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Bijak, M., R. Ziewiecki, J. Saluk, M. Ponczek, I. Pawlaczyk, H. Krotkiewski, B. Wachowicz, and P. Nowak. "Thrombin inhibitory activity of some polyphenolic compounds." Medicinal Chemistry Research 23, no. 5 (October 16, 2013): 2324–37. http://dx.doi.org/10.1007/s00044-013-0829-4.
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Wang, Xiaoyan, Zhen Yang, Feifei Su, Jin Li, Evans Owusu Boadi, Yan-xu Chang, and Hui Wang. "Study on Structure Activity Relationship of Natural Flavonoids against Thrombin by Molecular Docking Virtual Screening Combined with Activity Evaluation In Vitro." Molecules 25, no. 2 (January 20, 2020): 422. http://dx.doi.org/10.3390/molecules25020422.
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Thrombin, a key enzyme of the serine protease superfamily, plays an integral role in the blood coagulation cascade and thrombotic diseases. In view of this, it is worthwhile to establish a method to screen thrombin inhibitors (such as natural flavonoid-type inhibitors) as well as investigate their structure activity relationships. Virtual screening using molecular docking technique was used to screen 103 flavonoids. Out of this number, 42 target compounds were selected, and their inhibitory effects on thrombin assayed by chromogenic substrate method. The results indicated that the carbon-carbon double bond group at the C2, C3 sites and the carbonyl group at the C4 sites of flavones were essential for thrombin inhibition, whereas the methoxy and O-glycosyl groups reduced thrombin inhibition. Noteworthy, introduction of OH groups at different positions on flavonoids either decreased or increased anti-thrombin potential. Myricetin exhibited the highest inhibitory potential against thrombin with an IC50 value of 56 μM. Purposively, the established molecular docking virtual screening method is not limited to exploring flavonoid structure activity relationships to anti-thrombin activity but also usefully discovering other natural active constituents.
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KINOSHITA, Akemi, and Noboru HORIE. "Inhibitory Activity of Green Tea Catechins on Thrombin." Japanese Journal of Thrombosis and Hemostasis 4, no. 6 (1993): 417–22. http://dx.doi.org/10.2491/jjsth.4.417.
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Žula, Aleš, Izabela Będziak, Danijel Kikelj, and Janez Ilaš. "Synthesis and Evaluation of Spumigin Analogues Library with Thrombin Inhibitory Activity." Marine Drugs 16, no. 11 (October 27, 2018): 413. http://dx.doi.org/10.3390/md16110413.
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Spumigins are marine natural products derived from cyanobacteria Nodularia spumigena, which mimics the structure of the d-Phe-Pro-Arg sequence and is crucial for binding to the active site of serine proteases thrombin and factor Xa. Biological evaluation of spumigins showed that spumigins with a (2S,4S)-4-methylproline central core represent potential lead compounds for the development of a new structural type of direct thrombin inhibitors. Herein, we represent synthesis and thrombin inhibitory activity of a focused library of spumigins analogues with indoline ring or l-proline as a central core. Novel compounds show additional insight into the structure and biological effects of spumigins. The most active analogue was found to be a derivative containing l-proline central core with low micromolar thrombin inhibitory activity.
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Panda, Subhamay, and Iman Ehsan. "MOLECULAR DOCKING STUDIES OF SNAKE VENOM SERINE PROTEASE OF SHARP-NOSED PIT VIPER WITH HESPERETIN." Asian Journal of Pharmaceutical and Clinical Research 11, no. 6 (June 7, 2018): 457. http://dx.doi.org/10.22159/ajpcr.2018.v11i6.25531.
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Objective: The management of snake bite envenomation is a global challenge affecting millions of people. Immunotherapy is still regarded as the treatment of choice; however, their subsequent adverse effects restrict their potential use for therapy against snake venom poisoning. In recent years, more attention has been given to the exploration of indigenous medicinal plants for their outstanding benefits for the treatment of several diseases and disorders, including snake venom poisoning. Hesperetin is a naturally occurring compound derived from a flavanone glycoside, hesperidin and is obtained from various citrus fruits. It is known to possess significant inhibitory activity against snake venom serine proteases. The aim of our present study was to investigate the significant inhibitory action of hesperetin on thrombin-like serine protease from sharp-nosed pit viper (Deinagkistrodon acutus) snake venom.Methods: We have employed molecular docking analysis by implementing the state-of-the-art docking program to validate the hypothesis of the prospective inhibitory properties of hesperetin on thrombin-like serine proteases of snake venom. AutoDock 4.2, InterProSurf, MGLTools, and MTiAutoDock were utilized for the molecular docking analysis of thrombin-like serine protease obtained from the snake venom of sharp-nosed pit viper with the natural compound hesperetin.Results: The results generated from in silico based approach reveals the significant inhibitory role of hesperetin against thrombin-like snake venom proteases, which might lead to the drug designing of the inhibitors of snake venom serine proteases.Conclusion: The implementation of molecular docking analysis by the employment of state-of-the-art docking program supports the potential of inhibitory activity of naturally obtained hesperetin compound on thrombin-like serine proteases of snake venom. The generated in silico results suggests that the novel structure hesperetin - flavanone might act as a potent inhibitor of thrombin-like snake venom proteases, and unlocks the possibilities for designing drugs of the inhibitors of snake venom serine proteases. Moreover, the investigation of the novel compound obtained from natural sources for their inhibitory activity against snake venom serine proteases would lead to the discovery of newer inhibitory compound from a highly uninvestigated research arena.
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ohishi, Rika, Naoko watanabe, Masaharu Aritomi, Komakazu Gomi, Takao Kiyota, Shuji Yamamoto, Torao lshida, and Ikuro Maruyama. "Evidence that the Protein C Activation Pathway Amplifies the lnhibition of Thrombin Generation by Recombinant Human Thrombomodulin in Plasma." Thrombosis and Haemostasis 70, no. 03 (1993): 423–26. http://dx.doi.org/10.1055/s-0038-1649598.
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SummaryThrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.
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Danihelová, Martina, Miroslav Veverka, and Ernest Šturdík. "Inhibition of pathophysiological proteases with novel quercetin derivatives." Acta Chimica Slovaca 6, no. 1 (April 1, 2013): 115–22. http://dx.doi.org/10.2478/acs-2013-0018.
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Abstract Novel quercetin derivatives were prepared to change its physicochemical properties and effects on activity of proteolytic enzymes. For them preparation, the selective protection procedures some of the quercetin hydroxyl groups and acylation of the others with acylchlorides were used. The ability of these compounds to inhibit the activity of serine proteases e.g. trypsin, thrombin, urokinase and elastase was studied. In micromolar range, tested derivatives were the most potent inhibitors of thrombin. There was estimated better inhibition of thrombin for prenylated, acetylated, feruloyl and caffeoyl quercetin esters. Slight inhibitory effect of all quercetin derivatives on elastase was found. Among tested derivatives only diquercetin displayed better inhibiton. Trypsin and urokinase were inhibited by quercetin at comparable level. Slight improvement in inhibitory effect of trypsin and urokinase was seen for chloronaphtoquinone quercetin that revealed enhanced inhibiton of thrombin, too. However, no influence on elastase activity was determined for this compound. Obtained results indicate that certain modifications of quercetin structure could improve its biological properties.
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Gerotziafas, Grigorios T., Galea Vasso, Jeanine M. Walenga, Evi Kalodiki, Mourad Chaari, Hatmi Mohamed, Jawed Fareed, and Elalamy Ismail. "Thrombin Generation Assessment in Platelet Rich Plasma Offers Additional Criteria for Low Molecular Weight Heparin Antithrombotic Fingerprint Characterization." Blood 114, no. 22 (November 20, 2009): 4177. http://dx.doi.org/10.1182/blood.v114.22.4177.4177.
