Comparison of Activity and Safety of DSPAα1 and Its N-Glycosylation Mutants
Abstract
:1. Introduction
2. Methods and Materials
2.1. Preparation of Plasmids and Strains
2.2. Mutagenesis of DSPAα1
2.3. Expression and Purification of Wild-Type and Mutant DSPAα1
2.4. SDS-PAGE Analysis
2.5. Fibrin Assay of DSPAα1 Activity
2.6. Kinetics of S-2765 Hydrolysis
2.7. Detection of Thermal Stability and Acid-Base Stability
2.8. Rat Modelling and Drug Delivery
2.9. Thrombus Detection and Hemostatic Factors Analysis
2.10. Thrombopathological Biopsy Analysis
3. Results
3.1. Prediction of N-Glycosylation Sites and Structural Predictive Analysis
3.2. Expression, Purification, and SDS-PAGE Analysis of DSPAα1 and Mutants
3.3. Effect of N-Glycosylation on Thermostability and Acid-Base Tolerance
3.4. Effect of N-Glycosylation on Fibrinolytic Activity and Fibrin Selectivity of DSPAα1 and Its Mutants In Vitro
3.5. Effects of N-Glycosylation Mutations on Thrombolytic Effects of DSPAα1 and Its Mutants in Rats
3.6. Effects of N-Glycosylation Mutations on the Thrombolysis Safety of DSPAα1 and Its Mutants in Rats
4. Discussions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
Abbreviations | Full Name |
Å | Ångström unit, the smallest unit representing the spatial distance between two sites or atoms |
D-D | D-dimer |
F | Fibrin |
Fbn | Fibrinogen |
FDP | Fibrin/Fibrinogen Degradation Products |
HRP | Horseradish peroxidase containing 16% glycoprotein |
Kcat | Turnover number |
Km | Michaelis constant |
MPV | The mean of platelet volume |
Plg | Plasminogen |
PLT | The platelet count |
PDW | The width of platelet distribution |
PCT | Thrombocytosis |
PAI | Plasminogen activator inhibitor |
PNGase F | The most effective enzyme for removing N-linked glycans from glycoproteins |
rt-PA | Alteplase |
S-2765 | A chromogenic substrate for determination of Factor Xa activity |
STI | Soybean Trypsin Inhibitor, unglycosylated |
SPF | Specific pathogen-free |
t-PA | Tissue plasminogen activator |
u-PA | Urokinase-type plasminogen Activator |
α2-AP | α2-Antiplasmin |
References
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Administration | Before | After | |||||
---|---|---|---|---|---|---|---|
Sampling time (min) | −5.000 | 10.000 | 30.000 | 60.000 | 90.000 | 120.000 | 180.000 |
blood volume (μL) | 300.000 | 300.000 | 300.000 | 300.000 | 300.000 | 300.000 | 300.000 |
TBT (min) | −5.000 | 10.000 | 30.000 | 60.000 | 90.000 | 120.000 | 180.000 |
Enzyme | Motifs | Modification | Mutation Sites | N-Glycosylation Sites |
---|---|---|---|---|
rDSPAα1 | N153-N184(A186)-K368-N398 | / | / | N153-N398 |
