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1
Péč, Martin Jozef, Jakub Benko, Jakub Jurica, Monika Péčová, Marek Samec, Tatiana Hurtová, Tomáš Bolek, et al. "The Anti-Thrombotic Effects of PCSK9 Inhibitors." Pharmaceuticals 16, no. 9 (August 22, 2023): 1197. http://dx.doi.org/10.3390/ph16091197.
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Atherosclerosis is the primary process that underlies cardiovascular disease. The connection between LDL cholesterol and the formation of atherosclerotic plaques is established by solid evidence. PCSK9 inhibitors have proven to be a valuable and practical resource for lowering the LDL cholesterol of many patients in recent years. Their inhibitory effect on atherosclerosis progression seems to be driven not just by lipid metabolism modification but also by LDL-independent mechanisms. We review the effect of PCSK9 inhibitors on various mechanisms involving platelet activation, inflammation, endothelial dysfunction, and the resultant clot formation. The main effectors of PCSK9 activation of platelets are CD36 receptors, lipoprotein(a), oxidised LDL particles, tissue factor, and factor VIII. Many more molecules are under investigation, and this area of research is growing rapidly.
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De Meyer, Simon. "ADAMTS13, an Anti-thrombotic Protein: Evidence Outside of Thrombotic Thrombocytopenic Purpura." Blood 134, Supplement_1 (November 13, 2019): SCI—41—SCI—41. http://dx.doi.org/10.1182/blood-2019-121114.
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von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that plays a crucial role in hemostasis and thrombosis. VWF recruits platelets at sites of vascular injury by acting as a molecular bridge between circulating platelets and the site of injured or activated blood vessels. Biosynthesis of VWF is restricted to endothelial cells and megakaryocytes. Endothelial VWF is constitutively secreted into plasma and subendothelium, or is stored as "ultra-large" (UL)-VWF multimers in endothelial Weibel-Palade bodies. VWF produced in megakaryocytes is packaged as UL-VWF in the a-granules of platelets. VWF stored in endothelial and platelet storage organelles is secreted in a regulated process in response to stimulation by secretagogues. Absence or dysfunction of VWF results in bleeding symptoms, as observed in patients with von Willebrand disease. An abnormally high activity of VWF can lead to thrombotic events. Interestingly, the activity of VWF is determined by the size of its multimers and UL-VWF can spontaneously form platelet aggregates. An important regulator of VWF size is the metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which digests large thrombogenic VWF molecules into smaller, less reactive multimers via cleavage of the Y1605-M1606 bond in the VWF A2 domain. ADAMTS13 is mainly synthesized in the liver by hepatic stellate cells, but other sites of synthesis, including renal podocytes, tubular epithelial cell, platelets and endothelial cells, have also been described. ADAMTS13 is released as an active enzyme into the circulation with no physiological inhibitors. Reduced or absent ADAMTS13 activity causes the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), characterized by VWF and platelet-rich microthrombi that cause multiple organ failure and even death when left untreated. Besides its clear role in the pathophysiology of TTP, the anti-thrombotic and even anti-inflammatory properties of ADAMTS13 have also become apparent in various other thrombotic conditions. High VWF levels and low ADAMTS13 levels are associated with increased risk or even worse outcome of cardiovascular disease, including ischemic stroke and myocardial infarction. Preclinical studies in mouse models showed the beneficial effect of ADAMTS13 in both cerebral and myocardial ischemia/ reperfusion injury by decreasing both thrombosis and inflammation. In addition, ADAMTS13 was shown to also exert a direct thrombolytic effect on VWF-rich thrombi. In a mouse model of ischemic stroke, this thrombolytic activity resulted in efficient lysis of intracranial thrombi that were resistant to standard treatment with tissue plasminogen activator. Hence, ADAMTS13, as a therapeutic agent, could become an interesting avenue, not only to manage TTP, but also to treat other thrombotic complications. Disclosures De Meyer: Fonds voor Wetenschappelijk Onderzoek: Research Funding; KU Leuven: Employment, Research Funding; Ablynx: Consultancy, Research Funding; Cerenovus: Membership on an entity's Board of Directors or advisory committees; WhiteSwell: Consultancy.
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Fuentes, Eduardo, and Iván Palomo. "Extracellular ATP metabolism on vascular endothelial cells: A pathway with pro-thrombotic and anti-thrombotic molecules." Vascular Pharmacology 75 (December 2015): 1–6. http://dx.doi.org/10.1016/j.vph.2015.05.002.
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Muro, S., and V. Muzykantov. "Targeting of Antioxidant and Anti-Thrombotic Drugs to Endothelial Cell Adhesion Molecules." Current Pharmaceutical Design 11, no. 18 (July 1, 2005): 2383–401. http://dx.doi.org/10.2174/1381612054367274.
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Zaki, Magdi E. A., Sami A. Al-Hussain, Vijay H. Masand, Manoj K. Sabnani, and Abdul Samad. "Mechanistic and Predictive QSAR Analysis of Diverse Molecules to Capture Salient and Hidden Pharmacophores for Anti-Thrombotic Activity." International Journal of Molecular Sciences 22, no. 15 (August 3, 2021): 8352. http://dx.doi.org/10.3390/ijms22158352.
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Thrombosis is a life-threatening disease with a high mortality rate in many countries. Even though anti-thrombotic drugs are available, their serious side effects compel the search for safer drugs. In search of a safer anti-thrombotic drug, Quantitative Structure-Activity Relationship (QSAR) could be useful to identify crucial pharmacophoric features. The present work is based on a larger data set comprising 1121 diverse compounds to develop a QSAR model having a balance of acceptable predictive ability (Predictive QSAR) and mechanistic interpretation (Mechanistic QSAR). The developed six parametric model fulfils the recommended values for internal and external validation along with Y-randomization parameters such as R2tr = 0.831, Q2LMO = 0.828, R2ex = 0.783. The present analysis reveals that anti-thrombotic activity is found to be correlated with concealed structural traits such as positively charged ring carbon atoms, specific combination of aromatic Nitrogen and sp2-hybridized carbon atoms, etc. Thus, the model captured reported as well as novel pharmacophoric features. The results of QSAR analysis are further vindicated by reported crystal structures of compounds with factor Xa. The analysis led to the identification of useful novel pharmacophoric features, which could be used for future optimization of lead compounds.
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Shiels, Katie, Alexandros Tsoupras, Ronan Lordan, Constantina Nasopoulou, Ioannis Zabetakis, Patrick Murray, and Sushanta Kumar Saha. "Bioactive Lipids of Marine Microalga Chlorococcum sp. SABC 012504 with Anti-Inflammatory and Anti-Thrombotic Activities." Marine Drugs 19, no. 1 (January 10, 2021): 28. http://dx.doi.org/10.3390/md19010028.
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Microalgae are at the start of the food chain, and many are known producers of a significant amount of lipids with essential fatty acids. However, the bioactivity of microalgal lipids for anti-inflammatory and antithrombotic activities have rarely been investigated. Therefore, for a sustainable source of the above bioactive lipids, the present study was undertaken. The total lipids of microalga Chlorococcum sp., isolated from the Irish coast, were fractionated into neutral-, glyco-, and phospho-lipids, and were tested in vitro for their anti-inflammatory and antithrombotic activities. All tested lipid fractions showed strong anti-platelet-activating factor (PAF) and antithrombin activities in human platelets (half maximal inhibitory concentration (IC50) values ranging ~25–200 μg of lipid) with the highest activities in glyco- and phospho-lipid fractions. The structural analysis of the bioactive lipid fraction-2 revealed the presence of specific sulfoquinovosyl diacylglycerols (SQDG) bioactive molecules and the HexCer-t36:2 (t18:1/18:1 and 18:2/18:0) cerebrosides with a phytosphingosine (4-hydrosphinganine) base, while fraction-3 contained bioactive phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules. These novel bioactive lipids of Chlorococcum sp. with putative health benefits may indicate that marine microalgae can be a sustainable alternative source for bioactive lipids production for food supplements and nutraceutical applications. However, further studies are required towards the commercial technology pathways development and biosafety analysis for the use of the microalga.
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Shiels, Katie, Alexandros Tsoupras, Ronan Lordan, Constantina Nasopoulou, Ioannis Zabetakis, Patrick Murray, and Sushanta Kumar Saha. "Bioactive Lipids of Marine Microalga Chlorococcum sp. SABC 012504 with Anti-Inflammatory and Anti-thrombotic Activities." Marine Drugs 19, no. 1 (January 10, 2021): 28. http://dx.doi.org/10.3390/md19010028.
