,3) = 10.38 Hz), 3.862 (1H, dd, HF-2,J (1,2) = 4.88 Hz, J (2,3) = 10.50 Hz), 3.825 (1H, dd, H∆U-2,J (1,2) = 8.30 Hz, J (2,3) = 8.16 Hz), 3.729 (1H, d, HrU-1, J (1,2) = 11.92 Hz), 3.691 (1H, d, HrU-1’, J (1’,2) = 6.72 Hz), 3.632 (1H, d, HU-5, J (4,5) = 9.42 Hz), 3.616 (1H, dd, HU-3,J (2,3) = 8.24 Hz, J (3,4) = 8.61 Hz), 3.542 (1H, dd, HU-2,J (1,2) = 8.16 Hz, J (2,3)=8.24 Hz), 1.998 (3H, s, HrA/A-8), 1.988 (3H, s, HdA-8), 1.328 (3H, d, HF-6,J (5,6) = 6.36 Hz),1.239 (3H, d, HrF-6, J (5,6) = 6.64 Hz), 1.230 (3H, d, HdF-6, J (5,6) = 6.48 Hz);13C NMR (200 MHz, D2O) δ 180.08 (CrU-6), 177.72 (C(r/d)A-7), 177.72 (CU-6), 171.60 (C∆U-6), 149.64 (C∆U-5), 109.38 (C∆U-4), 106.50 (CU-1),105.78 (C∆U-1), 104.16 (CrA-1), 104.16 (CrF-1), 102.33 (CdA/A-1), 101.98 (CF-1), 100.99 (CdF-1), 84.03 (CrU-4), 82.14 (CrU-3), 82.11 (CU-3), 81.95 (CF-4), 81.70 (Cr/dF-4), 79.85 (CU-5), 79.11 (C∆U-3), 78.99 (C(d/r) A-4), 78.78 (C(r/d)A-3), 78.11 (CF-3), 77.98 (CU-4), 77.94 (Cr/dF-3), 76.28 (CU-2), 75.04 (CrA-5), 74.68 (CrU-5), 74.61(C(d)A-5), 73.00 (C(r/d)U-2), 70.53 (CrA-6), 69.85 (CdA-6), 70.03 (CA-6), 69.55 (CrF-5),69.24 (CdF-5), 69.39 (CrF-2), 69.39 (CdF-2), 69.13 (CF-2), 69.02 (CF-5), 65.25 (CrU-1), 54.23 (C(r/d)A-2), 25.29 (C(r/d)A-8), 18.83 (CF-6), 18.79 (CrF-6), 18.56 (CdF-6); HRMS (ESI-Q-TOF) m/z: [M-3Na]3- calculated for C72H95N3Na15O97S143-1115.2388; Found for 1115.2299.
oHG-14, tetradecasaccharide,L-Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-{D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-β(1,3)-}3-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-(α1,]3)-D-GlcA-ol:1H NMR (800 MHz, D2O) δ 5.681 (1H, d, H∆U-4), 5.279 (1H, d, HF-1,J (1,2) = 4.08 Hz), 5.200 (1H, d, HdF-1, J (1,2) = 4.00 Hz), 5.034 (1H, d, HrF-1, J (1,2) = 4.08 Hz), 4.956 (1H, d, HF-4, J (3,4) = 2.64 Hz), 4.905 (1H, m, HdA-4), 4.845 (1H, d, H∆U-1, J (1,2) = 8.30 Hz), 4.832 (1H, d, HrF-4, J (3,4) =4.30 Hz), 4.826 (1H, d, HdF-4, J (3,4) = 2.82 Hz), 4.782 (1H, m, HF-5,J (5,6) = 6.36 Hz), 4.753 (1H, m, HrA-4), 4.754 (1H, HA-4), 4.621 (1H, d, HrA-1, J (1,2) =6.80 Hz), 4.545 (1H, d, HrF-3, J (3,4) = 4.30 Hz), 4.528 (1H, d, HdF-3, J (3,4) = 2.82 Hz), 4.500 (1H, d, HdA/A-1,J (1,2) = 8.52 Hz), 4.492 (1H, d, HA-1, J (1,2) = 8.52 Hz), 4.443 (1H, d, HF-3, J (3,4) = 2.64 Hz), 4.430 (1H, dd, H∆U-3, J (2,3) = 8.16 Hz, J (3,4) = 2.72 Hz), 4.415 (1H, d, HU-1, J (1,2) = 8.16 Hz), 4.355 (1H, m, HrF-5, J (5,6) = 6.56 Hz), 4.313 (1H, d, HdA-6, J (6,6’)=12.06 Hz), 4.282 (1H, dd, HdF-5,J (5,6) = 6.48 Hz), 4.226 (1H, m, HrA-6, J (6,6’) = 10.68 Hz), 4.280 (1H, d, HA-6, J (6,6’) = 12.06 Hz), 4.093 (1H, m, HrA-6’), 4.189 (1H, m, HdA/A-6’, J (6,6’) = 12.06 Hz), 4.078 (1H, dd, HdA-2, J (1,2) = 8.52 Hz), 4.076 (1H, dd, HdA-3), 4.073 (1H, m, HrU-2, J (1,2) = 3.76 Hz), 3.977 (1H, m, HA-2, J (1,2) = 8.52 Hz), 4.009 (1H, m, HdA-5, J (5,6’) = 5.34 Hz), 4.009 (1H, m, HrU-3), 4.009 (1H, m, HrU-4), 4.009 (1H, m, HrA-5), 3.996 (1H, m, HrA-2, J (1,2) = 6.80 Hz), 3.982 (1H, m, HrA-3), 3.934 (1H, m, HA-5, J (5,6’) = 5.34 Hz), 3.935 (1H, d, HU-4, J (4,5) = 9.42 Hz), 3.866 (1H, m, HA-3), 3.886 (1H, dd, HrF-2, J (1,2) = 4.08 Hz,J (2,3) = 10.88 Hz), 3.885 (1H, dd, HdF-2, J (1,2) = 4.00 Hz,J (2,3) = 10.38 Hz), 3.873 (1H, dd, HF-2, J (1,2) = 4.08 Hz, J (2,3) = 10.50 Hz), 3.826 (1H, dd, H∆U-2, J (1,2) = 8.30 Hz,J (2,3) = 8.16 Hz), 3.729 (1H, d, HrU-1, J (1,2) = 11.84 Hz), 3.691 (1H, d, HrU-1’,J (1,1’) = 11.84 Hz, J (1’,2) = 6.80 Hz), 3.632 (1H, d, HU-5, J (4,5) = 9.42 Hz), 3.620 (1H, dd, HU-3, J (2,3) = 8.00 Hz,J (3,4) = 8.64 Hz), 3.540 (1H, dd, HU-2, J (1,2) = 8.16 Hz,J (2,3) =8.00 Hz), 1.998 (3H, s, HrA/A-8), 1.988 (3H, s, HdA-8), 1.323 (3H, d, HF-6, J (5,6) = 6.36 Hz),1.239 (3H, d, HrF-6, J (5,6) = 6.56 Hz), 1.230 (3H, d, HdF-6,J (5,6) = 6.48 Hz); 13C NMR (200 MHz, D2O) δ 180.06 (CrU-6), 177.82 (C(r/d)A-7), 177.72 (CU-6), 171.60 (C∆U-6), 149.65 (C∆U-5), 109.38 (C∆U-4), 106.51 (CU-1),105.78 (C∆U-1), 104.09 (CrA-1), 104.09 (CrF-1), 102.33 (CdA/A-1), 101.97 (CF-1), 101.00 (CdF-1), 84.03 (CrU-4), 82.39 (CrU-3), 82.11 (CU-3), 81.98 (CF-4), 81.73 (Cr/dF-4), 79.75 (CU-5), 79.23 (C∆U-3), 79.15 (C(d/r) A-4), 78.54 (C(r/d)A-3), 78.12 (CF-3), 77.98 (CU-4), 77.98 (Cr/dF-3), 76.29 (CU-2), 75.05 (CrA-5), 74.69 (CrU-5), 74.61(C(d)A-5), 73.01 (Cr/dU-2), 70.53 (CrA-6), 69.89 (CdA-6), 70.03 (CA-6), 69.55 (CrF-5),69.24 (CdF-5), 69.40 (CrF-2), 69.40 (CdF-2), 69.13 (CF-2), 69.03 (CF-5), 65.26 (CrU-1), 54.23 (C(r/d)A-2), 25.29 (C(r/d)A-8), 18.80 (CF-6), 18.83 (CrF-6), 18.56 (CdF-6); HRMS (ESI-Q-TOF) m/z: [M-6Na]6- calculated for C92H121N4Na17O124S186-705.2759; Found for 705.2753.
