DISSCUSSION
Assay for potencies and selectivity of anti-iXase activities of dHG-5
and its containing oliogosaccharides showed that dHG-5 showed potent
anti-iXase activity and f.IXa-binding activity without obvious effects
on the other coagulation factor targets, which indicated its high
selectivity in anti-iXase. Silmilar to our previous report (Xiao, Zhaoet al. , 2019), it was notable that dHG-5 and its containing
oligosaccharides seemed to enhance the activity of f.IXa in hydrolyzing
its chromogenic substrate in the absence of f.VIIIa. This phenomena
might be attributed to the conformational change of f.IXa induced by
oligosaccharides. In fact, under physiological conditions, f.IXa
combines its cofactor f.VIIIa, phospholipid and Ca2+to form iXase complex, which enhances the catalytic efficiency of f.IXa
with million times (Duffy & Lollar, 1992; Mertens, Wijngaarden et
al. , 1985). Thus, the direct effects of dHG-5 and its containing
oligosaccharides on f.IXa activity was very faint when compared with
their inhibition on f.IXa-f.VIIIa complex assembling, i.e., anti-iXase
activities.
Assay for anticoagulant activities of dHG-5 and its containing
oliogosaccharides showed that the anticoagulant mechanisms of dHG-5 and
its containing oligosaccharides were AT-III- and HCII-independent, which
were different from that of LMWH and DS. At the equivalent anticoagulant
potency’s doses, these oligosaccharides showed approximate thrombosis
inhibition, suggesting their antithrombotic activities were closely
related to their anticoagulant activities. The results of
pharmacodynamics of dHG-5 showed the linear kinetics, suggesting the
predictable pharmacodynamic characteristics.
Through a series of studies, the pharmacological properties of dHG-5 and
its containing oligosaccharides in anti-iXase, f.IXa-binding,
anticoagulant and antithrombotic activities were clearly illuminated.
dHG-5 showed strong anti-iXase activity, f.IXa-binding, anticoagulant
and antithrombotic activities without activating f.XII and platelets.
Within bounds, the anti-iXase, f.IXa-binding and anticoagulant
activities of these oligosaccharides increased with the increase of dp,
oHG-17 to -29 closed to the full activity. It indicated that the
increase of dp obviously increased these activities of these
oligosaccharides to a certain extent, even octasaccharide was the
minimum structural unit required for the potent anti-iXase activity
(Yin, Zhou et al. , 2018). Moreover, the activity potencies of
dHG-5 in anti-iXase, f.IXa-binding, anticoagulation and anti-thrombosis
were close to the weighted average sum of that of its containing
oligosaccharides (Table 1). It indicated that there was no synergy or
antagonism among these oligosaccharides, the similarity of dHG-5 and
oHG-17 in Mw and activities also confirmed this. Thus pharmacological
activities of dHG-5 could be explained well by the contribution of its
containing oligosaccharides both qualitatively and quantitatively.
Besides, the relationships between Mw and f.IXa-binding, anti-iXase,
anticoagulant and antithrombotic potencies of dHG-5 containing
oligosaccharides all fitted well with similar power function, which
suggested the high dependency among these activities.
dHG-5 is an active ingredient consisting of a series of oligosaccharides
homologs. As a multi-component drug, it is a great challenge to clarify
the correlation of pharmacological activity between drug and its
containing components. For instance, heparins and LMWHs have been widely
used in clinic, but due to complexity of the composition, the types and
composition ratios of their containing oligosaccharides could not be
clarified (Loganathan, Wang et al. , 1990). Consequently, the
correlation of pharmacological activity between these preparations and
their containing oligosaccharides has not been reported. Our study
showed that the chemical composition of dHG-5 was relatively clear. And
the nine purified oligosaccharide components (oHG-5, -8, -11, -14, -17,
-20, -23, -26 and -29) accounted for about 95% of dHG-5. And spectral
analysis (1D/2D NMR and MS) confirmed that these oligosaccharide
components had the regular structures and shared the common formula.
Compared with heparins that have complex composition and diverse
substituents, the chemical composition of dHG-5 was clear, which enabled
the further study in structure-activity relationship and the correlation
of pharmacological activities between dHG-5 and its containing
oligosaccharides.