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Microalgae are at the start of the food chain, and many are known producers of a significant amount of lipids with essential fatty acids. However, the bioactivity of microalgal lipids for anti-inflammatory and antithrombotic activities have rarely been investigated. Therefore, for a sustainable source of the above bioactive lipids, the present study was undertaken. The total lipids of microalga Chlorococcum sp., isolated from the Irish coast, were fractionated into neutral-, glyco-, and phospho-lipids, and were tested in vitro for their anti-inflammatory and antithrombotic activities. All tested lipid fractions showed strong anti-platelet-activating factor (PAF) and antithrombin activities in human platelets (half maximal inhibitory concentration (IC50) values ranging ~25–200 μg of lipid) with the highest activities in glyco- and phospho-lipid fractions. The structural analysis of the bioactive lipid fraction-2 revealed the presence of specific sulfoquinovosyl diacylglycerols (SQDG) bioactive molecules and the HexCer-t36:2 (t18:1/18:1 and 18:2/18:0) cerebrosides with a phytosphingosine (4-hydrosphinganine) base, while fraction-3 contained bioactive phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules. These novel bioactive lipids of Chlorococcum sp. with putative health benefits may indicate that marine microalgae can be a sustainable alternative source for bioactive lipids production for food supplements and nutraceutical applications. However, further studies are required towards the commercial technology pathways development and biosafety analysis for the use of the microalga.
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Clemetson, Jeannine, and Kenneth Clemetson. "Platelet GPIb complex as a target for anti-thrombotic drug development." Thrombosis and Haemostasis 99, no. 03 (2008): 473–79. http://dx.doi.org/10.1160/th07-12-0718.
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SummarySpecific inhibition of platelet function is a major target of antithrombotic drug research. Platelet receptors are both accessible and specific but have multiple functions often linked to a wide range of ligands. GPIb complex is best known as a major platelet receptor for vonWillebrand factor essential for platelet adhesion under high shear conditions found in arteries and in thrombosis. Recent animal studies have supported inhibition of GPIb as a good candidate for anti-thrombotic drug development with several classes of proteins showing important specific effects and the required discrimination between roles in haemo- stasis and thrombosis is important to protect against bleeding complications.These include antibodies, several classes of snake venom proteins, mutant thrombin molecules and peptides affecting subunit interactions.However,due to the nature of its receptor- ligand interactions involving large protein-protein interfaces, the possibility of developing classic pharmaceutical inhibitors for long term (and perhaps oral) treatment is still unclear, and additional information about structural interactions and signalling mechanisms is essential.
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Bălașa, Adrian Florian, Cristina Chircov, and Alexandru Mihai Grumezescu. "Marine Biocompounds for Neuroprotection—A Review." Marine Drugs 18, no. 6 (May 31, 2020): 290. http://dx.doi.org/10.3390/md18060290.
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While terrestrial organisms are the primary source of natural products, recent years have witnessed a considerable shift towards marine-sourced biocompounds. They have achieved a great scientific interest due to the plethora of compounds with structural and chemical properties generally not found in terrestrial products, exhibiting significant bioactivity ten times higher than terrestrial-sourced molecules. In addition to the antioxidant, anti-thrombotic, anti-coagulant, anti-inflammatory, anti-proliferative, anti-hypertensive, anti-diabetic, and cardio-protection properties, marine-sourced biocompounds have been investigated for their neuroprotective potential. Thus, this review aims to describe the recent findings regarding the neuroprotective effects of the significant marine-sourced biocompounds.
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Lopez, Jose J., Mohammed El Haouari, Isaac Jardin, Nieves Alonso, Sergio Regodon, Raquel Diez-Bello, Pedro C. Redondo, and Juan A. Rosado. "Flavonoids and Platelet-Derived Thrombotic Disorders." Current Medicinal Chemistry 26, no. 39 (January 3, 2019): 7035–47. http://dx.doi.org/10.2174/0929867325666180417170218.
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: Thrombotic disorders are characterized by an increase in the probability of the formation of unnecessary thrombi that might be due to the activation of the coagulation cascade or the circulating platelets. Platelets or thrombocytes play an essential role in hemostasis but abnormal platelet function leads to the development of a number of cardiovascular complications, including thrombotic disorders. Under pathological conditions, platelets are associated with the development of different thrombotic disorders, including atherosclerosis, arterial thrombosis and stroke, deep venous thrombosis and pulmonary embolism; therefore, platelets are the target of a number of anti-thrombotic strategies. Flavonoids, a large group of polyphenols ubiquitously expressed in fruits and vegetables that have attracted considerable attention because of their benefits in human health, including the reduction of the risk of cardiovascular disease. Flavonoids have been reported to reduce platelet activity by attenuating agonist-induced GPIIb/IIIa receptor activation, mobilization of intracellular free Ca2+, granule exocytosis, as well as activation of different signaling molecules such as mitogen- activated protein kinases or phospholipases. This review summarizes the current studies concerning the modulation of platelet activation by flavonoids, giving especial attention to those events associated to thrombotic disorders.
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Wautier, Jean-Luc, Pierre Gane, Wassim El Nemer, Jean-Dider Rain, Caroline Le Van Kim, and Marie-Paule Wautier. "Increased Adhesion of Red Blood Cells from Patients with Polycythemia Vera Is Mediated by Lu/B-CAM and Laminin alpha 5." Blood 106, no. 11 (November 16, 2005): 3527. http://dx.doi.org/10.1182/blood.v106.11.3527.3527.
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Abstract Polycythemia vera (PV) is associated to a high risk of vascular thrombosis which is related to red blood cell (RBC) volume and platelet activation. We have investigated whether, beside high hematocrit, RBCs exhibit qualitative abnormalities. Along this line we have measured RBC adhesion to human umbilical vein endothelial cell (HUVEC) under static and flow conditions. A group of 38 PV patients and one of 36 normal subjects were explored. Adhesion molecule expression using specific antibodies (anti-CD36, CD49d, ICAM-4, Lu/B-CAM, CD147, CD47) and flow cytometry were determined on RBCs. In addition antibodies directed against adhesion molecules or recombinant Lu/B-CAM or VCAM molecules were tested in the adhesion assays. PV-RBC adhesion was found to be increased 3.33 folds (2–4.5) compared to normal RBCs (p<0.001). The extent of adhesion was correlated to Lu/B-CAM expression on RBC (p<0.001) and was inhibited by polyclonal or monoclonal anti-Lu/B-CAM antibodies and soluble Lu/B-CAM recombinant molecules but not VCAM molecules. Adhesion of PV-RBCs activated HUVEC and stimulated VCAM expression, but stimulation of HUVEC by TNF did not increase PV-RBC adhesion. The increase of adhesion was apparently limited to a population of RBCs which are not reticulocytes and which cannot be detached from HUVEC even by a shear stress above 2 Pa. On HUVEC side the adhesion can be blocked by anti-laminin alpha 5 chain antibodies but not anti-Lu/B-CAM. These results indicate that Lu/B-CAM on PV-RBC is responsible for the increased adhesiveness to laminin expressed on HUVEC. This may be one of the factors involved in thrombotic events.
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Varga, Zsuzsa. "Cardioprotective role of omega-3 polyunsaturated fatty acids." Orvosi Hetilap 149, no. 14 (April 2008): 627–37. http://dx.doi.org/10.1556/oh.2008.28296.
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Cardioprotective action of omega-3 polyunsaturated fatty acids such as eicosapentaenoic and docosahexaenoic acid in fish and α-linolenic acid in plants was demonstrated in primary and secondary clinical trials. Fish oil therapy causes a marked decrease in serum triacylglycerol and very low density lipoprotein levels and increases moderately high density lipoprotein levels without any adverse effects. Omega-3 fatty acids decrease slightly, but significantly blood pressure, enhance endothelial function, they have anti-aggregator, anti-thrombotic and anti-inflammatory effects as well. These beneficial effects are in connection with modification of gene transcription levels of some key molecules such as nuclear factor-κB and sterol element binding receptor protein-1c, which regulate for example expression of adhesion molecules or several receptors involved in triglyceride synthesis (hepatocyte X receptor, hepatocyte nuclear factor 4α, farnesol X receptor, and peroxisome proliferator-activated receptors). On the basis of these observations, the supplementation of the diet with omega-3 fatty acids (fish, fish oil, linseed, and linseed oil or canola oil) is advisable in primary and secondary prevention.
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Schaller, Monica, Sabine Hiltbrunner, Irmela Sulzer, Monique Vogel, Karim Kentouche, Bernhard Laemmle, and Johanna Kremer Hovinga. "Anti-idiotypic small molecules neutralize pathogenic anti-ADAMTS13 autoantibodies in autoimmune Thrombotic Thrombocytopenic Purpura in vitro - new treatment tools in vivo? (P5173)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 68.14. http://dx.doi.org/10.4049/jimmunol.190.supp.68.14.