QNGlyα1 | Q153-N184(A186)-K368-Q398 | Deletion | N153Q N398Q | / |
QNGlyα1-1 | Q153-N184(A186)-K368-N398 | Deletion | N153Q | N398 |
QNGlyα1-2 | N153-N184(A186)-K368-Q398 | Deletion | N398Q | N153 |
ANGlyα1 + 2 | N153-N184(T186)-K368-N398 | Addition | A186T | N153-N184-N398 |
ANGlyα1 + U | N153-N184(A186)-N368-N398 | Addition | K368N | N153-N368-N398 |
Enzyme | Cofactor | Kcat ×103 | Km mM | Kcat/Km mM−1S−1 | Stimulation Factor | Ratio Fbn/Fbg |
---|---|---|---|---|---|---|
rDSPAα1 | None | 0.094 ± 0.001 | 2.709 ± 0.155 | 34.830 ± 2.350 | 1.000 | |
Fbg | 0.097 ± 0.000 | 0.343 ± 0.015 | 282.240 ± 13.070 | 8.100 | ||
Fbn | 0.259 ± 0.024 | 0.045 ± 0.010 | 5883.440 ± 983.570 | 168.900 | 20.800 | |
QNGlyα1 | None | 0.084 ± 0.004 | 1.380 ± 0.257 | 62.240 ± 10.690 | 1.000 | |
Fbg | 0.118 ± 0.012 | 0.369 ± 0.077 | 328.740 ± 62.750 | 5.300 | ||
Fbn | 0.165 ± 0.017 | 0.763 ± 0.085 | 216.420 ± 5.840 | 3.500 | 0.700 | |
QNGlyα1-1 | None | 0.088 ± 0.001 | 2.258 ± 0.046 | 38.800 ± 0.780 | 1.000 | |
Fbg | 0.144 ± 0.019 | 0.493 ± 0.005 | 292.580 ± 1.780 | 7.500 | ||
Fbn | 0.149 ± 0.001 | 0.448 ± 0.030 | 333.760 ± 20.680 | 8.600 | 1.100 | |
QNGlyα1-2 | None | 0.138 ± 0.008 | 1.764 ± 0.111 | 78.240 ± 0.600 | 1.000 | |
Fbg | 0.126 ± 0.004 | 1.320 ± 0.033 | 95.230 ± 3.970 | 1.200 | ||
Fbn | 0.124 ± 0.008 | 0.153 ± 0.016 | 812.780 ± 54.960 | 10.400 | 8.500 | |
ANGlyα1 + 2 | None | 0.089 ± 0.001 | 3.688 ± 0.318 | 24.280 ± 1.990 | 1.000 | |
Fbg | 0.144 ± 0.003 | 0.577 ± 0.024 | 248.960 ± 6.330 | 10.300 | ||
Fbn | 0.168 ± 0.027 | 0.052 ± 0.005 | 3257.040 ± 561.250 | 134.100 | 13.100 | |
ANGlyα1 + U | None | 0.082 ± 0.002 | 0.674 ± 0.067 | 122.880 ± 13.680 | 1.000 | |
Fbg | 0.096 ± 0.005 | 0.453 ± 0.129 | 221.760 ± 60.490 | 1.800 | ||
Fbn | 0.097 ± 0.001 | 0.168 ± 0.028 | 590.130 ± 98.900 | 4.800 | 2.700 |
Samples | P1 | P2 | P3 | P4 | P5 | P6 | Total Score |
---|---|---|---|---|---|---|---|
CK | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
Mg | 20.000 ± 0.000 | 20.000 ± 0.000 | 19.375 ± 0.354 | 19.375 ± 0.354 | 18.750 ± 0.463 | 16.875 ± 0.744 | 114.375 ± 0.835 |
rt-PA | 15.833 ± 1.329 | 16.667 ± 1.211 | 12.500 ± 0.837 | 14.167 ± 0.753 | 10.833 ± 1.169 | 20.000 ± 0.000 | 90.000 ± 2.366 |
rDSPAα1 | 9.167 ± 1.169 | 14.167 ± 0.983 | 7.500 ± 0.548 | 7.500 ± 0.548 | 5.000 ± 0.000 | 18.333 ± 0.516 | 61.667 ± 1.966 |
QNGlyα1 | 15.833 ± 0.753 | 20.000 ± 0.000 | 13.333 ± 0.516 | 17.500 ± 0.547 | 20.000 ± 0.000 | 16.667 ± 0.816 | 103.333 ± 0.816 |
ANGlyα1 + 2 | 16.667 ± 0.816 | 20.000 ± 0.000 | 14.167 ± 0.753 | 15.000 ± 0.632 | 18.333 ± 0.516 | 8.333 ± 0.816 | 92.500 ± 1.049 |