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Abstract Abstract 4177 Introduction National and international organizations recognize that low molecular weight heparins (LMWHs) are distinct entities and that they should not be used interchangeably in clinical practice. Physicochemical properties, such as molecular weight anti-Xa/anti-IIa ratios, are used by health authorities to establish LMWHs differentiation. LMWHs are multitargeted drugs in which small differences in physicochemical properties may lead to significantly different inhibition of blood coagulation. In the present in vitro study we have investigated if thrombin generation assay, reflecting the overall coagulation process, is a suitable tool for the LMWH variability evalution. Materials and methods LMWHs (bemiparin, enoxaparin, nadroparin, dalteparin and tinzaparin) and unfractionated heparin (UFH) were added in vitro in normal citrated platelet rich plasma from 10 healthy individuals, at clinically relevant concentrations (ranging from 0.2 to 1 anti-Xa IU/ml). Thrombin generation was studied with Thrombogram-Thrombinoscope® assay using diluted thromboplastin (Innovin®, 1/1000 final dilution, Siemens France). The concentrations doubling the lag-time, the time to Peak, the IC50 for Peak, the endogenous thrombin potential (ETP) and the mean rate index of propagation phase (MRI) were determined for each compound. Results LMWHs compared on their anti-Xa activity basis showed variable inhibitory effect on thrombin generation. At equivalent anti-Xa concentrations tinzaparin was significantly more potent than the other LMWHs, being almost similar to UFH profile. Enoxaparin, nadroparin and dalteparin showed a similar inhibitory activity. Bemiparin had the lesser pronounced inhibitory effect on thrombin generation. The impact of anti-Xa and anti-IIa activities on each phase of thrombin generation process is different. Thrombogram chronometric parameters are mainly influenced by the anti-IIa activity. Similarly, ETP inhibition depends more on anti-IIa rather than anti-Xa activity. The MRI of the propagation phase is more sensitive to the anti-Xa activity of LMWHs. Conclusion Thrombin generation assessment in platelet rich plasma allows to evaluate the anticoagulant fingerprint of LMWHs and to differentiate them on global and functional criteria. Each LMWH has a particular inhibitory impact on each phase of thrombin generation process. These functional criteria need to be standardized and probably required for a better characterization of LMWHs heterogeneity by health authorities. They could be used also to evaluate bioequivalence of generic and original LMWHs. Disclosures: No relevant conflicts of interest to declare.
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Kang, Kyu-Tae, Byoung-In Jung, Ok-Nam Bae, Moo-Yeol Lee, Seung-Min Chung, Joo-Young Lee, Sang-Koo Lee, In-Chul Kim, and Jin-Ho Chung. "Antithrombotic activity of LB30057, a newly synthesized direct thrombin inhibitor." Thrombosis and Haemostasis 89, no. 01 (2003): 104–11. http://dx.doi.org/10.1055/s-0037-1613549.
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SummaryAn amidrazonophenylalanine derivative, LB30057, inhibits the catalytic activity of thrombin potently by interaction with the active site of thrombin, and has high water solubility. In the present study, we evaluated the effect of LB30057 on the biological activities of thrombin at various tissues, and determined whether thrombin inhibition by LB30057 could reduce the incidence of occlusive thrombosis in an in vivo animal model. Treatment with LB30057 to human plasma prolonged clotting times in a concentration-dependent manner. LB30057 suppressed significantly thrombin-induced phosphatidylserine (PS) exposure in platelets, suggesting that LB30057 could inhibit blood coagulation accelerated by PS exposure. In human platelets, soluble thrombin- and clot-induced platelet aggregation was inhibited by LB30057 potently. Consistent with this finding, LB30057 showed concentration-dependent inhibitory effects on serotonin secretion and P-selectin expression induced by thrombin in platelets. In the blood vessel isolated from the guinea pig, treatment with LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular contraction. In vivo study revealed that LB30057 following oral administration significantly increased the time to occlusion and improved carotid arterial patency using rat carotid artery thrombosis model. All these results suggest that LB30057 is a potent inhibitor of biological activities of thrombin at various target tissues and, therefore, might be developed as an anti-thrombotic agent for treatment and prevention of thrombotic diseases.
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Maclean, Glenn, Stephanie Brister, and Michael Buchanan. "Selective and Sustained Inhibition of Surface-bound Thrombin Activity by Intimatan/Heparin Cofactor II and Its Relevance to Assessing Systemic Anticoagulation In Vivo, Ex Vivo and In Vitro." Thrombosis and Haemostasis 86, no. 09 (2001): 909–13. http://dx.doi.org/10.1055/s-0037-1616149.
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SummaryWe compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001.We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.
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Römisch, Jürgen, Hans-Arnold Stöhr, Harald Stauß, Rainer Koschinsky, Werner Stüber, and Eric-Paul Pâques. "Inhibition of In Vitro Clot Growth by r-Hirudin Is More Effective and Longer Sustained than by an Analogous Peptide." Thrombosis and Haemostasis 71, no. 03 (1994): 320–24. http://dx.doi.org/10.1055/s-0038-1642437.
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SummaryThe specific thrombin inhibitors r-hirudin and a synthetic peptide (I) D -FPRP(G)4-NGDFEEIPEEYL were compared in in vitro tests, r-hirudin proved to be the superior compound with respect to inhibition of amidolytic small substrate turnover that is catalysed by soluble and immobilised thrombin as well as to inhibition of fibrinogen activation. In an in vitro clot model significantly higher molar concentrations of peptide I are needed to achieve fibrin bound thrombin inhibition equivalent to that of r-hirudin. Stable complexes consisting of thrombin and hirudin oppose labile complexes containing the synthetic peptide. The latter leads to a regaining of thrombin activity with subsequent additional fibrin accretion. Analyses of the mixtures of thrombin and peptide I display a time dependent release of amino-terminal D -FPR peptide (III) exhibiting, similar to the residual fragment (peptide II), only weak inhibitory activity. Peptide I and the carboxy-terminal fragment induce, within a certain concentration range, an increase in thrombin activity and clot growth.
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Clayton, Daniel, Sameer S. Kulkarni, Jessica Sayers, Luke J. Dowman, Jorge Ripoll-Rozada, Pedro José Barbosa Pereira, and Richard J. Payne. "Chemical synthesis of a haemathrin sulfoprotein library reveals enhanced thrombin inhibition following tyrosine sulfation." RSC Chemical Biology 1, no. 5 (2020): 379–84. http://dx.doi.org/10.1039/d0cb00146e.
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Swedenborg, Jesper, Maciej Dryjski, Siw Frebelius, and Per Olsson. "Uptake and Inactivation of Thrombin on Rabbit Aortic Endothelium Studied with Two Different Substrates." Thrombosis and Haemostasis 54, no. 04 (1985): 828–32. http://dx.doi.org/10.1055/s-0038-1660142.
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SummaryThe endothelium is an important compartment for uptake and inhibition of thrombin. The amount of enzymatically active bound thrombin can be detected with both small synthetic substrates and with aid of fibrinogen as substrate. The present study was designed to investigate the relation between endothelially bound thrombin with amidolytic activity towards a synthetic substrate (S-2238) and thrombin capable of converting fibrinogen by measuring generation of fibrinopeptide A (FPA). The luminal surfaces of rabbit aortae (2 cm2) were exposed in vitro to thrombin (0.625-5.0 NIH units/ml). Thrombin disappeared from the solution and a certain fraction was recovered on the surface. There was a linear relationship between the amount of thrombin on the surface and the concentration of thrombin in the incubation mixture. Approximately one third of the thrombin measured with S-2238 was also able to cleave fibrinogen. After incubation with defibrinogenated plasma almost total inhibition of fibrinogen splitting activity occurred within 30 sec. The inhibition of the amidolytic activity was less complete. When endothelially bound thrombin was exposed to plasma much less FPA was generated than in a fibrinogen solution. A minor fraction of endothelially bound thrombin was inhibited also upon incubation with Tyrode without recovery of any enzymatic activity in the solution. The results indicate that a fraction of thrombin bound to the endothelium has retained enzymatic activity and that a fraction of the enzymatically active thrombin is capable of converting fibrinogen. Inhibition of thrombin enzymatic activity occurs rapidly upon exposure to plasma. The endothelium itself has a minor inhibitory effect also in the absence of plasma. Thrombin with retained fibrinogen splitting activity may contribute to thrombotic events particularly if plasma inhibitors are insufficient. Thrombin with activity towards fibrinogen could be bound to GAG:s whereas thrombin active towards S-2238 could be bound to GAGrs TM or possibly other receptors.
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Longstaff, Colin, Man-Yu Wong, and Patrick J. Gaffney. "An International Collaborative Study to Investigate Standardisation of Hirudin Potency." Thrombosis and Haemostasis 69, no. 05 (1993): 430–35. http://dx.doi.org/10.1055/s-0038-1651628.
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SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.
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Glusa, Erika, Ellen Bretschneider, Joachim Daum, and Christiane Noeske-Jungblut. "Inhibition of Thrombin-mediated Cellular Effects by Triabin, a Highly Potent Anion-binding Exosite Thrombin Inhibitor." Thrombosis and Haemostasis 77, no. 06 (1997): 1196–200. http://dx.doi.org/10.1055/s-0038-1656137.