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Abstract A therapy aiming at neutralization of inhibitory autoantibodies (Abs) causing autoimmune thrombotic thrombocytopenic purpura (aTTP) would be highly desirable. We screened a large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins, Molecular Partners AG, Switzerland; library size 1015-23) for anti-idiotypic binders to an equimolar pool of three spleen-derived inhibitory anti-ADAMTS13 monoclonal Abs of two aTTP patients containing 1 of 4 shared CDR3 motifs. Four unique anti-idiotypic DARPins were purified and their neutralization potential tested against 8 spleen-derived anti-ADAMTS13 Abs (200 nM), belonging to the same, shared CDR3 motif used for selection. Anti-idiotypic DARPins restored ADAMTS13 activity up to 100% (2000 nM). By ELISA we found Abs in plasma of 27 of 37 randomly selected aTTP patients that bound specifically to the anti-idiotypic DARPins. Pre-incubation with anti-idiotypic DARPins (960 nM) prevented binding of plasma anti-ADAMTS13 Ab to recombinant ADAMTS13 in a competition ELISA (n=5). Our findings of anti-idiotypic DARPins able to neutralize several, although similar (belonging to the same CDR3 motif) pathogenic anti-ADAMTS13 Abs and the fact that the DARPins are also specifically recognized by plasma-derived anti-ADAMTS13 Abs of different aTTP patients are promising and suggest that possibly only a limited number of the selected DARPins are required to develop a general treatment for aTTP.
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Solitano, Virginia, Gionata Fiorino, Ferdinando D’Amico, Laurent Peyrin-Biroulet, and Silvio Danese. "Thrombosis in IBD in the Era of JAK Inhibition." Current Drug Targets 22, no. 1 (December 31, 2020): 126–36. http://dx.doi.org/10.2174/1389450121666200902164240.
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: Patients with inflammatory bowel diseases (IBD) have an increased risk of thrombosis. The interaction between inflammation and coagulation has been extensively studied. It is well-known that some drugs can influence the haemostatic system, but several concerns on the association between therapies and increased risk of thrombosis remain open. While biologics seem to have a protective role against thrombosis via their anti-inflammatory effect, some concerns about an increased risk of thrombosis with JAK inhibitors have been raised. We conducted a literature review to assess the association between biologics/small molecules and venous/arterial thrombotic complications. An increased risk of venous and arterial thrombosis was found in patients treated with corticosteroids, whereas anti-TNF were considered protective agents. No thromboembolic adverse event was reported with vedolizumab and ustekinumab. In addition, thromboembolic events rarely occurred in patients with ulcerative colitis (UC) after therapy with tofacitinib. The overall risk of both venous and arterial thrombosis was not increased based on the available evidence. Finally, in the era of JAK inhibitors, treatment should be individualized by evaluating the pre-existing potential thrombotic risk balanced with the intrinsic risk of the medication used.
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Allen, Kristi L., Mukesh K. Jain, and Keith R. McCrae. "KLF2 and KLF4 Are Essential Mediators of the Anti-Thrombotic Effects of Statins in the Presence of Antiphospholipid/Anti-ß2GPI Antibodies,." Blood 118, no. 21 (November 18, 2011): 3272. http://dx.doi.org/10.1182/blood.v118.21.3272.3272.
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Abstract Abstract 3272 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLA). The majority of APLA are directed against phospholipid binding proteins, particularly β2GPI. Anti-ß2GPI antibodies activate endothelial cells and monocytes in a β2GPI-dependent manner through a pathway that involves NF-κB and leads to increased expression of adhesion molecules, tissue factor and proinflammatory cytokines. Krüppel-like factors (KLFs) regulate endothelial cell and monocyte responses to inflammatory stimuli; increased expression of these transcription factors inhibits proinflammatory and procoagulant gene expression, and maintains vascular homeostasis. We recently reported that anti-ß2GPI antibodies decrease the expression of KLF2 and KLF4 in endothelial cells (Allen et al, Blood 2011), promoting endothelial cell activation. Subsequent studies demonstrate that these antibodies decrease expression of KLF2 in monocytes as well. Statins have been proposed as a potential alternative to anticoagulation for APS patients, and stimulate the expression of KLFs. We hypothesized that the ability of statins to block endothelial cell activation in response to anti-β2GPI antibodies was mediated by KLFs. Treatment of endothelial cells and monocytes with 100 nM fluvastatin, lovastatin, or simvastatin upregulated KLF2 and KLF4 mRNA, even in the presence of anti-ß2GPI antibodies. In parallel, statin treatment inhibited the anti-β2GPI antibody-mediated induction of E-selectin, VCAM-1, and TF mRNA in endothelial cells, and ICAM-1 and TF mRNA in human monocytes. To assess the dependence of these effects on KLF expression, endothelial cells were pretreated with KLF2 or KLF4 siRNA prior to treatment with statins. siRNA-mediated inhibition of KLF expression completely blocked the ability of statins to prevent anti-β2GPI antibody-induced endothelial cell activation, as measured by adhesion molecule and TF mRNA levels and expression of E-selectin on the endothelial cell surface. Taken together, these data demonstrate that KLFs are critical modulators of the effects of statins on endothelial cells, and that increased expression of KLFs may represent a mechanism by which these drugs inhibit the activation of endothelial cells and monocytes by APLA/anti-β2GPI antibodies. Disclosures: No relevant conflicts of interest to declare.
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Bhavika Kunwar, Vartika Jain, and Surendra Kumar Verma. "Qualitative phytochemical screening and in vitro thrombolytic activity of Capparis decidua Edgew. Fruit." GSC Biological and Pharmaceutical Sciences 19, no. 3 (June 30, 2022): 160–67. http://dx.doi.org/10.30574/gscbps.2022.19.3.0232.
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Abnormal thrombosis plays an important role in development of ischemic heart disease and stroke. Synthetic thrombolytic agents are effective but possess some adverse effects. Therefore, the need for development of comparatively safer anti-thrombotic drugs from natural resources such as plants arises. Capparis decidua Edgew. (Family – Capparaceae) is a densely branched, xerophytic shrub and grows abundantly in arid and semiarid regions. Its fruits are major ingredient of the popular ‘Panchkuta’ vegetable of Rajasthan and also used to prepare pickle. Caper fruits are recommended for treatment of several diseases in traditional medicine including cardiac ailments and shown antioxidant and hypolipidemic potential in scientific studies and therefore, fruits of C. decidua were evaluated for their in vitro thrombolytic potential for the first time. Preliminary qualitative phytochemical screening of fruits of C. decidua; purchased from local market of Udaipur has shown the presence of flavonoids, terpenoids, phenol, phlobatanin, amino acids and carbohydrates and absence of saponin, tannins, steroids and cardiac glycosides. A significant percent clot lysis activity of 23.16±1.26 and 32.39±2.10 was exhibited by methanolic extracts (ME-I and ME-II) of fruits of C. decidua respectively as compared to the positive control streptokinase and negative control as distilled water. However, characterization of bioactive anti-thrombotic molecules as well as large scale, clinical studies is warranted to establish in vivo thrombolytic efficacy of its fruits. Thrombolytic potential of Caper berries as observed in the present study could be useful to recommend its consumption as a dietary health supplement in order to prevent from thrombotic cardiovascular diseases.
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Magamedov, I. D., L. P. Pivovarova, S. P. Nokhrin, V. V. Soroka, O. B. Ariskina, I. V. Osipova, I. M. Radjabov, et al. "Factors of inflammatory, adhesiveness and thrombosis in acute lower limb ischemia and dexamethasone therapy." Russian Journal of Immunology 25, no. 3 (September 20, 2022): 251–58. http://dx.doi.org/10.46235/1028-7221-1117-foi.
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Lymphocyte-to-platelet adhesion during hypoxia, tissue damage, activation of inflammation and coagulation is associated with expression of ICAM-1 membrane molecules by blood and tissue cells. At the same time, the platelet adhesion receptors determine their adherence to endothelium and recruited lymphocytes. Moreover, the role of platelets in pathogenesis of ischemic cardiovascular diseases comprises their ability to modulate both hemostasis and inflammatory reactions, which are accompanied by secretion of inflammatory mediators and some factors that promote recruitment of leukocytes to tissue damage sites. Creatine kinase activity is a sensitive marker of tissue damage and tissue ischemia. The purpose of the present study was to assess the effect of anti-inflammatory therapy with dexamethasone upon the intensity of inflammation and adhesive properties of lymphocytes, number of platelets in peripheral blood of the patients with acute lower limb ischemia (ALLI), as well as to evaluate the effectiveness of treatment.
To study the effect of anti-inflammatory therapy, a group of 32 patients treated with dexamethasone was selected; the control group was represented by 71 patients with basic therapy, the comparison group consisted of 15 volunteers. After revascularization, all patients received antiplatelet and anticoagulant therapy. Dexamethasone infusions were carried out as a course of 4 to 6 days after reconstructive surgery. In all patients, the content of C-reactive protein in blood, the activity of creatine kinase, the content of platelets and, especially, of enlarged platelets were determined. The numbers of lymphocytes expressing ICAM-1 (CD54+) adhesion molecules were counted using immunocytochemical technique. The studies were performed before surgery and 1, 3, 5, 7, 10 days after surgery.