oHG-17, heptadecasaccharide,L-Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-{D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-β(1,3)-}4-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-(α1,]3)-D-GlcA-ol:1H NMR (800 MHz, D2O) δ 5.681 (1H, d, H∆U-4), 5.279 (1H, d, HF-1,J (1,2) = 4.08 Hz), 5.200 (1H, d, HdF-1, J (1,2) = 4.00 Hz), 5.034 (1H, d, HrF-1, J (1,2) = 4.08 Hz), 4.956 (1H, d, HF-4, J (3,4) = 2.64 Hz), 4.905 (1H, m, HdA-4), 4.845 (1H, d, H∆U-1, J (1,2) = 8.30 Hz), 4.832 (1H, d, HrF-4, J (3,4) =4.30 Hz), 4.826 (1H, d, HdF-4, J (3,4) = 2.82 Hz), 4.782 (1H, m, HF-5,J (5,6) = 6.36 Hz), 4.753 (1H, m, HrA-4), 4.754 (1H, HA-4), 4.621 (1H, d, HrA-1, J (1,2) =6.80 Hz), 4.545 (1H, d, HrF-3, J (3,4) = 4.30 Hz), 4.528 (1H, d, HdF-3, J (3,4) = 2.82 Hz), 4.500 (1H, d, HdA/A-1,J (1,2) = 8.52 Hz), 4.492 (1H, d, HA-1, J (1,2) = 8.52 Hz), 4.443 (1H, d, HF-3, J (3,4) = 2.64 Hz), 4.430 (1H, dd, H∆U-3, J (2,3) = 8.16 Hz, J (3,4) = 2.72 Hz), 4.415 (1H, d, HU-1, J (1,2) = 8.16 Hz), 4.355 (1H, m, HrF-5, J (5,6) = 6.56 Hz), 4.313 (1H, d, HdA-6, J (6,6’)=12.06 Hz), 4.282 (1H, dd, HdF-5,J (5,6) = 6.48 Hz), 4.226 (1H, m, HrA-6, J (6,6’) = 10.68 Hz), 4.280 (1H, d, HA-6, J (6,6’) = 12.06 Hz), 4.093 (1H, m, HrA-6’), 4.189 (1H, m, HdA/A-6’, J (6,6’) = 12.06 Hz), 4.078 (1H, dd, HdA-2, J (1,2) = 8.52 Hz), 4.076 (1H, dd, HdA-3), 4.073 (1H, m, HrU-2, J (1,2) = 3.76 Hz), 3.977 (1H, m, HA-2, J (1,2) = 8.52 Hz), 4.009 (1H, m, HdA-5, J (5,6’) = 5.34 Hz), 4.009 (1H, m, HrU-3), 4.009 (1H, m, HrU-4), 4.009 (1H, m, HrA-5), 3.996 (1H, m, HrA-2, J (1,2) = 6.80 Hz), 3.982 (1H, m, HrA-3), 3.934 (1H, m, HA-5, J (5,6’) = 5.34 Hz), 3.935 (1H, d, HU-4, J (4,5) = 9.42 Hz), 3.866 (1H, m, HA-3), 3.886 (1H, dd, HrF-2, J (1,2) = 4.08 Hz,J (2,3) = 10.88 Hz), 3.885 (1H, dd, HdF-2, J (1,2) = 4.00 Hz,J (2,3) = 10.38 Hz), 3.873 (1H, dd, HF-2, J (1,2) = 4.08 Hz, J (2,3) = 10.50 Hz), 3.826 (1H, dd, H∆U-2, J (1,2) = 8.30 Hz,J (2,3) = 8.16 Hz), 3.729 (1H, d, HrU-1, J (1,2) = 11.84 Hz), 3.691 (1H, d, HrU-1’, J (1,1’) = 11.84 Hz, J (1’,2) = 6.80 Hz), 3.632 (1H, d, HU-5, J (4,5) = 9.42 Hz), 3.620 (1H, dd, HU-3, J (2,3) = 8.00 Hz,J (3,4) = 8.64 Hz), 3.540 (1H, dd, HU-2, J (1,2) = 8.16 Hz,J (2,3) =8.00 Hz), 1.998 (3H, s, HrA/A-8), 1.988 (3H, s, HdA-8), 1.323 (3H, d, HF-6, J (5,6) = 6.36 Hz),1.239 (3H, d, HrF-6, J (5,6) = 6.56 Hz), 1.230 (3H, d, HdF-6,J (5,6) = 6.48 Hz); 13C NMR (200 MHz, D2O) δ 180.08 (CrU-6), 177.82 (C(r/d)A-7), 177.72 (CU-6), 171.60 (C∆U-6), 149.64 (C∆U-5), 109.38 (C∆U-4), 106.51 (CU-1),105.78 (C∆U-1), 104.16 (CrA-1), 104.16 (CrF-1), 102.33 (CdA/A-1), 101.98 (CF-1), 100.99 (CdF-1), 84.03 (CrU-4), 82.14 (CrU-3), 82.11 (CU-3), 81.95 (CF-4), 81.73 (Cr/dF-4), 79.85 (CU-5), 79.11 (C∆U-3), 78.99 (C(d/r) A-4), 78.78 (C(r/d)A-3), 78.11 (CF-3), 77.98 (CU-4), 77.91 (Cr/dF-3), 76.28 (CU-2), 75.04 (CrA-5), 74.68 (CrU-5), 74.61(C(d)A-5), 72.77 (CrU-2), 73.00 (CdU-2), 70.53 (CrA-6), 69.85 (CdA-6), 70.03 (CA-6), 69.55 (CrF-5),69.24 (CdF-5), 69.39 (CrF-2), 69.39 (CdF-2), 69.13 (CF-2), 69.02 (CF-5), 65.25 (CrU-1), 54.23 (C(r/d)A-2), 25.29 (C(r/d)A-8), 18.83 (CF-6), 18.79 (CrF-6), 18.56 (CdF-6); HRMS (ESI-Q-TOF) m/z: [M-4Na]4- calculated for C112H147N5Na24O151S224-1308.8784; Found for 1308.3858.
dHG-5,L-Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-{D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-β(1,3)-}n-D-GalNAc4S6S-β(1,4)-[L-Fuc3S4S-(α1,]3)-D-GlcA-ol, \(\overset{\overline{}}{\mathbf{n}}\)=4: 1H NMR (800 MHz, D2O) δ 5.685 (1H, d, H∆U-4), 5.283 (1H, d, HF-1,J (1,2) = 2.72 Hz), 5.201 (1H, d, HdF-1, J (1,2) = 3.36 Hz), 5.030 (1H, d, HrF-1, J (1,2) =3.24 Hz), 4.970 (1H, d, HF-4), 4.908 (1H, m, HdA-4), 4.843 (1H, d, H∆U-1,J (1,2) = 8.80 Hz), 4.834 (1H, d, HrF-4, J (3,4) =2.40 Hz), 4.830 (1H, d, HdF-4, J (3,4) = 2.40 Hz), 4.786 (1H, m, HF-5, J (5,6) = 6.00 Hz), 4.763 (1H, m, HrA-4), 4.751 (1H, HA-4), 4.620 (1H, d, HrA-1,J (1,2) =6.88 Hz), 4.544 (1H, d, HrF-3, J (3,4) = 2.40 Hz), 4.530 (1H, d, HdF-3, J (3,4) = 2.40 Hz), 4.506 (1H, d, HdA-1, J (1,2) = 8.80 Hz), 4.485 (1H, d, HA-1,J (1,2) = 8.80 Hz), 4.437 (1H, d, HF-3), 4.423 (1H, dd, H∆U-3,J (2,3) = 8.16 Hz), 4.398 (1H, d, HU-1, J (1,2) = 8.80 Hz), 4.368 (1H, m, HrF-5, J (5,6) = 6.48 Hz), 4.326 (1H, d, HdA-6), 