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SummaryTriabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 μM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF2γ, thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endothelium- denuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used.These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects.
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Lindhout, Theo, Ron Blezer, and H. Coenraad Hemker. "The Anticoagulant Mechanism of Action of Recombinant Hirudin (CGP 39393) in Plasma." Thrombosis and Haemostasis 64, no. 03 (1990): 464–68. http://dx.doi.org/10.1055/s-0038-1647337.
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SummaryWe studied the inhibitory action of recombinant desulphatohirudin (CGP 39393) on thrombin generation in whole plasma. Human plasma was activated either with thromboplastin or factor IXa. Hirudin delayed thrombin generation, but it was unable to prevent the explosive appearance of thrombin. The dose-dependent prolongation of the lag phase of the intrinsic and extrinsic thrombin generation curve was not the result of titration of thrombin activity by hirudin but the result of a delayed formation of the prothrombin converting complex (prothrombinase). In case of extrinsic activation, hirudin did not affect factor Xa generation, but prolonged the lag phase of the factor Va generation curve, causing its appearance when factor Xa generation was already in the decay phase. Because of its inhibitory action on the thrombin-mediated activation of factor VIII, hirudin prolonged the lag phase of the factor X converting complex that consists of factor IXa and factor VIIIa. Our observations with hirudin are in keeping with the notion that inhibition of the thrombin-mediated amplification reactions in blood coagulation is a very efficient way to delay or inhibit completely thrombin generation. However, although hirudin neutralizes stoichiometric amounts of thrombin, the interaction between in situ generated thrombin and hirudin appears not to be fast enough to prevent trace amounts of thrombin to activate factors VIII and V. Consequently, an explosive thrombin generation is observed even when free hirudin is present.
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JUšKA, Alfonsas, and Richard W. FARNDALE. "Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data." Biochemical Journal 340, no. 1 (May 10, 1999): 245–53. http://dx.doi.org/10.1042/bj3400245.
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Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 25-32] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC.
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Taira, Kenichi, Koichi Abe, Takayuki Ishibasi, Katsuaki Sato, and Kazunori Ikebukuro. "Control of Aptamer Function Using Radiofrequency Magnetic Field." Journal of Nucleic Acids 2011 (2011): 1–6. http://dx.doi.org/10.4061/2011/103872.
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Remote control of aptamer function has allowed us to control protein function in space and time. Here, we propose a novel control system for aptamer function by radiofrequency magnetic field- (RFMF-) induced local heating of a gold nanoparticle conjugated with an aptamer. In this study, we used a 31-mer thrombin-binding aptamer (TBA), which can inhibit thrombin activity, as a model aptamer. We evaluated the RFMF control of the inhibitory activity of a gold nanoparticle-conjugated TBA. To evaluate the effect of RFMF on enzymatic activity, we utilized a complementary DNA strand that maintains the broken structure during the activity assay. We observed a decrease in the inhibitory activity of TBA after RFMF irradiation. It indicates that RFMF is capable of controlling the TBA structure. Because RFMF allows noninvasive control of aptamer function, this strategy is expected to be novel way of controlling aptamer drug activity.
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GABAZZA, Esteban C., Tatsuya HAYASHI, Masaru IDO, Yukihiko ADACHI, and Koji SUZUKI. "Adenosine inhibits thrombin-induced expression of tissue factor on endothelial cells by a nitric oxide-mediated mechanism." Clinical Science 102, no. 2 (January 3, 2002): 167–75. http://dx.doi.org/10.1042/cs1020167.
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Enhanced expression of tissue factor (TF) is associated with the occurrence of coronary disease, strokes and arterial thrombosis. We demonstrated previously that adenosine inhibits TF expression in human umbilical vein endothelial cells (HUVECs) stimulated with inflammatory mediators. In the present study, we evaluated the mechanism of adenosine-induced inhibition of TF expression in HUVECs. The adenosine inhibitory activity on thrombin-induced TF expression in HUVECs was potentiated by the NO precursor, l-arginine, but it was significantly suppressed by the NO scavenger, 2(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, and by endothelial NO synthase inhibitors, NG-monomethyl-l-arginine and NG-nitro-l-arginine methyl ester, in a dose-dependent manner. The concentrations of nitrites, cGMP and cAMP in the culture medium of HUVECs treated with a mixture of thrombin and adenosine were significantly higher compared with the culture medium of HUVECs treated with thrombin alone. Northern blotting showed that thrombin decreases and adenosine increases the eNOS mRNA expression in HUVECs. A cAMP-dependent protein kinase inhibitor suppressed NO-mediated TF inhibition in a dose-dependent manner. Overall, these results suggest that adenosine inhibits thrombin-induced TF expression in endothelial cells by a NO-mediated mechanism, and that increased intracellular formation of cAMP is implicated in this inhibitory activity of NO.
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Berry, Christopher N., Christine Girardot, Catherine Lecoffreo, and Catherine Lunven. "Effects of the Synthetic Thrombin Inhibitor Argatroban on Fibrin- or Clot-Incorporated Thrombin: Comparison with Heparin and Recombinant Hirudin." Thrombosis and Haemostasis 72, no. 03 (1994): 381–86. http://dx.doi.org/10.1055/s-0038-1648875.
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SummaryThe inhibitory effects of argatroban on clot- or fibrin-bound human thrombin were studied using the thrombin-specific chromogenic substrate S2238 (200 μM). These effects were compared to those of recombinant hirudin (rHV2 Lys 47) and the heparin/antithrombin III complex. Argatroban concentration-dependently inhibited the cleavage of S2238 by a thrombin solution, which had been titrated to give the same change in OD405 nm as fibrin-bound thrombin, with an IC50 of 1.1 μM with 90% inhibition at 8 μM. rHV2 Lys 47 and heparin had IC50 values of 1.2 nM and 0.003 U/ml respectively under these conditions. However, when the compounds were tested against fibrin-bound thrombin, argatroban had an IC50 of 2.8 μM with 65% inhibiton at 8 μM, whereas rHV2 Lys 47 had an IC50 of 23 nM (with only 56% inhibition at 200 nM), and heparin had an IC50 of 0.5 α 0.38 U/ml (with only 58% inhibition at 5 U/ml); i. e. the two compounds were 19 and 168 times less active against fibrin-bound thrombin than against thrombin in solution. The differences between the inhibitory effects of the compounds against thrombin bound to a plasma clot were even more striking in that the IC50 of argatroban was increased from 1.1 (vs. thrombin in solution) to 2.7 μM, while, although rHV2 Lys 47 and heparin had IC50 values of 2.8 nM and 0.004 U/ml against thrombin in solution, they had little (32% inhibition by 4 pM rHV2 Lys 47) or no effect (even at 5.0 U/ml heparin) against the amidolytic activity of a plasma clot. We conclude that argatroban could present advantages over hirudin and heparin in the treatment of pathologies where the enzymatic activity of clot-bound thrombin may play a significant role.
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Houslay, M. D., D. Bojanic, D. Gawler, S. O'Hagan, and A. Wilson. "Thrombin, unlike vasopressin, appears to stimulate two distinct guanine nucleotide regulatory proteins in human platelets." Biochemical Journal 238, no. 1 (August 15, 1986): 109–13. http://dx.doi.org/10.1042/bj2380109.
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The thrombin-stimulated GTPase activity of human platelets was additive with respect to the GTPase stimulation effected by prostaglandin E1, but not with that stimulated by adrenaline, vasopressin and platelet-activating factor (PAF). Treatment of platelet membranes with pertussis toxin partially inhibited the thrombin-stimulated GTPase, but had no effect on the vasopressin-stimulated GTPase activity, whereas cholera toxin treatment had no effect on either of these stimulated GTPase activities. Thrombin, adrenaline and PAF, but not vasopressin, inhibited the adenylate cyclase activity of isolated plasma membranes through the action of Ni only, this being inhibited by pertussis toxin. It is suggested that thrombin exerts effects through both the inhibitory guanine nucleotide regulatory protein Ni and through the putative guanine nucleotide regulatory protein, Np, involved in regulating receptor-stimulated inositol phospholipid metabolism. However, vasopressin appears to exert its effects solely through the putative Np.
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Tripodi, A., A. Krachmalnicoff, and P. M. Mannucci. "Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding." Thrombosis and Haemostasis 56, no. 03 (1986): 349–52. http://dx.doi.org/10.1055/s-0038-1661681.
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SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.
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Pahl, M. V., N. D. Vaziri, F. Oveisi, J. Wang, and Y. Ding. "Antithrombin III inhibits mesangial cell proliferation." Journal of the American Society of Nephrology 7, no. 10 (October 1996): 2249–53. http://dx.doi.org/10.1681/asn.v7102249.