During exacerbation of the limb ischemia and damage to endothelium, the accumulation of cytolysis products was noted. Expression of adhesion molecules was increased both on endotheliocytes and on inflammation effector cells, i.e., leukocytes and platelets. The adhesion molecules transmit the activating signal inside the cell, thus promoting adhesion of leukocytes and platelets to endothelium, lymphocytic-platelet adhesion, formation of parietal thrombi, and possible occlusion of damaged vessels. Increased expression of adhesion molecules is associated with activation of metabolism, inflammation, coagulation and oxidative stress. It may stimulate all hematopoietic lineages, including platelets. The involvement level of cellular reactions in pathogenesis of the disease affects effectiveness and duration of treatment, risk of recurrent thrombosis and lethal outcome. Anti-inflammatory therapy with dexamethasone contributed to earlier remission, it was associated with lower frequency of infectious and thrombotic complications, decreased mortality, and reduced duration of treatment.
Inflammation, adhesiveness of effector cells and thrombosis are important factors in pathogenesis of acute lower limb ischemia. Additional anti-inflammatory therapy with dexamethasone contributes to earlier remission, decreased proportion of infectious and thrombotic complications, lower frequency of deaths, and reduced duration of treatment.
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Wagener, Frank A. D. T. G., Peter Pickkers, Stephen J. Peterson, Stephan Immenschuh, and Nader G. Abraham. "Targeting the Heme-Heme Oxygenase System to Prevent Severe Complications Following COVID-19 Infections." Antioxidants 9, no. 6 (June 19, 2020): 540. http://dx.doi.org/10.3390/antiox9060540.
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SARS-CoV-2 is causing a pandemic resulting in high morbidity and mortality. COVID-19 patients suffering from acute respiratory distress syndrome (ARDS) are often critically ill and show lung injury and hemolysis. Heme is a prosthetic moiety crucial for the function of a wide variety of heme-proteins, including hemoglobin and cytochromes. However, injury-derived free heme promotes adhesion molecule expression, leukocyte recruitment, vascular permeabilization, platelet activation, complement activation, thrombosis, and fibrosis. Heme can be degraded by the anti-inflammatory enzyme heme oxygenase (HO) generating biliverdin/bilirubin, iron/ferritin, and carbon monoxide. We therefore postulate that free heme contributes to many of the inflammatory phenomena witnessed in critically ill COVID-19 patients, whilst induction of HO-1 or harnessing heme may provide protection. HO-activity not only degrades injurious heme, but its effector molecules possess also potent salutary anti-oxidative and anti-inflammatory properties. Until a vaccine against SARS-CoV-2 becomes available, we need to explore novel strategies to attenuate the pro-inflammatory, pro-thrombotic, and pro-fibrotic consequences of SARS-CoV-2 leading to morbidity and mortality. The heme-HO system represents an interesting target for novel “proof of concept” studies in the context of COVID-19.
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Ivanov, Daniil G., Son N. Ngyuen, Si-Hung Le, Yi Du, Nikola Ivetic, Ishac Nazy, and Igor A. Kaltashov. "Structure of pathogenic anti-PF4 autoantibodies reveals the mechanism of immune response formation in vaccine-induced thrombotic thrombocytopenia (VITT)." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 234.07. http://dx.doi.org/10.4049/jimmunol.210.supp.234.07.
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Abstract Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe, but rare, side effect of adenoviral-vectored COVID-19 vaccines. It is attributed to formation of autoantibodies recognizing Platelet Factor 4 (PF4) and capable of triggering platelet activation, which leads to thrombosis. We used mass spectrometry (MS) to determine the structure of a VITT patient-derived anti-PF4 antibodies (VITT IgG) and identified their unique properties underlying pathogenicity. The VITT IgGs studied in this work were extracted from the plasma of a ChAdOx1 vaccine recipient, who developed VITT. Intact-mass MS analysis of these antibodies revealed one major clone, and its complete structural characterization was carried out using MS-based de novo sequencing. This allowed us to establish its subclass (IgG2), determine the amino acid sequence, and identify an N-glycan in the variable region. This information was used to build a 3D model of the VITT IgG, which revealed the presence of a large polyanionic patch within the paratope involving four CDR regions. Molecular dynamics simulations highlight the role of electrostatics as the major driver of the VITT IgG binding to PF4. The large equatorial belt of the positive charge circumscribing the PF4 tetramer serves as a distributed epitope, allowing it to cross-link up to three VITT IgG molecules, giving rise to large immune complexes capable of FcyRIIa-mediated platelet activation. The molecular mechanism of VITT pathogenesis emerging from this work explains how VITT IgGs and PF4 form large immune complexes in the absence of heparin, which is essential to for platelet activation in a similar disease, heparin-induced thrombocytopenia, and sheds light on the etiology of this devastating condition. Supported by grant from NIH R01 GM112666
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Ucci, F. M., F. Villani, A. Capozzi, M. De Michele, S. Truglia, S. Mancuso, L. Rapino, et al. "AB0127 ANTIGENIC ASSESSMENT FOR THE Β2GPIOX-PF4 COMPLEX IN A MONOCENTRIC COHORT OF PATIENTS WITH APS, THROMBOSIS DURING SARS-COV-2 INFECTION AND VITT." Annals of the Rheumatic Diseases 82, Suppl 1 (May 30, 2023): 1243.1–1243. http://dx.doi.org/10.1136/annrheumdis-2023-eular.6134.
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BackgroundPlatelet factor 4 (PF4) is a protein with a pro-clotting function expressed by activated platelets with a high affinity for anionic glycosaminoglycans present on the platelet surface. It has been shown that the positively charged surface of PF4 tetramer interacts with the negatively charged regions of β2glycoprotein I (β2GPI) domains, stabilising the link between the antigen and the phospholipid surface, thus increasing the possibility of binding with the respective antibodies. In particular, a tetramer of PF4 selectively binds two molecules of β2GPI, favouring the dimerization of the same, which is crucial in platelet activation and therefore in thrombotic manifestations of antiphospholipid syndrome (aPS). PF4 may be a common denominator in syndromes such as aPS and heparin-induced thrombocytopenia (HIT), which share similar clinical manifestations as thrombocytopenia and thrombosis. Other syndromes, which share the same clinical and laboratory features of HIT despite not having previously received heparin, appear to be associated with the presence of anti-PF4 antibodies. Such pathologies could only be explained by HIT antibodies with heparin-independent platelet-activating properties. One of these could be vaccine-induced Immune thrombotic thrombocytopenia (VITT) post-somministration of ChAdOx1 nCoV-19 vaccine. Recent studies have shown structural similarities between heparin and β2GPI, which may be responsible for thrombotic events in those infected with SARS-Cov-2 and VITT, who never had heparin. In particular, oxidised-β2GPI (β2GPIox) may mimic heparin by structural analogy and link to PF4. Considering the structural similarities between heparin and β2GPIox, and demonstrating the immunogenicity of the hypothesised complex in APS, the alternative molecule could be represented by β2GPIox itself, thus explaining the thrombotic events following vaccination in subjects who have never received heparin.ObjectivesThe aim of the study is to test the potential immunogenicity of the β2GPIox-PF4 complex and the presence of antibodies against this complex in patients with aPS, thrombosis during infection with SARS-CoV-2 or VITT.Methods34 patients with proven diagnosis of APS, 17 patients with thrombosis related to infection SARS CoV-2 and 3 patients with VITT were enrolled. Only one aPS patient received heparin prior to testing. Antibodies to the β2GPIox-PF4 complex were evaluated by home made ELISA immunoenzyme testing. Competitive inhibition (homologous) experiments of the bond of PF4 with β2GPIox in the fluid phase were conducted in order to verify the binding specificity.ResultsAnti-β2GPIox-PF4 antibodies were detected in 11 of 34 aPS patients (32%) and all VITT patients (100%), while none of covid-19 patients tested positive. In addition, Anti-β2GPIox-PF4 antibodies positivity significantly correlated with the presence of anti-β2GPI antibodies (p=0.0259) and with a triple positive antibody profile (p=0.0017) (Figure 1). There was no significant association between anti-β2GPIox-PF4 antibodies’ title and clinical features of aPS patients.ConclusionThe results of this study show a new antibody positivity in aPS and VITT. In particular we have identified a high prevalence of anti-β2GPIox-PF4 antibodies which may be involved in the pathophysiology of both aPS and VITT. In addition the absence of anti-β2GPIox-PF4 antibodies in covid-19 patients suggest that the thrombotic mechanism that underlies VITT is different from covid-19 and, on the contrary, could resemble aPS.Figure 1.References[1]Alessandri C et al. New autoantigens in the antiphospholipidsyndrome. Autoimmun Rev. 2011[2]Sikara MP et al. {beta}2 Glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome. Blood. 2010 Jan 21[3] Favaloro EJ et al. Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT/HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT/TTS. Antibodies (Basel). 2022 Jan 21Acknowledgements:NIL.Disclosure of InterestsNone Declared.
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Caso, Francesco, Luisa Costa, Donato Rigante, Orso Maria Lucherini, Paolo Caso, Vittoria Bascherini, Bruno Frediani, et al. "Biological Treatments in Behçet’s Disease: Beyond Anti-TNF Therapy." Mediators of Inflammation 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/107421.