4.287 (1H, d, HA-6), 4.284 (1H, dd, HdF-5,J (5,6) = 6.96 Hz), 4.239 (1H, m, HrA-6), 4.221 (1H, m, HdA-6’), 4.183 (1H, m, HA-6’), 4.104 (1H, m, HrA-6’), 4.091 (1H, dd, HdA-3), 4.081 (1H, dd, HdA-2, J (1,2) = 8.80 Hz), 4.075 (1H, m, HrU-2, J (1,2) = 3.36 Hz), 4.021 (1H, m, HA-2, J (1,2) = 8.80 Hz), 4.012 (1H, m, HrA-5), 4.010 (1H, m, HrU-3), 4.007 (1H, m, HdA-5), 4.005 (1H, m, HrU-4), 3.993 (1H, m, HrA-3), 3.974 (1H, m, HrA-2, J (1,2) = 6.88 Hz), 3.935 (1H, d, HU-4, J (4,5) = 8.00 Hz), 3.932 (1H, m, HA-5), 3.895 (1H, m, HA-3), 3.885 (1H, dd, HrF-2,J (1,2) = 3.24 Hz, J (2,3) = 10.88 Hz), 3.885 (1H, dd, HdF-2,J (1,2) = 3.36 Hz, J (2,3) = 10.88 Hz), 3.872 (1H, dd, HF-2,J (1,2) = 2.72 Hz, J (2,3) = 9.04 Hz), 3.827 (1H, dd, H∆U-2,J (1,2) = 8.80 Hz, J (2,3) = 8.12 Hz), 3.729 (1H, d, HrU-1,J (1,2) = 11.68 Hz), 3.692 (1H, d, HrU-1’, J (1,1’) = 11.68 Hz,J (1’,2) = 6.72 Hz), 3.63 (1H, d, HU-5, J (4,5) = 8.00 Hz), 3.620 (1H, dd, HU-3, J (2,3) = 8.56 Hz), 3.538 (1H, dd, HU-2, J (1,2) = 8.80 Hz, J (2,3) =8.56 Hz), 1.998 (3H, s, HrA/A-8), 1.988 (3H, s, HdA-8), 1.341 (3H, d, HF-6, J (5,6) = 6.00 Hz),1.239 (3H, d, HrF-6, J (5,6) = 6.48 Hz), 1.230 (3H, d, HdF-6,J (5,6) = 6.96 Hz); 13C NMR (200 MHz, D2O) δ 180.08 (CrU-6), 177.90 (C(r/d)A-7), 177.74 (CU-6), 171.63 (C∆U-6), 149.62 (C∆U-5), 109.43 (C∆U-4), 106.55 (CU-1),105.81 (C∆U-1), 104.16 (CrA-1), 104.16 (CrF-1), 102.34 (CA-1), 102.29 (CdA -1), 101.93 (CF-1), 100.95 (CdF-1), 82.33 (CrU-4), 82.32 (CrU-3), 82.09 (CU-3), 82.08 (CF-4), 81.70 (Cr/dF-4), 79.65 (CU-5), 79.20 (C∆U-3), 79.11 (C(r)A-3), 78.98 (CrA-3), 78.90 (C(d/r) A-4), 78.56 (CdA-3), 78.07 (CF-3), 77.96 (CU-4), 77.92 (Cr/dF-3), 76.36 (CU-2), 75.03 (CrA-5), 74.62 (CrU-5), 74.62(CA-5), 74.55(CdA-5), 72.97 (CdU-2), 72.61 (CrU-2), 70.52 (CrA-6), 70.03 (CdA-6), 69.88 (CA-6), 69.52 (CrF-5), 69.24 (CdF-5), 69.36 (CrF-2), 69.36 (CdF-2), 69.24 (CdF-5), 69.09 (CF-2), 69.00 (CF-5), 65.21 (CrU-1), 54.22 (C(r/d)A-2), 25.26 (C(r/d)A-8), 18.77 (CF-6), 18.84 (CrF-6), 18.56 (CdF-6); HRMS (ESI-Q-TOF) m/z: [M-4Na]4- calculated for C112H147N5Na24O151S224-1308.8784; Found for 1308.3858.