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Thrombin stimulates and heparin and heparan sulfate inhibit mesangial cell proliferation. In addition, heparin has been shown to inhibit thrombin-stimulated smooth muscle cell proliferation. The anticoagulant action of heparin is mediated by antithrombin III. This study investigated whether heparin's antiproliferative action is also mediated by antithrombin III. To this end, the effect of antithrombin III on thrombin-stimulated mesangial cell growth was examined. As expected, thrombin stimulated DNA synthesis and cell growth in cultured human mesangial cells. The effect of thrombin on DNA synthesis and mesangial cell proliferation was inhibited by standard heparin and antithrombin III, separately or together. The magnitude of the inhibitory action of antithrombin III was equal to that of equimolar concentrations of heparin and that observed with the combination of the two. Experiments carried out in serum (hence antithrombin III)-free medium revealed that heparin's inhibitory effects are independent of antithrombin III. It was concluded that antithrombin III, an endogenous inhibitor of thrombin's coagulant activity, is an equally effective inhibitor of thrombin's mitogenic action.
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Krošlák, Erik, Tibor Maliar, Mária Maliarová, Peter Nemeček, Peter Hozlár, Miroslav Ondrejovič, Michaela Havrlentová, and Ján Kraic. "Antioxidant and protease-inhibitory potential of extracts from grains of oat." Open Chemistry 14, no. 1 (January 1, 2016): 324–34. http://dx.doi.org/10.1515/chem-2016-0035.
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AbstractThe most of important crops cultivated for production of foods and feeds could be considered as plants possessing nutraceutical or medically interesting compounds, especially if can be eaten without processing. Chemical and biological parameters that were evaluated in 100 oat (Avena sativa L.) genotypes were others than those that are important in food and feed production. Contents of polyphenols and flavonoids, radical scavenging activity (DPPH), and inhibitory activities against five proteases (trypsin, thrombin, urokinase, elastase, cathepsin B) were analyzed in extracts from mature grains. The antioxidant activity (DPPH) correlated to the content of total polyphenols. Only a minority (15 from 100) of analyzed genotypes created separate subgroup with a high content of polyphenols, flavonoids, and high antioxidant activity. The best in these parameters were genotypes CDC-SOL-FI, Saul, and Avesta, respectively. Fifteen other genotypes assembled another minority subgroup (also 15 from 100) on the basis of their high inhibitory activities against tested proteases. The highest trypsin-, urokinase-, and elastase-inhibitory activities were in genotype Racoon, the best in thrombin-, and cathepsin B-inhibitory activities were genotypes Expression and SW Kerstin, respectively. Three oats genotypes – Rhea, AC Percy, and Detvan appeared in both subgroups.
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Gao, Yuye, Xie-an Yu, Bing Wang, Guo Yin, Jue Wang, Tiejie Wang, and Kaishun Bi. "Based on Multi-Activity Integrated Strategy to Screening, Characterization and Quantification of Bioactive Compounds from Red Wine." Molecules 26, no. 21 (November 8, 2021): 6750. http://dx.doi.org/10.3390/molecules26216750.
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According to French Paradox, red wine was famous for the potential effects on coronary heart disease (CHD), but the specific compounds against CHD were unclear. Therefore, screening and characterization of bioactive compounds from red wine was extremely necessary. In this paper, the multi-activity integrated strategy was developed and validated to screen, identify and quantify active compounds from red wine by using ultra high performance liquid chromatography-fraction collector (UHPLC-FC), ultra fast liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UFLC-Q-TOF/MS) and bioactive analysis. UHPLC-FC was employed to separate and collect the components from red wine, which was further identified by UFLC-Q-TOF/MS to acquire their structural information. Furthermore, the active fractions were tested for antioxidant activity, inhibitory activity against thrombin and lipase activities in vitro by the activity screening kit. As the results, there were 37 fractions had antioxidant activity, 22 fractions had thrombin inhibitory activity and 28 fractions had lipase inhibitory activity. Finally, 77 active components from red wine were screened and 12 ingredients out of them were selected for quantification based on the integration of multi-activity. Collectively, the multi-activity integrated strategy was helpful for the rapid and effective discovery of bioactive components, which provided reference for exploring the health care function of food.
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Kovalishyn, Vasyl, Vsevolod Tanchuk, Iryna Kopernyk, Volodymyr Prokopenko, and Larysa Metelytsia. "Prediction of Thrombin and Factor Xa Inhibitory Activity with Associative Neural Networks." Current Computer Aided-Drug Design 10, no. 3 (March 2, 2015): 259–65. http://dx.doi.org/10.2174/157340991003150302231419.
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Wang, Min, Yuanyuan Wang, Jinfeng Wang, Minji Zou, Shen Liu, Tao Xu, Xin Cai, Chen Wu, Jiaxi Wang, and Donggang Xu. "Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity." Biotechnology Letters 31, no. 12 (August 15, 2009): 1923–27. http://dx.doi.org/10.1007/s10529-009-0094-2.
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Ren, Weixin, Yujie Ren, Minghui Dong, and Yonghong Gao. "Design, Synthesis, and Thrombin Inhibitory Activity Evaluation of Some Novel Benzimidazole Derivatives." Helvetica Chimica Acta 99, no. 4 (April 2016): 325–32. http://dx.doi.org/10.1002/hlca.201500527.
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Cateni, Francesca, Marina Zacchigna, Bojan Doljak, Marko Anderluh, Giuseppe Procida, and Andrej Piltaver. "Bioactive Lipids Metabolites in Amanita Virosa." Natural Product Communications 7, no. 11 (November 2012): 1934578X1200701. http://dx.doi.org/10.1177/1934578x1200701121.
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Thrombin is the key serine proteinase of the coagulation cascade and, therefore, a suitable target for inhibition of blood coagulation. An extract of Amanita virosa considerably inhibited thrombin (48%), but showed no inhibitory activity on trypsin. On the basis of inhibition selectivity between thrombin and trypsin and potency of thrombin inhibition, A. virosa constitutes a good starting material for the isolation of further compounds that are active against thrombin. Bioassay oriented fractionation of the extract of A. virosa led to the isolation of a complex mixture of triglycerides (TGs), monoacylglycerols (MAGs), free fatty acids (FAs) and ergosterol. The structures of the isolated lipids metabolites were determined on the basis of chemical and spectroscopic evidences.
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Farrell, David H., Rehana S. Lovely, and Lynn K. Boshkov. "Inhibition of Thrombin Cleavage of Factor VIII by Fibrinogen γ’ Chain Carboxyl Terminus." Blood 106, no. 11 (November 16, 2005): 1957. http://dx.doi.org/10.1182/blood.v106.11.1957.1957.
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Fibrinogen can contain two types of γ chains, γA and γ’, that have identical sequences for the first 407 amino acids, but differ at their carboxyl termini. The γA chain contains four amino acids from γA 408–411, whereas the γ’ chain contains a different, highly anionic sequence of twenty amino acids from γ’ 408–427. This small change results in profound differences in the fibrinogen isoforms containing these distinct chains. The γ’ chain contains high affinity binding sites for zymogen factor XIII and for thrombin, and fibrinogen containing γ’ chains forms fibrin clots that are resistant to fibrinolysis. We have shown previously that the fibrinogen γ’ chain carboxyl terminus binds to thrombin exosite II, an anion-binding exosite known for its heparin binding activity. Recent reports show that the presence of the γ’ chain in fibrinogen reduces prothrombin activation in whole plasma assays. This inhibition may correspond to an activity originally described by Seegers in 1945 as “antithrombin I”, the adsorption of thrombin to fibrin. We have therefore investigated the enzymatic effects of γ’ peptide binding to thrombin in order to determine the role of the γ’ chain in the inhibition of prothrombin activation. In whole plasma, the γ’410–427 peptide prolonged the activated partial thromboplastin time (aPTT) in a dose-dependent manner, causing a delay in clotting from 29 seconds to 47 seconds at a concentration of 500 μM. A scrambled control peptide at the same concentration had little effect, increasing the clotting time to only 31.4 seconds. A series of γ’ deletion peptides were tested for their ability to prolong the aPTT. The inhibitory effect of the peptides corresponded in rank order to their thrombin binding affinity. In contrast to the aPTT, the prothrombin time increased only slightly from 11.7 seconds to 12.8 seconds (INR from 1.04 to 1.14) with the γ’410–427 peptide. This suggested that the γ’ peptide exerted its inhibitory effect on (a) component(s) of the intrinsic pathway, rather than the extrinsic pathway. We therefore investigated the effect of the γ’ peptide on thrombin activation of factor VIII, a thrombin substrate that, unlike factor V, is unique to the intrinsic pathway. Using purified factor VIII and thrombin in vitro, we showed that the γ’ peptide inhibited thrombin cleavage of factor VIII. However, this inhibitory effect was not due to direct inhibition of the thrombin active site, since the free γ’ peptide had little effect on thrombin cleavage of a small peptidyl substrate, tosyl-glycyl-prolyl-arginine-4-nitranilide acetate. These results suggest that factor VIII interactions with thrombin exosite II are essential for efficient cleavage of factor VIII, and support recent findings by others that interactions between thrombin exosite II and the A2 domain of factor VIII facilitate thrombin-catalyzed cleavage. These findings may explain, at least in part, the inhibitory effect of the γ’ chain on prothrombin activation in plasma.