According to pharmacoeconomics, dHG-5 is suitable for developing a noval
anticoagulant. Though the activities of oHG-17 are similar to that of
dHG-5, the preparation process of pure oligosaccharide is complex, and
not feasible for large-scale industrial production. While it is more
feasible for the scale preparation of dHG-5. What’s more, the
composition and proportion of dHG-5 containing oligosaccharides could be
controlled well in the preparation process. Therefore, dHG-5 may be more
suitable as an iXase inhibitor to be a novel anticoagulant applied in
clinic.
dHG-5 may be more effective than other intrinsic coagulation inhibitors
in antithrombosis. The effects of common coagulation pathway inhibitors
and intrinsic coagulation pathway inhibitors on hemostatic function are
different (Colman, 2006; Qiufang, Tucker et al. , 2010; Wheeler &
Gailani, 2016; Woodruff, Xu et al. , 2013). Compared with
available clinical drugs, selective intrinsic coagulation pathway
inhibitors may have the characteristics of antithrombosis with low
bleeding tendency (Lin, Zhao et al. , 2020). According to the
cell-based coagulation model, f.XIIa and f.XIa inhibitors may have
limited effects on coagulation amplification and propagation during
thrombosis, while iXase inhibitors should be able to exhibitie more
effective antithrombotic activity (Hoffman, 2003; Lin, Zhao et
al. , 2020).
In conclusion, our data demonstrate the anti-iXase, f.IXa-binding,
anticoagulant and antithrombotic activities of dHG-5 are contributed by
that of its containing oligosaccharides in terms of a weighted average
sum. These activities of dHG-5 containing oligosaccharides were
positively correlated with their chain length to a certain degree, among
which, the molecular weight of oHG-17 may be necessary to achieve full
activity. Not only dHG-5 has the characteristic of antithrombosis with
low bleeding tendency, but also clear chemical composition. This paper
makes an important supplement to the preclinical study of dHG-5, which
makes a good preparation for the entry into the clinical study.
Reference
Ahmad, S. S.,Rawala-Sheikh, R.Walsh, P. N. (1992). Components and
Assembly of the Factor X Activating Complex. Seminars in
Thrombosis and Hemostasis, 18 (03), 311-323.
Becattini, C.,Cohen, A. T.,Agnelli, G.,Howard, L.,Castejon,
B.,Trujillo-Santos, J., et al. (2016). Risk stratification of
patients with acute symptomatic pulmonary embolism based on presence or
absence of lower extremity DVT systematic review and meta-analysis.Chest, 149 (1), 192-200.
Benjamin, E. J.,Virani, S. S.,Callaway, C. W.,Chamberlain, A. M.,Chang,
A. R.,Cheng, S., et al. (2018). Heart Disease and Stroke
Statistics-2018 Update: A Report from the American Heart Association.Circulation, 137 (12), e67-e492.
Cai, Y.,Yang, W. J.,Li, X. M.,Zhou, L. T.,Wang, Z. J.,Lin, L. S.,
et al. (2019). Precise Structures and Anti-intrinsic Tenase Complex
Activity of Three Fucosylated Glycosaminoglycans and Their Fragments.Carbohydrate Polymers, 224 , 115146.
Campello, E.,Henderson, M. W.,Noubouossie, D. F.,Simioni, P.Key, N. S.
(2018). Contact system activation and cancer: new insights in the
pathophysiology of cancer-associated thrombosis. Thrombosis and
Haemostasis, 118 (2), 251-265.
Carpenter, S. L.,Richardson, T.Hall, M. (2018). Increasing Rate of
Pulmonary Embolism Diagnosed in Hospitalized Children in the United
States from 2001 to 2014. Blood Advances, 2 (12), 1403-1408.
Cazenave, J. P.,Ohlmann, P.,Cassel, D.,Eckly, A.,Hechler, B.Gachet, C.
(2004). Preparation of Washed Platelet Suspensions from Human and Rodent
Blood. Methods Mol. Biol. (Clifton, N.J.), 272 , 13-28.
Colman, R. W. (2006). Are hemostasis and thrombosis two sides of the
same coin? Journal of Experimental Medicine, 203 (3), 493-495.
Duffy, E. J.Lollar, P. (1992). Intrinsic pathway activation of factor X
and its activation peptide-deficient derivative, factor Xdes-143-191.Journal of Biological Chemistry, 267 (11), 7821-7827.
