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)-L-Δ4,5GlcA-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)-L-Δ4,5GlcA-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)-L-Δ4,5GlcA-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)-L-Δ4,5GlcA-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)-L-Δ4,5GlcA-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)-L-Δ4,5GlcA-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).