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Boross, Peter, Cafer Yildiz, Kamran Bakhtiari, Joost C. M. Meijers, and C. Erik Hack. "Towards Restoring Coagulation in Hemophilia By Antibody-Mediated Inhibition of Antithrombin." Blood 126, no. 23 (December 3, 2015): 550. http://dx.doi.org/10.1182/blood.v126.23.550.550.
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Abstract Currently hemophilia A is mainly treated with factor VIII (FVIII). However, 20-25% of the hemophiliacs develop inhibiting antibodies (inhibitors) against FVIII. Hemophiliacs with inhibitors are treated with high dose FVIII or with therapies that bypass the need for FVIII such as activated FVII (rFVIIa, NovoSeven) or activated prothrombin concentrate. These have some disadvantages including rapid clearance, price and a risk of thrombo-embolism. In hemophilia, the balance between coagulation and anticoagulation is shifted towards the latter. This balance can be restored by administration of clotting factors but also by reducing anticoagulation mechanisms. Antithrombin (AT) constitutes a main anticoagulant mechanism as it inhibits thrombin, FXa and other activated clotting factors. In silico studies as well as observations that AT deficiency reduces bleeding in hemophilic mice and humans strongly support the concept that lowering functional AT levels bypasses the need for FVIII to generate thrombin. A Phase 1 clinical trial by Alnylam Pharmaceuticals showed that lowering AT activity by RNA-interference can boost thrombin generation in hemophilia. However, RNA-interference is an experimental therapy with unknown long term effects and is not suitable for acute treatment of bleeding as it takes weeks to become effective. We hypothesize that monoclonal antibodies (mAbs) that block AT activity can be used as FVIII bypass therapy. Such a mAb-based therapy is suitable for on demand and for prophylactic therapy, is relatively cheap and does not require administration of activated clotting factors. We have generated a panel of mouse anti-AT mAbs exhibiting varying binding characteristics to human AT. To assess the inhibitory activity of anti-AT mAbs, FXa activity assay was performed after FXa was incubated with normal plasma supplemented with the different anti-AT mAbs. Five anti-AT mAbs were identified that inhibit AT activity at around equimolar ratio. Inhibitory anti-AT mAbs were able to dose-dependently enhance thrombin generation in normal and hemophilic plasma. Heparin caused a marked prolongation of aPTT of normal and FVIII-deficient plasma, which was almost completely normalized by addition of the inhibitory mAb anti-AT 5G1.1, but not by anti-AT 6E7.2, indicating that these mAbs exert their inhibitory activity via different mechanisms. These data provide initial in vitro proof-of-concept that inhibitory anti-AT mAbs can be used to improve coagulation and may be used as FVIII bypass therapy. Disclosures Boross: Prothix B.V.: Employment. Hack:Prothix B.V.: Equity Ownership, Patents & Royalties.
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Ono, Mayumi, Katsuhiko Nawa, and Yasumasa Marumoto. "Antithrombotic Effects of Recombinant Human Soluble Thrombomodulin in a Rat Model of Vascular Shunt Thrombosis." Thrombosis and Haemostasis 72, no. 03 (1994): 421–25. http://dx.doi.org/10.1055/s-0038-1648882.
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SummaryThrombomodulin on endothelial cells is a cofactor for thrombin-catalyzed activation of protein C. We have investigated the anticoagulant function of recombinant human soluble thombomodulin (rsTM) in a rat model of arterio-venous (AV)-shunt thrombosis. A bolus injection of rsTM 30 s before the induction of AV-shunt thrombosis inhibited the thrombus formation in a dose-dependent manner. The dose of anticoagulant that inhibited thrombus formation by 50% was 0.4 mg/kg rsTMα, 0.15 mg/kg rsTMβ, and 13 U/kg heparin. Recently, we characterized three monoclonal antibodies (moAbs) against human TM whose epitopes are located in the TM epidermal growth factor-like domain (Nawa et al., 1994). moAb 2A2 inhibited thrombin binding to rsTM, and abolished both TM functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation. moAb 1F2 preserved the latter activities as an anticoagulant, but inhibited cofactor activity. moAb 10A3 had no inhibitory effect on either activity. Analysis of the in vivo anticoagulant mechanism of rsTM was facilitated by the availability of these moAbs. After incubation at rsTM/moAb molar ratios of 1:1.25, the effect of the mixtures were examined in the AV-shunt thrombosis model. An injection of 0.8 mg/kg rsTMα or 0.4 mg/kg rsTMβ resulted in a significant reduction on thrombus formation, as expected. moAb 10A3 had no effect on rsTM activity. However, co-injection of rsTM with moAb 1F2 resulted in a significant decrease of the inhibitory activity on thrombus formation. moAb 2A2 essentially abolished the inhibitory effect of rsTM. These moAb effects were observed for both rsTMα and β. These results suggest that the protein C activation accelerated by the injection of rsTM plays an important role for inhibiting thrombus formation in the AV-shunt thrombosis model.
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Gerotziafas, Grigoris T., Marie-Paule Roman, Elisabeth Verdy, Mohamed Hatmi, Meyer M. Samama, and Ismail Elalamy. "Platelets and Heparin Induced Thrombocytopenia Antibodies Do Not Influence the Inhibitory Activity of Argatroban on Thrombin Generation." Blood 110, no. 11 (November 16, 2007): 929. http://dx.doi.org/10.1182/blood.v110.11.929.929.
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Abstract Argatroban is a synthetic, reversible, direct thrombin inhibitor (DTI) used in patients with heparin induced thrombocytopenia (HIT). Argatroban as other DTIs prolongs PT, aPTT and ecarin clotting time. Argatroban added in vitro in normal platelet poor plasma (PPP) inhibits tissue factor (TF) triggered thrombin generation (TG) in a concentration dependent manner. We studied the influence of platelets, HIT antibodies and residual heparin, on the inhibitory effect of argatroban on TG. Argatroban (0 to 2 μM) was added in normal platelet rich plasma (PRP) and PPP, in pool PPP from three consecutive HIT patients (HIT-PPP) and in HIT-PRP prepared by mixing normal PRP with HIT-PPP (v/v). HIT-PRP and HIT-PPP were containing residual heparin (0,035 and 0,07 anti-Xa IU/ml respectively). All experiments were repeated 3 times. TG was triggered in the presence of TF (Dade-Behring Innovin; 1/1000 final dilution in plasma) and assessed with Calibrated Automated Thrombinoscope®. TG in PPP was triggered by adding PPP reagent (Thrombogram-Thrombinoscope®). Lag time (LT), time to peak (ttP), peak (P), endogenous thrombin potential (ETP) and mean velocity index (MVI) of thrombin generation were measured. The concentrations of argatroban which prolonged 2-fold the LT and the ttP and which inhibited 50% (IC50) the P, ETP and MVI were calculated. In the presence of low argatroban concentrations (0,1 an 0,2 μM) an artifactual increase of TG was observed. This is probably due to the interference of alpha2macroglobulin-bound thrombin with the fluorogenic substrate (as shown by Hemker’s group for other reversible DTIs). Argatroban at 1 μM inhibited TG by 50% in both normal PRP and PPP. Argatroban at 1 μM induced a 2-fold prolongation of aPTT (Table 1). HIT antibodies did not modify the inhibitory activity of argatroban on TG in HIT-PRP and HIT-PPP. The presence of traces of heparin in plasma from HIT patients had a synergistic effect with argatroban on the inhibition of TG (Table 1). Our in vitro study shows that argatroban at concentration achieved in clinical practice (about 1 μM) induces 50% inhibition of TG. The inhibitory activity of argatroban on TG is not modified by the presence of platelets or HIT antibodies. In contrast, traces of residual heparin in plasma from HIT patients amplify the inhibition of thrombin generation induced by argatroban. This finding has to be confirmed in a prospective clinical study since it implicates an increased vigilance during the switch from heparin to argatroban in acute HIT patients. Table 1. Inhibitory activity of argatroban on thrombin generation. normal PRP normal PPP HIT-PRP (0,03 aXa/ml) HIT-PPP (0,07 aXa/ml) Lag-time x2 0,7 μM 0,7 μM 0,9 μM 0,1 μM ttPeak x2 1 μM 1 μM 1 μM 0,2 μM Peak IC50 1 μM 1 μM 0,7 μM 0,3 μM ETP IC50 1 μM 1 μM 0,7 μM 0,3 μM MRI IC50 1 μM 1 μM 0,5 μM 0,3 μM PTx2 - 1 μM - - aPTT x2 - 1 μM - -
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Zavyalova, Elena, Valeriia Legatova, Rugiya Alieva, Arthur Zalevsky, Vadim Tashlitsky, Alexander Arutyunyan, and Alexey Kopylov. "Putative Mechanisms Underlying High Inhibitory Activities of Bimodular DNA Aptamers to Thrombin." Biomolecules 9, no. 2 (January 24, 2019): 41. http://dx.doi.org/10.3390/biom9020041.