Figueralosada, M.Lograsso, P. V. (2012). Enzyme Kinetics and Interaction
Studies for Human JNK1β1 and Substrates Activating Transcription Factor
2 (ATF2) and c-Jun N-terminal Kinase (c-Jun). Journal of
Biological Chemistry, 287 (16), 13291-13302.
Fonseca, R. J. C.,Santos, G. R. C.Mour, O. P. A. S. (2009). Effects of
Polysaccharides Enriched in 2,4-Disulfated Fucose Units on Coagulation,
Thrombosis and Bleeding. Practical and Conceptual Implications.Thrombosis and Haemostasis, 102 (05), 829-836.
Gao, N.,Lu, F.,Xiao, C.,Yang, L.,Chen, J.,Zhou, K., et al.(2015). Beta-eliminative Depolymerization of the Fucosylated Chondroitin
Sulfate and Anticoagulant Activities of Resulting Fragments.Carbohydrate Polymers, 127 , 427-437.
Gao, N.,Wu, M.,Liu, S.,Lian, W.,Li, Z.Zhao, J. (2012). Preparation and
Characterization of O-acylated Fucosylated Chondroitin Sulfate from Sea
Cucumber. Marine Drugs, 10 (8), 1647-1661.
Henry, B. L.,Monien, B. H.,Bock, P. E.Desai, U. R. (2007). A Novel
Allosteric Pathway of Thrombin Inhibition: Exosite II Mediated Potent
Inhibition of Thrombin by Chemo-enzymatic, Sulfated Dehydropolymers of
4-hydroxycinnamic Acids. Journal of Biological Chemistry,
282 (44), 31891-31899.
Hoffman, M. (2003). A cell-based model of coagulation and the role of
factor VIIa. Blood Reviews, 17 (1), S1-S5.
Jiménez, D.,Bikdeli, B.,Quezada, A.,Muriel, A.Monreal, M. (2019).
Hospital Volume and Outcomes for Acute Pulmonary Embolism: Multinational
Population Based Cohort Study. BMJ Clinical Research, 366 , l4416.
Kitazato, K.,Kitazato, K. T.,Nagase, H.Minamiguchi, K. (1996). DHG, a
new depolymerized holothurian glycosaminoglycan, exerts an
antithrombotic effect with less bleeding than unfractionated or low
molecular weight heparin, in rats. Thrombosis Research, 84 (2),
111-120.
Lawson, J. H.,Butenas, S.,Ribarik, N.Mann, K. G. (1993).
Complex-dependent inhibition of factor VIIa by antithrombin III and
heparin. Journal of Biological Chemistry, 268 (2), 767-770.
Li, B.,Suwan, J.,Martin, J. G.,Zhang, F.,Zhang, Z.,Hoppensteadt,
D., et al. (2009). Oversulfated chondroitin sulfate interaction
with heparin-binding proteins: new insights into adverse reactions from
contaminated heparins. Biochemical Pharmacology, 78 (3), 292-300.
Li, J. Z.,Bao, C. X.,Chen, G. Z.,Zhang, G. Z.,Fan, H. Z.Chen, J. D.
(1985). Antithrombin activity and platelet aggregation by acid
mucopolysaccharides isolated from Stichopus Japonicus Selenka.Acta Pharmacologica Sinica, 6 (2), 107-110.
Li, X. M.,Luo, L.,Cai, Y.,Yang, W. J.,Lin, L. S.,Li, Z., et al.(2017). Structural Elucidation and Biological Activity of A Highly
Regular Fucosylated Glycosaminoglycan from the Edible Sea CucumberStichopus Herrmanni . Journal of Agricultural and Food
Chemistry, 65 (42), 9315-9323.
Likui, Y.,Mao-Fu, S.,David, G.Rezaie, A. R. (2009). Characterization of
a heparin-binding site on the catalytic domain of factor XIa: mechanism
of heparin acceleration of factor XIa inhibition by the serpins
antithrombin and C1-inhibitor. Biochemistry, 48 (7), 1517-1524.
Lin, L.,Zhao, L.,Gao, N.,Yin, R.,Li, S.,Sun, H., et al. (2020).
From multi-target anticoagulants to DOACs, and intrinsic coagulation
factor inhibitors. Blood Reviews, 39 , 100615.
Loganathan, D.,Wang, H. M.,Mallis, L. M.Linhardt, R. J. (1990).
Structural Variation in the Antithrombin III Binding Site Region and its
Occurrence in Heparin from Different Sources. Biochemistry,
29 (18), 4362-4368.