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Behçet’s disease (BD) is universally recognized as a multisystemic inflammatory disease of unknown etiology with chronic course and unpredictable exacerbations: its clinical spectrum varies from pure vasculitic manifestations with thrombotic complications to protean inflammatory involvement of multiple organs and tissues. Treatment has been revolutionized by the progressed knowledge in the pathogenetic mechanisms of BD, involving dysfunction and oversecretion of multiple proinflammatory molecules, chiefly tumor necrosis factor- (TNF-)α, interleukin- (IL-) 1β, and IL-6. However, although biological treatment with anti-TNF-αagents has been largely demonstrated to be effective in BD, not all patients are definite responders, and this beneficial response might drop off over time. Therefore, additional therapies for a subset of refractory patients with BD are inevitably needed. Different agents targeting various cytokines and their receptors or cell surface molecules have been studied: the IL-1 receptor has been targeted by anakinra, the IL-1 by canakinumab and gevokizumab, the IL-6 receptor by tocilizumab, the IL12/23 receptor by ustekinumab, and the B-lymphocyte antigen CD-20 by rituximab. The aim of this review is to summarize all current experiences and the most recent evidence regarding these novel approaches with biological drugs other than TNF-αblockers in BD, providing a valuable addition to the actually available therapeutic armamentarium.
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Nozaki, Kazuya, Hiroaki Hikiami, Hirozo Goto, Takako Nakagawa, Naotoshi Shibahara, and Yutaka Shimada. "Keishibukuryogan (Gui-Zhi-Fu-Ling-Wan), a Kampo Formula, Decreases Disease Activity and Soluble Vascular Adhesion Molecule-1 in Patients with Rheumatoid Arthritis." Evidence-Based Complementary and Alternative Medicine 3, no. 3 (2006): 359–64. http://dx.doi.org/10.1093/ecam/nel025.
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An increasing death rate due to cardiovascular disease in patients with rheumatoid arthritis (RA) has been reported. Keishibukuryogan (KBG) is a traditional Chinese/Japanese (Kampo) formula that has been administered to patients with blood stagnation, e.g. thrombotic disease and atherosclerosis. The objective of this study was to evaluate the efficacy of KBG on disease activity and endothelial dysfunction in RA patients. Sixteen RA patients were enrolled and administered KBG (12 g per day) for 12 weeks in addition to continuing other drugs. The disease activity of RA was assessed by modified disease activity scores for 28 joints (DAS28). Plasma levels of adhesion molecules, soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were evaluated. C-reactive protein (CRP), inflammatory cytokines (IL-1β, IL-6 and TNF-α) and lipid peroxide (LPO) were also evaluated. Fourteen patients completed the study. The disease activity of RA, tender joint count, swollen joint count and DAS28 decreased significantly. Among adhesion molecules, only sVCAM-1 decreased significantly. LPO also decreased significantly, whereas CRP and inflammatory cytokines remained unchanged. These results suggest that KBG has insufficient anti-inflammatory or immunomodulating effect but does have a beneficial effect on articular symptoms and a protective effect against endothelial dysfunction in RA patients.
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Sturgeon, Sharelle A., Catherine Jones, James A. Angus, and Christine E. Wright. "Adaptation of the Folts and electrolytic methods of arterial thrombosis for the study of anti-thrombotic molecules in small animals." Journal of Pharmacological and Toxicological Methods 53, no. 1 (January 2006): 20–29. http://dx.doi.org/10.1016/j.vascn.2005.06.006.
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Wang, Chaoming, Mengrou Chen, Xiaoyu Lu, Shuo Yang, Min Yang, Yaqun Fang, Ren Lai, and Zilei Duan. "Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech." International Journal of Molecular Sciences 24, no. 13 (July 4, 2023): 11097. http://dx.doi.org/10.3390/ijms241311097.
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When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl3-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.
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Schaller, Monica, Sabine Hiltbrunner, Irmela Sulzer, Monique Vogel, Karim Kentouche, Bernhard Laemmle, and Johanna A. Kremer Hovinga. "High-Throughput Screening of a Large Combinatorial Library of Small Proteins Against Human Spleen-Derived Monoclonal Anti-ADAMTS13 Antibodies Yields Specific Anti-Idiotypic Molecules – Potential Tools for New Treatment Strategy in Acquired TTP." Blood 120, no. 21 (November 16, 2012): 487. http://dx.doi.org/10.1182/blood.v120.21.487.487.
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Abstract Abstract 487 Background. Acquired Thrombotic Thrombocytopenic Purpura (TTP) is the result of autoantibodies (Abs) neutralizing and/or accelerating clearance of ADAMTS13. Despite the first-line treatment with daily plasma exchange (PEX), acute TTP is still associated with a mortality rate of ∼20% leaving survivors at high risk of relapse. Availability of molecules for neutralization of pathogenic anti-ADAMTS13 Abs during acute bouts as well as for the targeted elimination of anti-ADAMTS13 specific memory B- and plasma-cells to prevent relapses are thus highly desirable tools to improve patient care and/or treatment of TTP. The primary epitope of pathological relevant, inhibitory anti-ADAMTS13 Abs was identified to lay in the ADAMTS13 spacer domain and to be conformational compromising amino acid residues R568, F592, Y660, Y661 and Y665. We have previously generated 29 specific human monoclonal anti-ADAMTS13 Abs from 2 patients (A and B) splenectomized for frequently relapsing TTP using spleen-derived mononuclear cells (ASH 2011, abstract #194). These anti-ADAMTS13 Abs, characterized by a 5–6 fold increased inhibitory capacity than previously reported as well as eight unique CDR3 motifs in their variable heavy chain genes, four of which are common to both acquired TTP patients, are useful tools to select small anti-idiotypic molecules, mimicking the conformational epitope on ADAMTS13. For this purpose we chose the large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins), characterized by: (1) a high diversity with a library size of 1015–23, allowing to find molecules to any target including conformational epitopes, (2) expressing molecules with a high stability, and (3) being immune tolerant when administered. Methods. Two DARPin libraries, N2C (diversity of 1015) and N3C (1023) coding for two or three randomized ankyrin repeat modules respectively, provided by Molecular Partners AG (Schlieren, Switzerland), were screened against three biotinylated inhibitory anti-ADAMTS13 Abs from patient A belonging to one CDR3 motif (CDR3 motif-1) and pooled in equimolar ratio as targets. Selected DARPins from the fourth round of ribosomal display were cloned, and single clones producing anti-ADAMTS13 idiotypes were purified, tested separately for their specificity towards 29 single, spleen-derived monoclonal anti-ADAMTS13 Abs by ELISA and their DNA sequence analyzed. Results. Seven of 192 (N2C-library) and 2/192 (N3C) single DARPin clones selected against a pool of three inhibitory anti-ADAMTS13 Abs, of a the single CDR3 motif-1, were highly specific for their target. Detailed DNA sequence analysis showed that 4/7 (N2C) and 2/2 (N3C) of the anti-ADAMTS13 specific DARPins were unique whereas 3/7 N2C DARPins consisted of repeats of the unique clones. So far, four (3 N2C and 1 N3C) unique DARPins were purified and their reactivity at equimolar concentration tested towards 200 nM of each of the 29 anti-ADAMTS13 Abs, individually coated on a microtiter plate by ELISA assay. All four DARPins were highly specific for the pool of Abs used for selection as well as other Abs belonging to the same CDR3 motif-1 (n=5). Anti-ADAMTS13 Abs containing the remaining three of the four common (patient A and B) CDR3 motifs, not used for selection, were recognized by 3/4 unique DARPins although to a lesser extent and more anti-ADAMTS13 Abs originating from patient A, were recognized than from patient B, namely 94%, 69% and 56% for CDR3 motif1-3 compared to only 31 %, 54% and 15% from patient B, respectively. Conclusions. Using inhibitory anti-ADAMTS13 of one patient (A) and one CDR3 motif, we were able to select a number of highly specific anti-idiotypic DARPins binding to different monoclonal anti-ADAMTS13 Abs of two genetically and geographically unrelated acquired TTP patients. The fact that several although similar (belonging to the same CDR3 motif) antibodies can be recognized by one anti-idiotypic molecule is promising and hints at a limited number of different anti-idiotypic molecules necessary to neutralize inhibitory anti-ADAMTS13 Abs in the plasma of acquired TTP patients. The analysis of their affinity and the neutralizing potential are ongoing. Disclosures: No relevant conflicts of interest to declare.
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Jayakumar, Thanasekaran, Chun-Ming Yang, Ting-Lin Yen, Chia-Yuan Hsu, Joen-Rong Sheu, Chih-Wei Hsia, Manjunath Manubolu, Wei-Chieh Huang, Cheng-Ying Hsieh, and Chih-Hsuan Hsia. "Anti-Inflammatory Mechanism of An Alkaloid Rutaecarpine in LTA-Stimulated RAW 264.7 Cells: Pivotal Role on NF-κB and ERK/p38 Signaling Molecules." International Journal of Molecular Sciences 23, no. 11 (May 24, 2022): 5889. http://dx.doi.org/10.3390/ijms23115889.