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Nucleic acid aptamers are prospective molecular recognizing elements. Similar to antibodies, aptamers are capable of providing specific recognition due to their spatial structure. However, the apparent simplicity of oligonucleotide folding is often elusive, as there is a balance between several conformations and, in some cases, oligomeric structures. This research is focused on establishing a thermodynamic background and the conformational heterogeneity of aptamers taking a series of thrombin DNA aptamers having G-quadruplex and duplex modules as an example. A series of aptamers with similar modular structures was characterized with spectroscopic and chromatographic techniques, providing examples of the conformational homogeneity of aptamers with high inhibitory activity, as well as a mixture of monomeric and oligomeric species for aptamers with low inhibitory activity. Thermodynamic parameters for aptamer unfolding were calculated, and their correlation with aptamer functional activity was found. Detailed analysis of thrombin complexes with G-quadruplex aptamers bound to exosite I revealed the similarity of the interfaces of aptamers with drastically different affinities to thrombin. It could be suggested that there are some events during complex formation that have a larger impact on the affinity than the states of initial and final macromolecules. Possible mechanisms of the complex formation and a role of the duplex module in the association process are discussed.
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Okada, Chiaki, Noriyuki Hatae, and Eiko Toyota. "Structural study of the Schiff base copper(II) chelates as thrombin inhibitor." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1665. http://dx.doi.org/10.1107/s205327331408334x.
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Thrombin is a trypsin-like serine protease that plays a critical role in the blood coagulation cascade. Thus, studies on thrombin-specific inhibitor are useful for the design of clinical useful compounds. We have previously reported of the Schiff base metal chelates carrying benzamidine and amino acid moieties. More than 50 kinds of Schiff base copper(II) chelates were synthesized fromp- orm-amidinosalicylaldehyde and various L- or D-amino acids. These chelates have effective inhibitory activity for trypsin and thrombin (Ki= 10-5~ 10-6M). In this series of inhibitors, the copper(II) chelate derived fromp-amidinosalicylaldehyde and D-tryptophan (chelate1g'in original paper) have shown exceptionally potent inhibitory activity against thrombin (Ki= 2.7×10-8M). To elucidate the structural basis of this high efficient inhibition, we were planning to determine and compare with four protein-inhibitor complex structures, chelate1g'or2g'bound to trypsin or thrombin (chelate2g'is derived fromm-amidinosalicylaldehyde and D-Trp). The crystals of trypsin binding the chelate2g'was obtained by sitting-drop vapor diffusion method by mixing 2.5 μL of 20 mg/mL protein-chelate complex solution with 2.5 μL of 0.1 M HEPES, pH 7.4, containing 0.1 M Li2(SO4), 25.0% PEG 3350. In the crystal structure, the imidazole nitrogen of His57 is coordinated with the copper(II) ion (1.94 Å). This close contact is made possible by conformation change of the His57. The indole ring of the chelate2g'simultaneously interacts with the copper(II) and the His57 by cation-π and π-π stacking interaction, respectively. In addition to trypsin-chelate2g'complex structure, we will report on the other three complex structures of trypsin-chelate1g', thrombin-chelate1g'and thrombin-chelate2g'.
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Sassi, Mouna, Taher Chakroun, Elisabeth Mbemba, Patrick Van Dreden, Ismail Elalamy, Annette K. Larsen, and Grigoris T. Gerotziafas. "The Antithrombotic Potential of Tinzaparin and Enoxaparin Upon Thrombin Generation Triggered In Vitro by Human Ovarian Cancer Cells IGROV1." Clinical and Applied Thrombosis/Hemostasis 23, no. 2 (September 24, 2016): 155–63. http://dx.doi.org/10.1177/1076029616665922.
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Background: A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents. Objective: We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells. Methods: Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs. Results: IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs. Conclusion: This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.
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Mitchell, Christina A., Lena Hau, and Hatem H. Salem. "Control of Thrombin Mediated Cleavage of Protein S." Thrombosis and Haemostasis 56, no. 02 (1986): 151–54. http://dx.doi.org/10.1055/s-0038-1661630.
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SummaryThrombin has been shown to cleave the vitamin K dependent cofactor protein S with subsequent loss of its cofactor activity. This study examines the control mechanisms for thrombin cleavage of protein S.The anticoagulant activity of activated protein C (APC) is enhanced fourteen fold by the addition of protein S. Thrombin cleaved protein S is seven fold less efficient than the native protein, and this loss of activity is due to reduced affinity of cleaved protein S for APC or the lipid surface compared to the intact protein.In the absence of Ca++, protein S is very sensitive to minimal concentrations of thrombin. As little as 1.5 nM thrombin results in complete cleavage of 20 nM protein S in 10 min and loss of cofactor activity. Ca++, in concentrations greater than 0.5 mM, will inhibit this cleavage and in the presence of physiological Ca++ concentrations, no cleavage of protein S could be demonstrated in spite of high concentrations of thrombin (up to 1 μM) and prolonged incubations (up to two hours). The endothelial surface protein thrombomodulin is very efficient in inhibiting the cleavage of protein S by thrombin suggesting that any thrombin formed on the endothelial cell surface is unlikely to cleave protein S, thus allowing the intact protein to act as a cofactor to APC.We conclude that the inhibitory effects of Ca++ and thrombomodulin on thrombin mediated cleavage of protein S imply that this event, by itself, is unlikely to represent a physiological control of the activity of protein S.
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Ofosu, F. A., P. Sie, G. J. Modi, F. Fernandez, M. R. Buchanan, M. A. Blajchman, B. Boneu, and J. Hirsh. "The inhibition of thrombin-dependent positive-feedback reactions is critical to the expression of the anticoagulant effect of heparin." Biochemical Journal 243, no. 2 (April 15, 1987): 579–88. http://dx.doi.org/10.1042/bj2430579.
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Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and prothrombin. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH2Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH2Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of 125I-prothrombin to 125I-prothrombin fragment 1 + 2 and 125I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of prothrombin activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH2Cl also completely inhibited the intrinsic-pathway activation of prothrombin in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH2Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH2Cl. The inhibition of the activation of prothrombin by the three agents was also abolished with longer times with re-added Ca2+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of 125I-thrombin-antithrombin III and 125I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant effects of heparin and pentosan polysulphate are mediated primarily by their ability to inhibit the thrombin-dependent activation of Factor V, thereby inhibiting the formation of prothrombinase complex, the physiological activator of prothrombin.
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Jandrot-Perrus, Martine, Marie-Geneviève Huisse, John L. Krstenansky, Annie Bezeaud, and Marie-Claude Guillin. "Effect of the Hirudin Carboxy-Terminal Peptide 54-65 on the Interaction of Thrombin with Platelets." Thrombosis and Haemostasis 66, no. 03 (1991): 300–305. http://dx.doi.org/10.1055/s-0038-1646411.
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SummaryThe carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed β cleavage site, we have analyzed the consequences of a to β-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The β cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the β cleavage site at Arg 73 on the thrombin B chain.
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Hauptmann, J., B. Kaiser, G. Nowak, J. Stürzebecher, and F. Markwardt. "Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors." Thrombosis and Haemostasis 63, no. 02 (1990): 220–23. http://dx.doi.org/10.1055/s-0038-1645198.