Inhibition for iXase. Inhibition of compounds for iXase was determined using the Biophen f.VIII:C kits (containing R1, R2, and R3 solutions) by a Bio-Tek Microplate Reader (ELx 808, USA) as previously described (Wu, Wen et al. , 2015). Briefly, 30 μLeach compound solution at different concentrations (Tris-HCl solution as control), 30 μL f.VIII (2 IU/mL), and 30 μL R2 solution (containing 60 nM f.IXa, human thrombin, phosphatidylcholine/phosphatidylserine and Ca2+) were mixed and incubated at 37°C for 2 min. Then, 30 μL R1 solution (containing 50 nM f.X and thrombin inhibitor) was added. After incubation for 1 min at 37°C, the residual FXa activity was measured by the addition of 30 μL R3 solution (FXa chromogenic substrate SXa-11). Assays were conducted at 37°C in 96-well plates, compounds were dissolved in Tris buffer (0.02 M Tris/HCl, pH 7.4). Hydrolysis of the substrate resulted in the release of p-nitroaniline, the optical density (OD405nm) was measured at 405nm (kinetic method, read per 30 s for 5 min). The change rate of absorbance at 405nm (ΔOD405nm/min) in the presence of the test compound was normalized to that of the control to culculate the relative activity. The compound concentrations - relative activity were plotted and fitted by the following equation using the Origin 8.0 software (OriginLab, USA): B = [IC50]n/{[IC50] n+[I]n }, where B represented the relative activity, [I] represented the compound concentrations, n represented the pseudo-Hill coefficient and IC50 represented the concentrations of compounds that inhibited 50% protease activity. Assays were carried out in duplicate.
Solution competition BLI study. The activities of compounds to compete with the immobilized oHG-11 for binding to f.IXa were performed on an Octet Red 96 instrument (Fortebio, USA). oHG-11 was biotinylated by reacting with amine-PEG3-biotin, then biotinylated oHG-11 was immobilized onto SA biosensors as previously described (B. Li, Suwanet al. , 2009; Xiao, Zhao et al. , 2019).
The kinetic assay of f.IXa binding to immobilized oHG-11 was conducted as below (Xiao, Lian et al. , 2016; Xiao, Zhao et al. , 2019). Firstly, baseline was equilibrated for 300 s with running buffer (0.15 M NaCl, 20 mM HEPES, pH 7.4, 2 mM CaCl2, 0.05% Tween 20 and 0.1% BSA). Then, f.IXa with gradient concentrations associated with immobilized oHG-11 for 600 s. Next, the dissociation was conducted in running buffer for 900 s. Finally, the sensors were regenerated by regeneration buffer (2 M NaCl, 20 mM HEPES, pH 7.4, 2 mM CaCl2, 0.05% Tween 20 and 0.1% BSA) for 10 s (repeated for 5 times). Assays were conducted once at 30°C in black 96-well flat bottom plates with agitation set to 1,000 revolutions per minute and final solution volume of 200 µL. Systematic baseline drift was corrected by subtracting the shift recorded by a sensor loaded with ligand but associated with no analyte. BLI kinetic data were analyzed by the Octet software version 7.0 and the binding curves were globally fitted with a 2:1 model (Figueralosada & Lograsso, 2012).
The relative ability of compounds binding to f.IXa was determined by competition binding assay (Xiao, Lian et al. , 2016; Xiao, Zhaoet al. , 2019). f.IXa (100 nM) were pre-incubated with compounds with gradient concentrations prior to interaction with immobilized oHG-11. The competition binding assay including baseline equilibrium, association, dissociation and regeneration processes were performed as above. The relative response (f.IXa binding response with compound/ f.IXa binding response without compound) were calculated, and compound concentrations - relative response were fitted by the following equation with Origin 8.0 software (OriginLab, USA): B = (IC50)n / [(IC50)n + [I]n], where meaning of B, I, n and IC50 were the same as described above (John P Sheehan, Kobbervig et al. , 2003; John P. Sheehan & Walke, 2006).