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Lipoteichoic acid (LTA) is a key cell wall component and virulence factor of Gram-positive bacteria. LTA contributes a major role in infection and it mediates inflammatory responses in the host. Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from Evodia rutaecarpa, has shown a variety of fascinating biological properties such as anti-thrombotic, anticancer, anti-obesity and thermoregulatory, vasorelaxing activity. It has also potent effects on the cardiovascular and endocrine systems. Herein, we investigated rutaecarpine’s (Rut) anti-inflammatory effects in LTA-stimulated RAW macrophage cells. The Western blot and spectrophotometric results revealed that Rut inhibited the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin (IL)-1β in the LTA-induced macrophage cells. Successively, our mechanistic studies publicized that Rut inhibited LTA-induced phosphorylation of mitogen-activated protein kinase (MAPK) including the extracellular signal-regulated kinase (ERK), and p38, but not c-Jun NH2-terminal kinase (JNK). In addition, the respective Western blot and confocal image analyses exhibited that Rut reserved nuclear transcription factor kappa-B (NF-κB) by hindering inhibitor of nuclear factor κB-α (IκBα) and NF-κB p65 phosphorylation and p65 nuclear translocation. These results indicate that Rut exhibits its anti-inflammatory effects mainly through attenuating NF-κB and ERK/p38 signaling pathways. Overall, this result suggests that Rut could be a potential therapeutic agent for the treatment of Gram-positive bacteria induced inflammatory diseases.
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Stark, Júlia. "Oxidatív stressz és atherosclerosis." Orvosi Hetilap 156, no. 28 (July 2015): 1115–19. http://dx.doi.org/10.1556/650.2015.30201.
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Atherosclerosis is a leading cause of death in developed countries. Genetic susceptibility and environmental factors play a role in the pathogenesis. Most of these factors lead to endothelial dysfunction and other pro-atherogenic processes by causing oxidative stress. Atherosclerosis typically develops at the curved and branched regions of the arterial tree, where the laminar blood flow is disturbed. This leads to increased permeability of the endothel to low density lipoprotein molecules, which accumulate in the intima and are oxidised by vascular cells. Oxidised low density lipoprotein takes part in many phases of atherogenesis: stimulates the binding of monocytes to the endothel, foam cell formation, the development of plaques, plaque destabilization and thrombotic complications. Since oxidative stress plays an important role in atherogenesis, it has been suggested that antioxidant molecules might have anti-atherogenic function. Many clinical investigations have shown that antioxidants such as N-acetylcystein, vitamin E and C, folic acid, and estrogens can prevent atherosclerosis, however, randomized studies failed to confirm this effect. Orv. Hetil., 2015, 156(28), 1115–1119.
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Lamendola, R., P. Favia, F. Palumbo, and R. d'Agostino. "Plasma-modification of polymers: process control in PE-CVD of gas-barrier films and plasma-processes for immobilizing anti-thrombotic molecules." European Physical Journal Applied Physics 4, no. 1 (October 1998): 65–71. http://dx.doi.org/10.1051/epjap:1998244.
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Huang, Mei-Zhou, Xiao-Rong Lu, Ya-Jun Yang, Xi-Wang Liu, Zhe Qin, and Jian-Yong Li. "Cellular Metabolomics Reveal the Mechanism Underlying the Anti-Atherosclerotic Effects of Aspirin Eugenol Ester on Vascular Endothelial Dysfunction." International Journal of Molecular Sciences 20, no. 13 (June 28, 2019): 3165. http://dx.doi.org/10.3390/ijms20133165.
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Aspirin eugenol ester (AEE) possesses anti-thrombotic, anti-atherosclerotic and anti-oxidative effects. The study aims to clarify the mechanism underlying the anti-atherosclerotic effects of AEE on vascular endothelial dysfunction. Both the high-fat diet (HFD)-induced atherosclerotic rat model and the H2O2-induced human umbilical vein endothelial cells (HUVECs) model were used to investigate the effects of AEE on vascular endothelial dysfunction. UPLC/QTOF-MS coupled with a multivariate data analysis method were used to profile the variations in the metabolites of HUVECs in response to different treatments. Pretreatment of HUVECs with AEE significantly ameliorated H2O2-induced apoptosis, the overexpression of E-selectin and VCAM-1, and the adhesion of THP-1 cells. Putative endogenous biomarkers associated with the inhibition of endothelial dysfunction were identified in HUVECs pretreated with AEE in the absence or presence of H2O2, and these biomarkers were involved in important metabolic pathways, including amino acid metabolism, carbohydrate metabolism, and glutathione metabolism. Moreover, in vivo, AEE also significantly reduced vascular endothelial dysfunction and decreased the overexpression of VCAM-1 and E-selectin. Based on our findings, the mechanism underlying the anti-atherosclerotic effects of AEE might be related to a reduction in vascular endothelial dysfunction mediated by ameliorating alterations in metabolism, inhibiting oxidative stress, and decreasing the expression of adhesion molecules.
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Badimon, Lina, Cristina Rodríguez, María González-Díez, and José Martínez-González. "Sphingosine-1-phosphate: A bioactive lipid that confers high-density lipoprotein with vasculoprotection mediated by nitric oxide and prostacyclin." Thrombosis and Haemostasis 101, no. 04 (2009): 665–73. http://dx.doi.org/10.1160/th08-10-0675.
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SummarySphingosine-1-phosphate (S1P) is a bioactive lipid generated in the intracellular membranes from the metabolism of sphingomyelin. Once secreted/exported by cells of haematopoietic origin and vascular cells S1P interacts with plasma proteins and accumulates in high-density lipoprotein (HDL). Growing evidence indicates that HDL-associated S1P is responsible for the beneficial effects of these lipoproteins on vasorelaxation, cell survival, cell adhesiveness, angiogenesis and synthesis of two powerful endogenous anti-atherogenic and anti-thrombotic molecules such as nitric oxide (NO) and prostacyclin (PGI2). It is likely that vascular effects of HDL-S1P are regulated by the local expression of S1P receptors. Five G protein-coupled receptors (S1P1 to S1P5), with differential expression patterns and dissimilar coupling mechanism to G protein subunits, have been identified in the vasculature. This review is focused on the central role of S1P as a bioactive component that confers vasculoprotective properties to HDL by eliciting a wide range of biological responses on endothelial and smooth muscle cells largely dependent on the up-regulation of NO and prostacyclin.
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Maevskaya, Marina V., Yulia V. Kotovskaya, Vladimir T. Ivashkin, Olga N. Tkacheva, Ekaterina A. Troshina, Marina V. Shestakova, Valeriy V. Breder, et al. "The National Consensus statement on the management of adult patients with non-alcoholic fatty liver disease and main comorbidities." Terapevticheskii arkhiv 94, no. 2 (February 15, 2022): 216–53. http://dx.doi.org/10.26442/00403660.2022.02.201363.
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The National Consensus was prepared with the participation of the National Medical Association for the Study of the Multimorbidity, Russian Scientific Liver Society, Russian Association of Endocrinologists, Russian Association of Gerontologists and Geriatricians, National Society for Preventive Cardiology, Professional Foundation for the Promotion of Medicine Fund PROFMEDFORUM.
The aim of the multidisciplinary consensus is a detailed analysis of the course of non-alcoholic fatty liver disease (NAFLD) and the main associated conditions. The definition of NAFLD is given, its prevalence is described, methods for diagnosing its components such as steatosis, inflammation and fibrosis are described. The association of NAFLD with a number of cardio-metabolic diseases (arterial hypertension, atherosclerosis, thrombotic complications, type 2 diabetes mellitus, obesity, dyslipidemia, etc.), chronic kidney disease and the risk of developing hepatocellular cancer were analyzed. The review of non-drug methods of treatment of NAFLD and modern opportunities of pharmacotherapy are presented. The possibilities of new molecules in the treatment of NAFLD are considered: agonists of nuclear receptors, antagonists of pro-inflammatory molecules, etc. The positive properties and disadvantages of currently used drugs (vitamin E, thiazolidinediones, etc.) are described. Special attention is paid to the multi-target ursodeoxycholic acid molecule in the complex treatment of NAFLD as a multifactorial disease. Its anti-inflammatory, anti-oxidant and cytoprotective properties, the ability to reduce steatosis an independent risk factor for the development of cardiovascular pathology, reduce inflammation and hepatic fibrosis through the modulation of autophagy are considered. The ability of ursodeoxycholic acid to influence glucose and lipid homeostasis and to have an anticarcinogenic effect has been demonstrated. The Consensus statement has advanced provisions for practitioners to optimize the diagnosis and treatment of NAFLD and related common pathogenetic links of cardio-metabolic diseases.