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SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.
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Tanratana, Pansakorn, and John P. Sheehan. "Detection of Factor IXa Activity By the Enhanced Thrombin Generation Assay." Blood 124, no. 21 (December 6, 2014): 4242. http://dx.doi.org/10.1182/blood.v124.21.4242.4242.
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Abstract Introduction – Factor Xa generation by the factor IXa-factor VIIIa (FIXa-FVIIIa) complex is the rate-limiting step for plasma thrombin generation (TG). Modeling of blood coagulation suggests that subnanomolar concentrations of FIXa are sufficient to support plasma TG, however, the isolated enzyme is poorly reactive with both substrate and inhibitors. Thus, most detection methods for FIXa require at least nanomolar protease concentrations, which does not allow characterization of physiologically relevant enzyme levels. Our laboratory has developed a novel, highly sensitive assay to detect FIXa activity based on TG in FIX-deficient plasma. Method – The enhanced thrombin generation assay (ETGA) detects FIXa activity in test samples by dilution into citrated FIX-deficient plasma. Briefly, a standard curve was established by adding 10 µl of test plasma containing 0-80 pM plasma-derived FIXa (pFIXa) to 50 µl of FIX-deficient plasma. Simultaneously, human FVIII (19.2 nM) was activated with 12.8 nM thrombin for 30 sec, neutralized with a 1.25-fold molar excess of hirudin, and the resulting thrombin-activated FVIIIa was added to plasma (final plasma concentration 1.3 nM) immediately after addition of calcium and the fluorogenic substrate. Plasma TG was detected by cleavage of Z-Gly-Gly-Arg-AMC in a Biotek Synergy HT plate reader, and data exported to TECHNOTHROMBIN TGA evaluation software to generate TG curve parameters including lag time, peak thrombin concentration, time to thrombin peak and velocity index. FIXa concentration was plotted versus mean peak thrombin ± SEM (n=3) and the data fit to a parabolic function. Sample FIXa activity was obtained from this standard curve using mean peak thrombin concentration. The specificity of the TG response was confirmed by pre-incubation of test plasma with inhibitory antibodies. To prevent contact pathway-dependent FIXa generation, the monoclonal antibody (MoAb) O1A6 was added, which directly blocks FIX activation by factor XIa[PT1] . Plasma TG was ̴20-50% lower when the assay was performed in the presence of MoAb O1A6. The dependence of the remaining TG activity on FIXa was confirmed by pre-incubation of test plasma with an inhibitory anti-FIX Gla domain antibody. An inhibitory anti-TF antibody had no effect on plasma activity in this assay. Results – The ETGA was employed to: 1) measure FIXa activity in plasma and ascites samples from patients with gynecologic malignancies (ovarian and uterine) and healthy controls, and 2) determine the half-life of FIXa in human plasma. Plasma FIXa activity from 14 patients with gynecologic malignancies, and 12 healthy controls were analyzed by the ETGA. The mean plasma FIXa activity level in the cancer group was 7.6 ± 2.1 pM, and in the control group was 8.2 ± 2.5 pM, confirmed by preincubation with MoAb as above. Coagulant activity in 6 malignant ascites samples from these patients was similarly evaluated with the ETGA assay. The citrated ascites samples demonstrated substantial TG activity, however, preincubation with inhibitory MoAb controls demonstrated that this activity was strictly TF-dependent and FIX-independent. The EGTA was also employed to detect the half-life of FIXa in FIX-deficient citrated human plasma. pFIXa (35 pM) and recombinant FIXa (50pM) was incubated in FIX-deficient plasma for 0-120 min at 37°C, and the residual FIXa activity was determined at varying timepoints. pFIXa demonstrated a remarkably long half-life in FIX-deficient plasma (36.0 ± 3.6 min). rFIXa WT demonstrated a similar half-life (40.9 ± 1.4 min), while mutation in the antithrombin (AT) binding site (R150) substantially prolonged protease half-lives (> 2 hr). Conclusion – These results demonstrate that the ETGA is capable of detecting physiologically relevant levels of FIXa activity. Initial evaluation of plasma FIXa activity suggests no significant differences between controls and patients with gynecologic malignancies, and no significant FIXa activity in malignant ascites. While rFIXa WT and pFIXa have comparable plasma half-lives, the R150A mutation in the AT-binding exosite markedly prolonged protease half-life, consistent with the role of AT as the primary plasma FIXa inhibitor. This novel assay may have applications in the detection of physiologically relevant FIXa activity in plasma and other blood derived products, recombinant FIX preparations, and other body fluids. Disclosures No relevant conflicts of interest to declare.
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Takeyama, Masahiro, Keiji Nogami, Tomoko Matsumoto, Tetsuhiro Soeda, Tsukasa Suzuki, Kunihiro Hattori, and Midori Shima. "Characterisation of an antibody specific for coagulation factor VIII that enhances factor VIII activity." Thrombosis and Haemostasis 103, no. 01 (2010): 94–102. http://dx.doi.org/10.1160/th09-05-0338.
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SummaryMany reports have identified factor (F)VIII inhibitory antibodies with epitopes located in all subunits of the FVIII molecule. Antibodies that promote FVIII activity do not appear to have been reported. We characterised, for the first time, a unique anti-FVIII monoclonal antibody, mAb216, that enhanced FVIII coagulant activity. The mAb216 shortened the activated partial thromboplastin time and specifically increased FVIII activity by ~1.5-fold dose-dependently. FXa generation and thrombin generation were similarly increased by ~1.4- and ~2.5-fold, respectively. An A2 epitope, not overlapping the common A2 epitope, was identified and the antibody was shown to enhance thrombin (and FXa)-catalysed activation of FVIII by modestly accelerating cleavage at Arg372. The presence of mAb216 mediated an ~1.5-fold decrease in Km for the FVIII-thrombin interaction. Enhanced FVIII activity was evident to an equal degree, even the presence of anti-FVIII neutralising antibodies with epitopes in each subunit. In addition, mAb216 depressed the rates of heat-denatured loss of FVIII activity and FVIIIa decay by 2 to ~2.5-fold. We have developed an anti-A2, FVIII mAb216 that augmented procoagulant activity. This enhancing effect could be attributed to an increase in thrombin-induced activation of FVIII, mediated by cleavage at Arg372 and a tighter interaction of thrombin with the A2 domain. The findings may cast new light on new principles for improving the treatment of haemophilia A patients.
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Stern, D. M., I. Bank, P. P. Nawroth, J. Cassimeris, W. Kisiel, J. W. Fenton, C. Dinarello, L. Chess, and E. A. Jaffe. "Self-regulation of procoagulant events on the endothelial cell surface." Journal of Experimental Medicine 162, no. 4 (October 1, 1985): 1223–35. http://dx.doi.org/10.1084/jem.162.4.1223.
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Interleukin 1 (IL-1) is a potent mediator of inflammatory and immunologic phenomena. In addition, IL-1 may be intimately involved in the regulation of hemostasis, since interaction of IL-1 with endothelial cells has been reported to induce tissue factor activity. We demonstrate that perturbation of the endothelial cell induces augmented IL-1 release. Human umbilical vein endothelial cells perturbed by treatment with lipopolysaccharide produced enhanced amounts of IL-1 activity. IL-1 activity from lipopolysaccharide-treated endothelial cell supernatants could be absorbed by an antibody to IL-1 coupled to Sepharose. Elaboration of IL-1 activity was dependent on the dose of lipopolysaccharide and occurred in a time-dependent manner. Addition of cycloheximide blocked generation of IL-1 activity. A physiological vessel wall perturbant, the coagulation enzyme thrombin, induced comparable amounts of IL-1 activity in endothelial cell cultures. This effect was specific for the enzyme, since active site-blocked thrombin and prothrombin had no effect on IL-1. In addition, IL-1-containing supernatants from thrombin-stimulated endothelial cells induced tissue factor procoagulant activity in fresh endothelial cell cultures. Thus, in contrast to the multiple, known inhibitory mechanisms that block thrombin procoagulant activity, these data suggest a circle of interaction in which thrombin induces endothelial cell elaboration of IL-1, a mediator of endothelial cell procoagulant activity. Endothelial cell production of IL-1 in response to perturbation allows these cells to play an integral role in the regulation of the inflammatory and coagulation systems.
