Comparative effects on anticoagulant activity and degree of fibrin polymerization of extracts from the new peony «Ivan Gorozhankin» and "Paeonia lactiflora"

Введение. Актуальность темы исследования обусловлена проблемой борьбы с тромбозами и тромбоэмболиями безопасными для организма методами. Во многих растениях обнаружены антикоагулянты разной природы (гепариноподобные, пептиды). Цель исследования - изучение возможности проявления синергических эффектов на антикоагулянтную и фибринолитическую активность крови и процессы полимеризации фибрина экстракта из корней пиона «Иван Горожанкин» в сравнительном аспекте с действием экстракта из корней пиона «молочноцветковый». Методика. Объектом исследования служили корни пионов «Иван Горожанкин» и «молочноцветковый», произрастающих в Ботаническом саду МГУ. Пион «Иван Горожанкин» был создан скрещиванием пиона «молочноцветкового» и «лекарственного» Разработаны методы получения экстрактов из корней различных пионов. При различных разведениях экстрактов (0.1, 1, 5%) определены антикоагулянтная активность по тестам, характеризующим внутренний, внешний и общий пути свертывания крови, а также степень полимеризации фибрина плазмы крови крыс. Для сравнения был использован стандартный препарат низкомолекулярного гепарина (LMWH) животного происхождения фирмы «Celsus» (США). Проведены выделение и очистка активного начала (гепариноидов) из сухих препаратов и измерены их активности. Pезультаты. Показано, что экстракты из обоих препаратов пионов обладали антикоагулянтной и суммарной фибринолитической активностью на нестабилизированном фибрине, но в разной степени. В экстрактах из корней пиона «Иван Горожанкин» отмечались преимущественные синергические эффекты, а именно превышение антикоагулянтной активности на 20-30%, суммарной фибринолитической - на 18% по сравнению с таковыми, отмечаемыми в экстрактах из корней пиона «молочноцветковый». Подобные результаты выявлены и при изучении степени полимеризации фибрина под влиянием очищенных препаратов из пионов. Рассмотрены возможные механизмы активирующего действия экстракта из пиона «Иван Горожанкин» на антикоагулянтные свойства плазмы, суммарную фибринолитическую активность и степень полимеризации фибрина. Это связано с блокадой активности тромбина и факторов внутреннего механизма свертывания крови. При этом антикоагулянтный эффект от применения экстракта из пиона «Иван Горожанкин» по тесту APTT (activated partial thromboplastin time) превышал на 20-30% ту же активность, выявленную у пиона «молочноцветковый», которая соответствовала антикоагулянтной активности препарата сравнения LMWH. В экстракте из пиона «Иван Горожанкин» впервые обнаружено наличие антикоагулянтного гепариноподобного вещества. Заключение. Впервые установлена способность экстракта из корней пиона «Иван Горожанкин» проявлять синергические антикоагулянтные и фибриндеполимеризационные эффекты, превышающие таковые у экстракта из пиона «молочноветковый». На основе полученных данных возникает необходимость исследования пиона «Иван Горожанкин» в качестве антитромботического, а возможно, и антиатеросклеротического агента. Introduction. The research topic is relevant due to the problem of safely combating thrombosis and thromboembolism. Anticoagulants of various kinds, e.g., heparin-like and peptides, have been found in many plants. Aim. To investigate the possibility of synergistic effects on the blood anticoagulant and fibrinolytic activity and on processes of fibrin polymerization by an extract from the roots of the «Ivan Gorozhankin» peony compared with the root extract from «Paeonia lactiflora». Methods. The focus of the study was the roots of the “Ivan Gorozhankin” peony and the Paeonia lactiflora growing in the Botanical Garden of the Moscow State University. The “Ivan Gorozhankin” peony was created by crossing P. lactiflora and the “medicinal” peony. Methods for obtaining extracts from the roots of various peonies have been developed. In 1%, 3%, and 5% dilutions of the extracts, the anticoagulant activity was determined according to tests characterizing the internal, external and general blood coagulation pathways, as well as by the degree of polymerization of rat blood plasma fibrin. For comparison, we used a standard preparation of low molecular weight heparin (LMWH) of animal origin (Celsus, USA). Isolation and purification of the active substances, heparinoids, were isolated from dry preparations and purified, and their activities were measured. Results. Extracts from both peony preparations had anticoagulant and total fibrinolytic activity on unstabilized fibrin, but to different extents. In the extracts from the roots of the “Ivan Gorozhankin” peony, preferential synergistic effects were noted, namely, the anticoagulant activity was higher by 20-30%, and the total fibrinolytic activity was higher by 18% compared to those of extracts from Paeonia lactiflora roots. Similar results were obtained when studying the degree of fibrin polymerization as influenced by purified peony preparations. Possible mechanisms of the activating action of the «Ivan Gorozhankin» peony extract on the anticoagulant properties of plasma, the total fibrinolytic activity, and the degree of fibrin polymerization are considered. This action is due to the inhibition of thrombin activity and factors of the internal mechanism of blood coagulation. According to the activated partial thromboplastin time (APTT) test, the anticoagulant effect of extracts from the «Ivan Gorozhankin» peony exceeded by 20-30% the activity of Paeonia lactiflora extract, which corresponded to the anticoagulant activity of the LMWH comparator drug. Using the described biochemical methods, the presence of an anticoagulant heparin-like substance in an extract from the peony «Ivan Gorozhankin» has been discovered. Conclusion. For the first time, the ability of an extract from the roots of the «Ivan Gorozhankin» peony to exhibit synergistic anticoagulant and fibrin-depolymerization effects was demonstrated. These effects exceeded those of the Paeonia lactiflora extract. Based on these data, it appears necessary to study the «Ivan Gorozhankin» peony as an antithrombotic, and possibly as an anti-atherosclerotic agent.