Long, A. T.,Kenne, E.,Jung, R.,Fuchs, T. A.Renne, T. (2016). Contact
system revisited: an interface between inflammation, coagulation, and
innateimmunity. Journal of Thrombosis and Haemostasis, 14 (3),
427-437.
Mertens, K.,Wijngaarden, A. V.Bertina, R. M. (1985). The role of factor
VIII in the activation of human blood coagulation factor X by activated
factor IX. Thrombosis and Haemostasis, 54 (3), 654-660.
Mustard, J. F.,Packham, M. A.,Perry, D. W.Ardlie, N. G. (1972).
Preparation of Suspensions of Washed Platelets from Humans.British Journal of Haematology, 22 (2), 193-204.
Nagase, H.,Enjyoji, K.,Minamiguchi, K.,Kitazato, K. T.,Kitazato,
K.,Saito, H., et al. (1995). Depolymerized holothurian
glycosaminoglycan with novel anticoagulant actions: antithrombin III-
and heparin cofactor II-independent inhibition of factor X activation by
factor IXa-factor VIIIa complex and heparin cofactor II-dependent
inhibition of thrombi. Blood, 85 (6), 1527-1534.
Neuenschwander, P. F.,Branam, D. E.Morrissey, J. H. (1993). Importance
of substrate composition, pH and other variables on tissue factor
enhancement of factor VIIa activity. Thrombosis and Haemostasis,
70 (06), 0970-0977.
Pinto, D. J. P.,Orwat, M. J.,Smith II, L. M.,Quan, M. L.,Lam, P. Y.
S.,Rossi, K. A., et al. (2017). Discovery of a Parenteral Small
Molecule Coagulation Factor XIa Inhibitor Clinical Candidate
(BMS-962212). Journal of Medicinal Chemistry, 60 , 9703-9723.
Pomin, V. H. (2013). NMR Chemical Shifts in Structural Biology of
Glycosaminoglycans. Analytical Chemistry, 86 (1), 65-94.
Pomin, V. H. (2014). Holothurian fucosylated chondroitin sulfate.Marine Drugs, 12 (1), 232-254.
Qiufang, C.,Tucker, E. I.,Pine, M. S.,India, S.,Anton, M.,Mao-Fu,
S., et al. (2010). A role for factor XIIa-mediated factor XI
activation in thrombus formation in vivo. Blood, 116 (19),
3981-3989.
Quan, M. L.,Pinto, D. J. P.,Smallheer, J. M.,Ewing, W. R.,Rossi, K.
A.,Luettgen, J. M., et al. (2018). Factor XIa Inhibitors as New
Anticoagulants. Journal of Medicinal Chemistry, 61 , 7425-7447.
Quick, A. J. (1936). On Various Properties of Thromboplastin ( Aqueous
Tissue Extracts). American Journal of Physiology, 114 (2),
282-296.
Robert, S.,Bertolla, C.,Masereel, B.,Dogné, J. M.Pochet, L. (2008).
Novel 3-Carboxamide-coumarins as Potent and Selective FXIIa Inhibitors.Journal of Medicinal Chemistry, 51 (11), 3077-3080.
Shang, F.,Gao, N.,Yin, R.,Lin, L.,Xiao, C.,Zhou, L., et al.(2018). Precise Structures of Fucosylated Glycosaminoglycan and its
Oligosaccharides as Novel Intrinsic Factor Xase Inhibitors.European Journal of Medicinal Chemistry, 148 , 423-435.
Sheehan, J. P.,Kobbervig, C. E.Kirkpatrick, H. M. (2003). Heparin
Inhibits the Intrinsic Tenase Complex by Interacting with an Exosite on
Factor IXa. Biochemistry, 42 (38), 11316-11325.
Sheehan, J. P.Walke, E. N. (2006). Depolymerized Holothurian
Glycosaminoglycan and Heparin Inhibit the Intrinsic Tenase Complex by a
Common Antithrombin-independent Mechanism. Blood, 107 (10),
3876-3882.
Smith. (2010). The cell-based model of coagulation. Journal of
Veterinary Emergency & Critical Care, 19 (1), 3-10.
Timmis, A.,Townsend, N.,Gale, C.,Grobbee, R.,Maniadakis, N.,Flather,
M., et al. (2017). European society of cardiology: cardiovascular
disease statistics 2017. European Heart Journal, 39 (7), 508-579.