PLT (109/L) | MPV | PDW (%) | PCT | p Value | ||
---|---|---|---|---|---|---|
CK | No dosing | 67.000 ± 7.211 | 9.400 ± 0.265 | 14.167 ± 0.462 | 0.062 ± 0.005 | |
15 min after dosing | 65.000 ± 1.732 | 9.633 ± 0.058 | 13.833 ± 0.306 | 0.062 ± 0.001 | p > 0.05 | |
1 h after dosing | 60.333 ± 4.163 | 9.700 ± 0.265 | 14.133 ± 0.643 | 0.062 ± 0.005 | p > 0.05 | |
Mg | No dosing | 830.667 ± 37.072 | 7.700 ± 0.520 | 8.933 ± 0.666 | 0.640 ± 0.016 | |
15 min after dosing | 826.333 ± 50.143 | 7.600 ± 0.300 | 8.833 ± 0.551 | 0.628 ± 0.021 | p > 0.05 | |
1 h after dosing | 746.000 ± 42.673 | 7.600 ± 0.265 | 8.800 ± 0.520 | 0.567 ± 0.008 | p < 0.005 | |
rt-PA | No dosing | 872.667 ± 105.945 | 6.833 ± 0.058 | 7.567 ± 0.231 | 0.594 ± 0.078 | |
15 min after dosing | 716.333 ± 106.931 | 7.133 ± 0.153 | 7.767 ± 0.404 | 0.512 ± 0.066 | p > 0.05 | |
1 h after dosing | 576.333 ± 42.595 | 6.933 ± 0.416 | 7.900 ± 0.721 | 0.401 ± 0.052 | p < 0.005 | |
rDSPAα1 | No dosing | 728.333 ± 73.173 | 7.367 ± 0.153 | 8.333 ± 0.231 | 0.537 ± 0.065 | |
15 min after dosing | 585.667 ± 71.654 | 7.133 ± 0.231 | 8.133 ± 0.451 | 0.417 ± 0.038 | p < 0.0001 | |
1 h after dosing | 724.333 ± 50.817 | 7.000 ± 0.100 | 7.867 ± 0.289 | 0.506 ± 0.028 | p > 0.05 | |
QNGlyα1 | No dosing | 860.667 ± 25.146 | 6.567 ± 0.115 | 7.300 ± 0.000 | 0.563 ± 0.028 | |
15 min after dosing | 772.667 ± 54.775 | 7.333 ± 0.289 | 8.167 ± 0.751 | 0.547 ± 0.067 | p > 0.05 | |
1 h after dosing | 498.000 ± 21.284 | 6.900 ± 0.265 | 7.867 ± 0.493 | 0.357 ± 0.031 | p < 0.005 | |
ANGlyα1 + 2 | No dosing | 768.000 ± 120.104 | 6.967 ± 0.503 | 7.900 ± 0.721 | 0.532 ± 0.048 | |
15 min after dosing | 572.333 ± 18.502 | 7.100 ± 0.436 | 7.900 ± 0.889 | 0.405 ± 0.014 | p > 0.05 | |
1 h after dosing | 712.667 ± 16.010 | 7.033 ± 0.643 | 7.900 ± 0.889 | 0.502 ± 0.055 | p > 0.05 |
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Peng, H.; Wang, N.; Wang, M.; Yang, C.; Guo, W.; Li, G.; Huang, S.; Wei, D.; Liu, D. Comparison of Activity and Safety of DSPAα1 and Its N-Glycosylation Mutants. Life 2023, 13, 985. https://doi.org/10.3390/life13040985
Peng H, Wang N, Wang M, Yang C, Guo W, Li G, Huang S, Wei D, Liu D. Comparison of Activity and Safety of DSPAα1 and Its N-Glycosylation Mutants. Life. 2023; 13(4):985. https://doi.org/10.3390/life13040985
Chicago/Turabian StylePeng, Huakang, Nan Wang, Mengqi Wang, Caifeng Yang, Wenfang Guo, Gangqiang Li, Sumei Huang, Di Wei, and Dehu Liu. 2023. "Comparison of Activity and Safety of DSPAα1 and Its N-Glycosylation Mutants" Life 13, no. 4: 985. https://doi.org/10.3390/life13040985