Differential sialic acid content in adult and neonatal fibrinogen mediates differences in clot polymerization dynamics

Neonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared to adults. Despite these differences, adult fibrinogen is used for treatment of bleeding in neonates, with mixed efficacy. In order to determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compare to those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin like enzymes (SVTLEs) that preferentially cleave fibrinopeptide A or B. We determined that sialic acid content of neonatal fibrinogen is a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal specific treatments of bleeding.

Lignosulfonic Acid Sodium Is a Noncompetitive Inhibitor of Human Factor XIa

The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC50 value of ~8 μg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis–Menten kinetics, LSAS decreased the VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa–antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure–activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.

Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

Plasma fibrinogen is an important coagulation factor and susceptible to post-translational modification by oxidants. We have reported impairment of fibrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fibrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We utilized polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels. We have demonstrated that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma fibrinogen solution disrupted lateral aggregation of protofibrils similarly to competitive inhibition of fibrin polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of fibrinogen protons demonstrated that the diffusion coefficient of fibrinogen increased by 17.4%, suggesting the oxidized fibrinogen was more compact and fast motion in the prefibrillar state. 2D-NMR analysis reflected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fibrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fibrin gels was dominated by intermediate water. Oxidation induced thinner fibers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confirmed that T2 relaxation is affected by the self-assembly of fibers and stiffness of the plasma fibrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fibrin clot.

Fibers Generated by Plasma Des-AA Fibrin Monomers and Protofibril/Fibrinogen Clusters Bind Platelets: Clinical and Nonclinical Implications

Objective Soluble fibrin (SF) is a substantial component of plasma fibrinogen (fg), but its composition, functions, and clinical relevance remain unclear. The study aimed to evaluate the molecular composition and procoagulant function(s) of SF. Materials and Methods Cryoprecipitable, SF-rich (FR) and cryosoluble, SF-depleted (FD) fg isolates were prepared and adsorbed on one hydrophilic and two hydrophobic surfaces and scanned by atomic force microscopy (AFM). Standard procedures were used for fibrin polymerization, crosslinking by factor XIII, electrophoresis, and platelet adhesion. Results Relative to FD fg, thrombin-induced polymerization of FR fg was accelerated and that induced by reptilase was markedly delayed, attributable to its decreased (fibrinopeptide A) FpA. FR fg adsorption to each surface yielded polymeric clusters and co-cryoprecipitable solitary monomers. Cluster components were crosslinked by factor XIII and comprised ≤21% of FR fg. In contrast to FD fg, FR fg adsorption on hydrophobic surfaces resulted in fiber generation enabled by both clusters and solitary monomers. This began with numerous short protofibrils, which following prolonged adsorption increased in number and length and culminated in surface-linked three-dimensional fiber networks that bound platelets. Conclusion The abundance of adsorbed protofibrils resulted from (1) protofibril/fg clusters whose fg was dissociated during adsorption, and (2) adsorbed des-AA monomers that attracted solution counterparts initiating protofibril assembly and elongation by their continued incorporation. The substantial presence of both components in transfused plasma and cryoprecipitate augments hemostasis by accelerating thrombin-induced fibrin polymerization and by tightly anchoring the resulting clot to the underlying wound or to other abnormal vascular surfaces.

Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

Plasma fibrinogen is an important coagulation factor that is susceptible to post-translational modification by oxidants. We have reported altered fibrin polymerization and increased methionine oxidation in fibrinogen after exposure to hypochlorous acid (HOCl), and similarly in the fibrinogen of severely injured trauma patients. Molecular dynamics suggests that methionine oxidation offers a mechanistic link between oxidative stress and coagulation through fibrin protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We used polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to test the hypothesis that HOCl oxidation alters fibrinogen conformation in the prefibrillar state and T2 water surface relaxation of fibrin fiber assemblies. We found that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma disrupted fibrin polymerization similarly to competitive inhibition of polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of 1H fibrinogen at 25oC demonstrated that fibrinogen oxidation increased translational diffusion coefficient by 17.4%, suggesting a more compact and rapidly translational motion of the protein with oxidation. 2D-NMR analysis of control plasma fibrin gels indicated that water existed in two states, namely intermediate (T2i) in the hydration shell of fibrin fibers, and bulk (T2) within the gel. T2 relaxation of bulk water protons was decreased 2-fold in oxidized fibrin gels and was inversely proportional to gel fiber density (T2). The fast exchange of water protons between hydration shell (T2i) and bulk water, indicating oxidation increased fiber hydration and formed densely packed fibrin gels. We have confirmed experimentally that HOCl oxidation affected native fibrinogen and fibrin gel structures and have demonstrated that NMR can serve as a valuable tool to probe the oxidative rearrangement of fibrin clot structure.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region

Objective: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Conclusions: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.

Factor XIII cross-links fibrin(ogen) independent of fibrin polymerization in experimental acute liver injury

Intravascular fibrin clot formation follows a well-ordered series of reactions catalyzed by thrombin cleavage of fibrinogen leading to fibrin polymerization and cross-linking by factor XIIIa (FXIIIa). Extravascular fibrin(ogen) deposits are observed in injured tissues; however, the mechanisms regulating fibrin(ogen) polymerization and cross-linking in this setting are unclear. The objective of this study was to determine the mechanisms of fibrin polymerization and cross-linking in acute liver injury induced by acetaminophen (APAP) overdose. Hepatic fibrin(ogen) deposition and cross-linking were measured following APAP overdose in wild-type mice, mice lacking the catalytic subunit of FXIII (FXIII-/-), and in FibAEK mice, which express mutant fibrinogen insensitive to thrombin-mediated fibrin polymer formation. Hepatic fibrin(ogen) deposition was similar in APAP-challenged wild-type and FXIII-/- mice yet cross-linking of hepatic fibrin(ogen) was dramatically reduced (>90%) by FXIII deficiency. Surprisingly, hepatic fibrin(ogen) deposition and cross-linking were only modestly reduced in APAP-challenged FibAEK mice, suggesting that in the APAP-injured liver fibrin polymerization is not strictly required for the extravascular deposition of cross-linked fibrin(ogen). We hypothesized that the oxidative environment in the injured liver, containing high levels of reactive mediators (e.g., peroxynitrite), modifies fibrin(ogen) such that fibrin polymerization is impaired without impacting FXIII-mediated cross-linking. Notably, fibrin(ogen) modified with 3-nitrotyrosine adducts was identified in the APAP-injured liver. In biochemical assays, peroxynitrite inhibited thrombin-mediated fibrin polymerization in a concentration-dependent manner without affecting fibrin(ogen) cross-linking over time. These studies depict a unique pathology wherein thrombin-catalyzed fibrin polymerization is circumvented to allow tissue deposition and FXIII-dependent fibrin(ogen) cross-linking.