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Misra, Raj N., Yolanda F. Kelly, Bärbel R. Brown, Daniel G. M. Roberts, Saeho Chong, and Steven M. Seiler. "Argatroban analogs: Synthesis, thrombin inhibitory activity and cell permeability of aminoheterocyclic guanidine surrogates." Bioorganic & Medicinal Chemistry Letters 4, no. 18 (September 1994): 2165–70. http://dx.doi.org/10.1016/s0960-894x(00)80064-5.
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Forneris, Federico, B. Tom Burnley, and Piet Gros. "Ensemble refinement shows conformational flexibility in crystal structures of human complement factor D." Acta Crystallographica Section D Biological Crystallography 70, no. 3 (February 15, 2014): 733–43. http://dx.doi.org/10.1107/s1399004713032549.
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Human factor D (FD) is a self-inhibited thrombin-like serine proteinase that is critical for amplification of the complement immune response. FD is activated by its substrate through interactions outside the active site. The substrate-binding, or `exosite', region displays a well defined and rigid conformation in FD. In contrast, remarkable flexibility is observed in thrombin and related proteinases, in which Na+and ligand binding is implied in allosteric regulation of enzymatic activity through protein dynamics. Here, ensemble refinement (ER) of FD and thrombin crystal structures is used to evaluate structure and dynamics simultaneously. A comparison with previously published NMR data for thrombin supports the ER analysis. The R202A FD variant has enhanced activity towards artificial peptides and simultaneously displays active and inactive conformations of the active site. ER revealed pronounced disorder in the exosite loops for this FD variant, reminiscent of thrombin in the absence of the stabilizing Na+ion. These data indicate that FD exhibits conformational dynamics like thrombin, but unlike in thrombin a mechanism has evolved in FD that locks the unbound native state into an ordered inactive conformationviathe self-inhibitory loop. Thus, ensemble refinement of X-ray crystal structures may represent an approach alternative to spectroscopy to explore protein dynamics in atomic detail.
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Stavenuiter, Fabian, Nicole Davis, Erning Duan, Andrew Gale, and Mary Heeb. "Platelet protein S directly inhibits procoagulant activity on platelets and microparticles." Thrombosis and Haemostasis 109, no. 02 (2013): 229–37. http://dx.doi.org/10.1160/th12-08-0622.
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SummaryAnticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities (“PS-direct”). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PSdirect that was similar to or greater than that of Zn2+-containing pdPS and much greater than that of Zn2+-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticlebound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralising antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.
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Tsoupras, Alexandros B., Maria Roulia, Eleftherios Ferentinos, Ioannis Stamatopoulos, Constantinos A. Demopoulos, and Panayotis Kyritsis. "Structurally Diverse Metal Coordination Compounds, Bearing Imidodiphosphinate and Diphosphinoamine Ligands, as Potential Inhibitors of the Platelet Activating Factor." Bioinorganic Chemistry and Applications 2010 (2010): 1–8. http://dx.doi.org/10.1155/2010/731202.
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Metal complexes bearing dichalcogenated imidodiphosphinate[R2P(E)NP(E)R2′]-ligands (E = O, S, Se, Te), which act as (E,E) chelates, exhibit a remarkable variety of three-dimensional structures. A series of such complexes, namely, square-planar[Cu{(OPPh2)(OPPh2)N-O,O}2], tetrahedral[Zn{(EPPh2)(EPPh2)N-E,E}2], E = O, S, and octahedral[Ga{(OPPh2)(OPPh2)N-O,O}3], were tested as potential inhibitors of either the platelet activating factor (PAF)- or thrombin-induced aggregation in both washed rabbit platelets and rabbit platelet rich plasma. For comparison, square-planar[Ni{(Ph2P)2N-S-CHMePh-P,P}X2], X = Cl, Br, the corresponding metal salts of all complexes and the(OPPh2)(OPPh2)NHligand were also investigated.Ga(O,O)3showed the highest anti-PAF activity but did not inhibit the thrombin-related pathway, whereasZn(S,S)2, with also a significant PAF inhibitory effect, exhibited the highest thrombin-related inhibition.Zn(O,O)2andCu(O,O)2inhibited moderately both PAF and thrombin, being more effective towards PAF. This work shows that the PAF-inhibitory action depends on the structure of the complexes studied, with the bulkierGa(O,O)3being the most efficient and selective inhibitor.
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Wutt, Daqing, Jiong Shen, and Binggen Ru. "Expression and purification of human metallothionein-3 beta domain in e.coli." Protein & Peptide Letters 7, no. 4 (August 2000): 257–63. http://dx.doi.org/10.2174/092986650704221207123557.
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Abstract: J3-domain of metallothionein-3/growth inhibitory factor(MT-3/GIF) is crucial to the neuronal growth inhibitory activity of MT-3. The MT-3 J3 domain was efficiently expressed as the soluble fusion form with GST protein at a high level which accounted for over 30% total cellular proteins. After the thrombin cleavage and purification, the bioactive recombinant MT-3 J3 domain was recovered with the yield of 6 mg/L of medium.
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Sugimoto, Emika, Minako Sata, Toshio Asano, and Mitsunobu Mohri. "The Inhibitory Effect of Recombinant Human Soluble Thrombomodulin on Initiation and Extension of Coagulation." Thrombosis and Haemostasis 82, no. 12 (1999): 1687–93. http://dx.doi.org/10.1055/s-0037-1614900.
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SummaryRecombinant human soluble thrombomodulin (rhsTM) was compared with various anticoagulants for in vitro anticoagulant effects on thrombin generation, clotting time, and thromboelastography. rhsTM as well as APC reduced the level of the peak of the thrombin generation curve, but we did not observe any time-delay to reach the peak. This effect of rhsTM was diminished in PC-deficient plasma and was closely associated with the inhibitory effect on prothrombinase and factor Va. On the other hand, hirudin and argatroban delayed the time to reach the level of the peak, without reducing it. rhsTM and other anticoagulants except for activated protein C (APC) were found to have concentration-dependent anticoagulant activity by conventional clotting tests. However, the concentration of rhsTM for clotting time was slightly affected by anti-protein C antibody. Moreover, the concentration of rhsTM required to inhibit thrombin activity directly was 50 times higher than that needed to inhibit thrombin generation. The effect of rhsTM on clot development was compared with that of other anticoagulants by thromboelastography; rhsTM reduced the growth of the clot but had little effect on the time to activate clotting, while the other anticoagulants had the opposite effect. This effect of rhsTM was completely abolished by the addition of anti-protein C or anti-protein S antibody.These findings suggest that rhsTM attenuates blood clotting by reducing the level of generated thrombin through protein C activation and subsequent factor Va inactivation and prothrombinase inhibition.
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Tang, Wenwen, Yuan Chen, and Fengxia Guo. "Comparative Analysis of Roots from Vicatia thibetica de Boiss and Angelica sinensis Based on Chemical Composition, Antioxidant, Nitrite-Scavenging and Enzyme Inhibition Activities." Molecules 28, no. 4 (February 17, 2023): 1942. http://dx.doi.org/10.3390/molecules28041942.
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Radix Vicatia thibetica de Boiss (RVT) is locally known as “Xigui” or “Dujiao-danggui” in Tibetan medicine and is often used as a substitute for Radix Angelica sinensis (RAS) in daily nourishing diets and clinical applications. In this study, we determined and compared the contents of polysaccharides, total coumarins, ferulic acid, total phenols, total flavonoids, chlorogenic acid, protein, and amino acids, and the composition of volatile oil in RVT and RAS. Biological activities, including antioxidants, scavenging of nitrite, inhibition of tyrosinase, thrombin, and coagulation FXa, were comparatively evaluated. Results showed that RVT contains more polysaccharides, phenols, flavonoids, proteins, glutamic acid, and lysine as compared to RAS. Among volatile compounds, 14 species are similar, and 20 species are different in RVT and RAS. Overall, among volatile compounds, the content of 3-N-Butylphthalide was higher, whereas the content of ligustilide was lower in RVT volatile oil. A significant difference was reported in the bioactivity of RVT and RAS. The biological activity of RVT had higher antioxidant, nitrite scavenging, and tyrosinase inhibitory activities, whereas it showed much lower thrombin and FXa inhibitory activities. Correlation analysis showed that the antioxidant, nitrite scavenging, and tyrosinase inhibitory activities were related to the phenol and flavonoid content, whereas the thrombin and FXa inhibitory activities were related to ferulic acid and volatile oil content. This study presents a comparative analysis of RAS and RVT’s chemical compositions of antioxidant, nitrite-scavenging, inhibition of tyrosinase, thrombin, and coagulation FXa activities. It was found that both RVT and RAS have their unique advantages, and RVT has the potential to be utilized as functional foods, cosmetics, and medical products.