Inhibition for human coagulation protease. Effects of compounds on coagulation factors were tested at the concentrations for inhibiting 50% and 90% iXase activity, in the presence or absence of AT-III using chromogenic substrate hydrolysis assays. Buffers used in the assays included 20 mM Tris/HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl2, and 0.1% polyethylene glycol (PEG) 8000 for f.IIa and f.Xa assays (Henry, Monien et al. , 2007); 25 mM HEPES buffer, pH 7.4, containing 100 mM NaCl and 5 mM CaCl2for f.VIIa assays (Henry, Monien et al. , 2007; Lawson, Butenaset al. , 1993; Neuenschwander, Branam et al. , 1993); 100 mM HEPES buffer, pH 8.0, containing 100 mM NaCl and10 mM CaCl2 for f.IXa assays (Henry, Monien et al. , 2007; Tina, Richard et al. , 2003); 25 mM Tris/HCl buffer, pH7.4, containing 100 mM NaCl, 0.1 mg/mL bovine serum albumin (BSA), 0.1% PEG 8000 for f.XIa assays (Likui, Mao-Fu et al. , 2009; Wuillemin, Eldering et al. , 1996) and 50 mM Tris/HCl buffer, pH 7.6, containing 150 mM NaCl, 0.1% BSA for f.XIIa assays (Tong, G Michaelet al. , 2004).
The effects of compounds on coagulation factors in the presence of AT-III were assayed in 96-well plates in following conditions: 100 nM f.VIIa, 1000 nM AT-III and 1 mM Pefachrome f.VIIa for f.VIIa assay; 100 nM f.IXa, 1000 nM AT-III, 25% glycol and 1 mg/mL Pefchrome f.IXa for f.IXa assay; 5 nM f.XIa, 100 nM AT-III and 1 mM S-2366 for f.XIa assay; 10 nM f.XIIa, 100 nM AT-III and 1 mM CS 31(02) for f.XIIa assay. The f.IIa and f.Xa assays were conducted according to instructions of heparin anti-f.IIa and -f.Xa test kits. In the assays without AT-III, the AT-III solutions were replaced with the same volume of buffer. After incubating the mixture for 1 min, chromogenic substrate was added, the OD405nm was measured at 37°C (kinetic method, read per 15 s for 2 min).. The ΔOD405nm/min in the presence of the test compounds was normalized to that of the control (compounds were replaced by buffer) to obtain the relative activity. Assays were carried out in duplicate.
Anticoagulant activities. APTT, PT, and TT were determined with a coagulometer (TECO MC-4000, Germany) using APTT, PT and TT reagents and human plasma as previously described (N. Gao, Wu et al. , 2012). In brief, 5 µL sample and 45 µL plasma were pipetted to cuvette; incubated at 37°C for 2 min, then 50 µL APTT reagent was added; after incubating for 3 min, 50 µL CaCl2 reagent was added, and clotting time was recorded. The compound plasma concentrations-clotting times were fitted linearly by Origin 8.0 software (OriginLab, USA), and the EC2.0× (concentration required for doubling clotting time) were calculated from the fitting curves. Determinations were performed in duplicate.
Rats deep venous thrombosis. The antithrombotic activities of dHG-5 oligosaccharides were tested at doses with equivalent anticoagulant to dHG-5: dHG-5 antithrombotic 50% effective dose (ED50) × [oligosaccharide EC2.0× / dHG-5 EC2.0×]. Thrombus formation was induced by a combination of thromboplastin and stasis (Vogel, Meuleman et al. , 1989). Rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (3 mL/kg). The abdomen was opened and the vena cava was exposed and dissected free from surrounding tissue. A loose suture was prepared under the inferior vena cava. 2% rabbit brain tissue thromboplastin suspension (1.5 mL/kg) was injected into femoral vein, after 10 s, stasis was established by tightening the suture and maintained for 20 min. Then ligated vessel (from the ligation site to 2 cm below) was opened longitudinally and the formed thrombus was taken, blotted on filter paper, dried at 60°C for 1 h and weighted (Mettler balance). Compounds or vehicle were administered subcutaneously 1 hour prior to the injection of thromboplastin. The rabbit brain tissue thromboplastin was prepared as previously described (Quick, 1936).