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Navarrete, Ana-Maria, Caterina Casari, Paulette Legendre, Isabelle Marx, Jiun-Ruey Hu, Peter J. Lenting, Olivier D. Christophe, and Cécile V. Denis. "A murine model to characterize the antithrombotic effect of molecules targeting human von Willebrand factor." Blood 120, no. 13 (September 27, 2012): 2723–32. http://dx.doi.org/10.1182/blood-2012-03-420042.
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Abstractvon Willebrand factor (VWF) is a promising target for developing antithrombotic drugs. The absence of accessible animal models impedes the study of specific human VWF (huVWF) targeting molecules in thrombosis. huVWF is not functional in the mouse because of a lack of interaction between huVWF and murine glycoprotein Ib. Using site-directed mutagenesis, we have replaced single or multiple amino acids in huVWF with their murine counterparts to eliminate species incompatibility. Using hydrodynamic injection, we have expressed the different chimeric VWF constructs into VWF−/− mice. Only huVWF with a complete murine A1 domain insertion was able to correct bleeding in vivo and form occlusive thrombi in mesenteric vessels after FeCl3 treatment. Using this model, we tested the antithrombotic effect of monoclonal antibodies against huVWF, blocking its interaction with collagens (mAbs 203 and 505) or with glycoprotein IIbIIIa (mAb 9). The 3 mAbs inhibited the thrombotic process in arterioles of VWF−/− mice expressing huVWFmuA1. Inhibiting VWF-interaction with collagens was more potent, emphasizing the potential of such a target as an antithrombotic tool. Our results validate our murine model as a simple in vivo tool to evaluate anti-huVWF agents.
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Flax, Sherri. "ADAMTS13 Immunohistochemical Expression in Normal and Diseased Equine Tissues." Blood 134, Supplement_1 (November 13, 2019): 4925. http://dx.doi.org/10.1182/blood-2019-127407.
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ADAMTS13 is a metallopeptidase primarily synthesized in the liver. Its main function is to cleave von Willebrand factor (VWF) on the endothelial surface, and thus regulate platelet adhesion at the sites of vascular injury; VWF acts as a scaffold at the site of injury for platelet activation, platelet binding, and clot formation. Platelet activation also results in the release of platelet-dense granule components (such as cytokines, pro- and anti-inflammatory factors and other bioactive molecules) that are essential regulators of coagulation, but are also associated with inflammation. Reduced activity of ADAMTS13 is found in human patients with acquired thrombotic thrombocytopenic purpura (TPP), sepsis, and DIC. Both human TTP and equine colic are histologically characterized by the presence of microthrombi. Cleavage of VWF is dependent upon allosteric activation of ADAMTS13, which is broadly conserved in many animals, including horses. Human TTP and equine idiopathic colic share several interesting clinical similarities. Both have seasonal variations, typically in summer, can be associated with viral and bacterial infections, may be hereditary, have autoimmune associations, and recur. Both TTP and equine colic are clinically characterized by fever, abdominal pain renal failure, and neurologic symptoms. Microangiopathy is the predominant pathology in both TTP and equine colic. Using tissues obtained from thirteen horses euthanized for non-thrombotic conditions, immunohistochemical stains indicate the presence of ADAMTS13 in equine liver and colon. Furthermore, in one horse with colic, immunohistochemical staining of the resected colon indicates the presence of ADAMTS13 in the aboral margin (Figure 1) and the absence ADAMTS13 staining in the diseased tissue. These findings suggest that equine colic may be an animal model of thrombotic microangiopathy, very similar to TTP. Future studies will investigate immunohistochemical detection of ADAMTS13 in human tissues. Figure 1 Disclosures No relevant conflicts of interest to declare.
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Schaller, Monica M., Monique Vogel, Magdalena Skowronska, Irmela Sulzer, and Johanna A. Kremer Hovinga Strebel. "Anti-Idiotypic-Designed-Ankyrin-Repeat-Molecules (DARPins) Universally Neutralize Plasma-Derived Autoantibodies in Acquired TTP Patients." Blood 126, no. 23 (December 3, 2015): 104. http://dx.doi.org/10.1182/blood.v126.23.104.104.
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Abstract Background. Autoantibodies (Abs) neutralizing and/or accelerating clearance of the von Willebrand factor-cleaving protease-ADAMTS13 are the major culprits in acquired Thrombotic Thrombocytopenic Purpura (aTTP). Despite the first-line treatment with daily plasma exchange (PEX), acute aTTP is still today associated with a mortality rate of ~20% leaving survivors at high risk of relapse. To improve patient care and/or treatment of aTTP we explored the capacity of anti-idiotypic small molecules to specifically bind and potentially neutralize pathogenic anti-ADAMTS13 autoantibodies thereby restoring plasma-derived ADAMTS13 activity in acquired TTP patients. Method: As selecting tools we used previously generated spleen-derived inhibitory human monoclonal anti-ADAMTS13 antibodies consisting of the 4 antigen-binding CDR3 motifs shared by the two aTTP patients (Schaller et al, Blood 2014;124(23):3469-79). Either 3 mAbs representative for CDR3 Motif-1 or 3 mAbs for CDR3 Motif-3 or 2 mAbs representing CDR3 Motif-4 were pooled at equimolar concentrations by ribosomal display to screen for binders in two combinatorial small protein libraries (DARPins from Molecular Partners, Schlieren, Switzerland) containing either two (N2C) or three (N3C) randomized ankyrin repeat modules, respectively From each of the 6 libraries single anti-idiotypic DARPins were cloned into a pMPAG6 expression vector and purified by His-tag affinity chromatography. In total 22 unique anti-idiotypic DARPins, 4 N2C-DARPins and 2 N3C-DARPins selected by Motif-1, 9 N2C-DARPins by Motif-3 and 7 N2C-DARPins by Motif-4 mAbs were identified. Inhibitor targeting Strategy: The neutralization potential towards plasma-derived anti-ADAMTS13 Abs, was tested by pre-incubating the DARPins prior measuring the residual activity by functional FRETS assay, in a cohort of 50 acute aTTP patients with pathological inhibitor titers >0.4 BU/ml (diagnosed at our Center 2006-2014). First, equimolar pools (1000 nM) of anti-idiotypic DARPins (Motif-1, Motif-3 or Motif-4) were tested in 11/50 patients, secondly the contribution of the single DARPins from the responsive pools was assessed in 18/50 patients to finally test the entire cohort with a combination of all neutralizing anti-idiotypic DARPins. Results: The pool of all Motif-1 anti-idiotypic DARPins (n=6) neutralized inhibitor titers below the pathological threshold in 9/11 (82 %) aTTP patients tested, whereas only a slight reduction of the inhibitor titers (in average 15%) was observed for Motif-3 and Motif-4 DARPin pools, revealing the Motif-1 DARPins as the most universal neutralizers. To test the contribution of each Motif-1 anti-idiotypic DARPin to the observed inhibitor neutralization, single DARPins were tested in 18/50 aTTP revealing a reduction of the inhibitor of 20-50% (N2C-19) or 20% (N3C-74) in 5/18 patients tested. In contrast 90% (45/50) aTTP patients incubated with the pool of all 6 Motif-1 DARPins caused inhibitor titers to drop either completely or below the pathological threshold. The inhibitor titer was reduced below 1BU/ml in 3/50, but was irresponsive in 2/50 patients. Conclusions: Our data clearly show that anti-idiotypic DARPins can potently and universally neutralize inhibitory anti-ADAMTS13 Abs of aTTP patients. However an effective neutralization, restoring ADAMTS13 activity above 10-15% in most patients was only achieved when pooling all Motif-1 DARPins. If a combination of Motif-3 and/or Motif-4 DARPins with Motif-1 DARPins can additionally increase neutralization capacity also in relapsing patients is currently under investigation. Disclosures No relevant conflicts of interest to declare.
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Zhao, Aizhi, Chunsheng Li, George Coukos, Don L. Siegel, and Nathalie Scholler. "Isolation of high affinity human scFv antibodies against mouse and human tumor endothelial marker 1 (TEM1) using yeast-display scFv library derived from an autoimmune patient. (42.4)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 42.4. http://dx.doi.org/10.4049/jimmunol.182.supp.42.4.