Tina, B.,Richard, S.,Yung-Jen, C.,Bock, P. E.,Ingemar, B. R.Olson, S. T.
(2003). Heparin and calcium ions dramatically enhance antithrombin
reactivity with factor IXa by generating new interaction exosites.Biochemistry, 42 (27), 8143-8152.
Tong, S.,G Michael, I.,Jian, C., Qi,Cockerill, K. A.,Linnik, M.
D.,Pamela, K., et al. (2004). Beta 2-Glycoprotein I binds factor
XI and inhibits its activation by thrombin and factor XIIa: loss of
inhibition by clipped beta 2-glycoprotein I. Proceedings of the
National Academy of Sciences of the United States of America, 101 (11),
3939-3944.
Vogel, G. M. T.,Meuleman, D. G.,Bourgondi?n, F. G. M.Hobbelen, P. M. J.
(1989). Comparison of Two Experimental Thrombosis Models in Rats Effects
of Four Glycosaminoglycans. Thrombosis Research, 54 (5), 399-410.
Wang, S.,Beck, R.,Burd, A.,Blench, T.,Marlin, F.,Ayele, T., et
al. (2010). Structure Based Drug Design: Development of Potent and
Selective Factor IXa (FIXa) Inhibitors. Journal of Medicinal
Chemistry, 53 (4), 1473-1482.
Wheeler, A. P.Gailani, D. (2016). The intrinsic pathway of coagulation
as a target for antithrombotic therapy. Hematology/Oncology
Clinics of North America, 30 (5), 1099-1114.
Woodruff, R. S.,Xu, Y.,Layzer, J.,Wu, W.,Ogletree, M. L.Sullenger, B. A.
(2013). Inhibiting the intrinsic pathway of coagulation with a factor
XII-targeting RNA aptamer. Journal of Thrombosis and Haemostasis,
11 (7), 1364-1373.
Wu, M.,Wen, D.,Gao, N.,Xiao, C.,Yang, L.,Xu, L., et al. (2015).
Anticoagulant and antithrombotic evaluation of native fucosylated
chondroitin sulfates and their derivatives as selective inhibitors of
intrinsic factor Xase. European Journal of Medicinal Chemistry,
92 , 257-269.
Wuillemin, W. A.,Eldering, E.,Citarella, F.,Ruig, C. P. D.,Cate, H.
T.Hack, C. E. (1996). Modulation of Contact System Proteases by
Glycosaminoglycans - Selective Enhancement of the Inhibition of Factor
XIa. Journal of Biological Chemistry, 271 (22), 12913-12918.
Xiao, C.,Lian, W.,Zhou, L.,Gao, N.,Xu, L.,Chen, J., et al.(2016). Interactions between depolymerized fucosylated glycosaminoglycan
and coagulation proteases or inhibitors. Thrombosis Research,
146 , 59-68.
Xiao, C.,Zhao, L. Y.,Gao, N.,Wu, M. Y.Zhao, J. H. (2019). Nonasaccharide
Inhibits Intrinsic Factor Xase Complex by Binding to Factor IXa and
Disrupting Factor IXa-Factor VIIIa Interactions. Thrombosis and
Haemostasis, 119 (5), 705-715. doi:10.1055/s-0039-1681047
Yin, R.,Zhou, L.,Gao, N.,Z., L.,Zhao, L.,Shang, F., et al.(2018). Oligosaccharides from depolymerized fucosylated
glycosaminoglycan: structures and minimum size for intrinsic factor Xase
complex inhibition. Journal of Biological Chemistry, 293 ,
14089-14099.
Zhao, L.,Wu, M.,Xiao, C.,Yang, L.,Lian, W.,Zhou, L., et al.(2015). Discovery of an Intrinsic Tenase Complex Inhibitor: Pure
Nonasaccharide from Fucosylated Glycosaminoglycan. Proceedings of
the National Academy of Sciences of the United States of America,
112 (27), 8284-8289.
Zhou, L.,Gao, N.,Sun, H.,Xiao, C.,Yang, L.,Lin, L., et al.(2020). Effects of Native Fucosylated Glycosaminoglycan, its
Depolymerized Derivatives on Intrinsic Factor Xase, Coagulation,
Thrombosis and Hemorrhagic Risk. Thrombosis and Haemostasis .
doi:10.1055/s-0040-1708480