Mice tail-bleeding model. The doses of dHG-5 (73.01 mg/kg) and LMWH (37.49 mg/kg) were designed as ten times of their antithrombotic ED50, respectively. Mice tail was cut 1 h after subcutaneous administration of compounds or vehicle. Mice were placed in prone position within an in-house device and a distal 5 mm segment of the tail was amputated. The tail was immediately immersed in a 50 mL pure water (37°C) for 1 h. After 1 h sample collection, the hemoglobin in the hemolyzed blood solution was measured using a UV-VIS spectrophotometer (UV-2450, Shimadzu) at 540 nm. Volume of blood loss was calculated from the OD540nm - blood volume standard curve.
f.XII activation. The activation effects of compounds on f.XII were determined using chromogenic substrate hydrolysis assays (Wu, Wenet al. , 2015). 40 µL diluted plasma (1:3, plasma: Tris/HCl buffer (v/v)) and 30 µL compounds dissolved in Tris/HCl buffer (0.02 M, pH 7.4) were mixed and incubated at 37℃ for 1 min. Then 30 µL of 0.3 mM CS-31(02) was added. The activation effects of compounds on f.XII were positively correlated with the release of p-nitroaniline from each substrate, which was monitored every 15 s for 2 min at 405 nm on a microplate reader. The activation effects of compounds on f.XII expressed as ΔOD405nm/min. OSCS was used as positive control. Determinations were performed in duplicate.
Platelet aggregation. Human blood was collected from elbow vein of healthy volunteers, anticoagulated with 3.2% sodium citrate, and centrifuged at 180 g for 10 min to obtain platelets rich plasma (PRP). The remaining components of the blood were centrifuged again at 1200 g for 15min to obtain platelets poor plasma (PPP). Rat blood were collected from abdominal aorta into tubes containing EDTAK2, and centrifuged at 180 g for 10 min to obtain PRP. Then rat washed platelets (WP) was prepared (Cazenave, Ohlmannet al. , 2004; Mustard, Packham et al. , 1972). Briefly, platelets pellet were washed twice by Ca2+-free Tyrode´s buffer (137 mM NaCl, 2.68 mM KCl, 0.2 mM MgSO4·7H2O, 0.42 mM NaH2PO4·2H2O, 11.9 mM NaHCO3, 5.05 mM Glucose, 0.2 mM EGTA, pH 6.53) for twice. After centrifugation (800 g, 10 min), platelet pellet was re-suspended in Tyrode´s buffer (137 mM NaCl, 2.68 mM KCl, 1.05 mM MgSO4·7H2O, 0.42 mM NaH2PO4·2H2O, 11.9 mM NaHCO3, 5.05 mM Glucose, 1.8 mM CaCl2, pH 7.35). Light transmission aggregation was measured using an aggregometer (Model 700, Chrono-log, America). 250 µL PRP or WP suspension was stimulated with 2.5 µL compounds dissolved in saline for 10 min at 37°C and continuous stirring at 1000 rpm. The baselines were adjusted by PPP and Tyrode´s buffer for PRP and WP suspension, respectively. Determinations were performed in duplicate.
Preliminary pharmacodynamics study on mice. Compounds dissolved in saline were injected subcutaneously to mice. Venous blood samples (approximately 0.15 mL) were collected before and after compounds administration 0.5 h, 1 h, 2 h, 4 h and 8 h from mice orbital venous plexus and centrifuged immediately at 1800 g for 10 min. Then, the plasma APTT at different time points was analyzed ex vivo. In brief, 25 µL plasma sample was pipetted to cuvette, incubated at 37°C for 2 min, then 25 µL APTT reagent was added; after incubating 3 min, 25 µL CaCl2 reagent was added and clotting time was recorded (MC-4000, MDC). All pharmacodynamics parameters were calculated with noncompartmental procedure using the Drug Analysis System 2.0 (DAS 2.0, BioVoice &BioGuider, Shanghai, China).