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Abstract TEM1 is predominantly expressed on neovasculature, thus ideally suited for targeted imaging and therapy for cancer. Yeast has emerged as an alternative technology for display and selection of recombinant antibody fragments (scFv). Because autoimmune disorders may produce self-reactive specificities, an autoimmune repertoire derived from a patient with thrombotic thrombocytopenic purpura (TTP) was screened to isolate anti-TEM1 scFv. A yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage. The library (10^9 members) was flow sorted with decreasing antigen concentration until a scFv pool detecting 2 nM of rhTEM1/GST was obtained. Secreted scFv were expressed and 470 scFv were screened by ELISA for binding to rhTEM1/GST. Nearly 50% of the scFv gave colorimetric signals higher than 3 standard deviations above background. No cross-reactivity with GST control protein was detected. Sequencing of 29 scFv identified 8 unique antibodies. Two of these clones exhibited Kd's in the 100 pM range and bound to both mouse and human TEM1. Such cross-reactive anti-mouse/human TEM will facilitate proof of concept for the development of novel anti-angiogenic targeted therapies. Our data suggest that the use of large scFv libraries derived from autoimmune patient repertoires may provide a rich source of high affinity antibody reagents to therapeutically relevant molecules. This work was supported by the Claneil Foundation
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Jang, Hyosun, Seo-Young Kwak, Sunhoo Park, Kyuchang Kim, Young-heon Kim, Jiyoung Na, Hyewon Kim, et al. "Pravastatin Alleviates Radiation Proctitis by Regulating Thrombomodulin in Irradiated Endothelial Cells." International Journal of Molecular Sciences 21, no. 5 (March 10, 2020): 1897. http://dx.doi.org/10.3390/ijms21051897.
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Although radiotherapy plays a crucial in the management of pelvic tumors, its toxicity on surrounding healthy tissues such as the small intestine, colon, and rectum is one of the major limitations associated with its use. In particular, proctitis is a major clinical complication of pelvic radiotherapy. Recent evidence suggests that endothelial injury significantly affects the initiation of radiation-induced inflammation. The damaged endothelial cells accelerate immune cell recruitment by activating the expression of endothelial adhesive molecules, which participate in the development of tissue damage. Pravastatin, a cholesterol lowering drug, exerts persistent anti-inflammatory and anti-thrombotic effects on irradiated endothelial cells and inhibits the interaction of leukocytes and damaged endothelial cells. Here, we aimed to investigate the effects of pravastatin on radiation-induced endothelial damage in human umbilical vein endothelial cell and a murine proctitis model. Pravastatin attenuated epithelial damage and inflammatory response in irradiated colorectal lesions. In particular, pravastatin improved radiation-induced endothelial damage by regulating thrombomodulin (TM) expression. In addition, exogenous TM inhibited leukocyte adhesion to the irradiated endothelial cells. Thus, pravastatin can inhibit endothelial damage by inducing TM, thereby alleviating radiation proctitis. Therefore, we suggest that pharmacological modulation of endothelial TM may limit intestinal inflammation after irradiation.
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Ishii, Kazuyoshi, Shosaku Nomura, Tomoki Ito, Yuta Katayama, Taiichi Kyo, Shuichi Ota, Masanori Seki, et al. "Recombinant Thrombomodurin For The Treatment Of Transplantation-Associated Coagulopathy After Allogeneic Stem Cell Transplantation." Blood 122, no. 21 (November 15, 2013): 5454. http://dx.doi.org/10.1182/blood.v122.21.5454.5454.
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Abstract Background Levels of cytokines, chemokines and soluble molecules fluctuate after allogeneic hematopoietic stem cell transplantation (HSCT). These biomarkers are possible to be a diagnostic and prognostic value for transplantation-associated coagulopathy (TAC). In the present study, we investigated the effects of recombinant thrombomodulin (rTM) on levels of the cytokines, chemokines, and soluble factors registered in the ‘SIGHT’ research. SIGHT comprises the capital letters of five complications after HSCT, namely sinusoidal obstruction syndrome (SOS same as VOD), infection, GVHD, hemophagocytic syndrome and thrombotic microangiopathy. Methods The subjects were 159 patients who underwent allogeneic HSCT (bone marrow = 83, peripheral blood stem cells = 31, cord blood = 45). Blood samples were collected before and after transplantation. Levels of cytokines (interleukin-6, tumor necrosis factor-α, high-mobility group box (HMGB) 1), chemokines (monocyte chemotactic protein (MCP)-1, RANTES), and soluble molecules (soluble vascular cell adhesion molecule (VCAM)-1, soluble E-selectin, plasminogen activator inhibitor-1, platelet-derived microparticles (PDMP)) were measured by enzyme-linked immunosorbent assay. The rTM was administered as a therapy for transplantation-associated coagulopathy (TAC). This protocol was completed in day 4-14 after HSCT and consisted of day doses of 380 unit/kg with every days. Control group was also used heparin or no anti-coagulation therapy. Results MCP-1, IL-6, and TNF-a exhibited more significant elevations on days 7–14 after HSCT. In contrast, the levels of HMGB1, sE-selectin, sVCAM-1, PAI-1 and PDMP exhibited significant changes on days 14–28. There were significant improvements in TNF-a, sE-selectin, sVCAM-1, HMGB1, PAI-1 and PDMP after rTM-treatment (n=73), but not after rTM-untreatment patients (n=86). Conclusion We believe one of causes for TAC is pro-inflammatory cytokine including HMGB1. For this reason, it is thought that the direct anti-inflammatory effect of rTM’s lectin domein plays an important role in therapeutic mechanism for TAC. The present findings suggest the possibility that rTM can play a therapeutic role for TAC after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.
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Natalucci, F., A. Di Filippo, F. Ceccarelli, I. Zizzari, G. Olivieri, V. Orefice, C. Pirone, et al. "AB0119 ROLE OF COSTIMULATORY MOLECULES IN SYSTEMIC LUPUS ERYTHEMATOSUS: FOCUS ON CD137." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 1190–91. http://dx.doi.org/10.1136/annrheumdis-2022-eular.3813.
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BackgroundSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by a wide autoantibodies production. The traditionally concept of a B-cell driven disease has been changed in the last years due to the evidence demonstrating the crucial role of T cells in SLE pathogenesis. In particular, regulatory (Treg) and memory T cells seem act through co-stimulatory and co-inhibitory molecules, such as CD137, PD1-1 and CTLA4. The over-expression of this molecules on lymphocytes may contribute to immune system dysregulation.ObjectivesThe primary objective of the present case-control study was to evaluate the expression of CD137, PD1-1 and CTLA4 on T cell surface of SLE patients by using flow-cytometry. Secondly, we evaluated the percentage of Treg and memory T cells.MethodsWe enrolled patients SLE patients (2019 ACR/EULAR criteria) and sex/age-matched healthy subjects (HS). Demographic, clinical, and laboratory data were collected in a standardized computerized electronically filled form. Disease activity was assessed by SLEDAI-2k. Each subject underwent peripheral blood sample collection. By using flow-cytometry we evaluated the expression of FOXP3, CD137, PD1-1 and CTLA4, CD45, CD25, CCR7 to determine the percentage of Treg and memory T cells.ResultsThe present analysis included 21 SLE patients [M/F 1/20 median age 48 years (IQR 17), median disease duration 144 months (IQR 204)]. The Treg percentage was significantly lower in SLE compared to HS [median 4.2 (IQR 0.32) versus 2.5 (IQR 2.44); p=0.001, Figure 1A]. Moving on effector Treg (eTreg), SLE patients with high disease activity (SLEDAI > 4) showed a significantly higher prevalence for these cells compared to patients with SLEDAI ≤ 4 [1.16 (IQR 0.51) versus 0.53 (IQR 0.8), p=0.014, Figure 1B]. Moreover, inverse correlation was found between eTreg percentage and SLEDAI-2k [p=0.029, r=-0.47 (CI 0.75 – 0.04) Figure 1C]. The evaluation of CD137 expression was significantly higher in SLE patients compared to HS on CD3+ cells [median 5.32 (IQR 6.11) versus 3.3 (IQR 1.7), p=0.001, Figure 1F]. On CD4+ cells, CD137 expression positively correlated with disease activity [p=0.0082, r=0.58 (CI 0.15-0.82)]. Finally, when analysing memory T cells subpopulations, inverse correlation has been found between effector memory T cells (TEM, CD45RA-CCR7-) and SLEDAI-2k when considering CD3+ [p=0.029, r=-0.56 (CI 0.81 – 0.12)] and CD4+ cells [p=0.016, R=-0.54 (CI -0.80 - -0.1)]. Of note, CD137 expression on T central memory cells (TCM, CD45RA-CCR7+) positively correlated with SLEDAI-2k [(p=0.019, r=0.52 (CI 0.09 – 0.79)].Figure 1.A) Comparison of the percentage of Treg in HS and SLE patients. B) Comparison of the percentage of eTreg in SLE patients with high disease activity and low disease activity C) Correlation between % eTreg and SLEDAI-2k. D) Comparison of the percentage of CD3+CD137+ cells in HS and SLE patients. E) Comparison of % of CD4+CD137+ cells in SLE patients with high disease activity and low disease activity F) Correlation between % of CD4+CD137+ cells and SLEDAI-2k.ConclusionOur results suggest a possible role of CD137-CD137L axis in SLE pathogenesis. The stimulatory role of this molecule is indicated by the positive correlation between SLEDAI-2k values and surface expression of CD137. Moreover, inverse correlation between SLEDAI-2k and eTreg percentage suggests a possible Treg dysregulation in SLE.Table 1.SLE cohort featuresClinical and Laboratory FeaturesMucocutaneous80.9%Articular76.1%Serositis19.0%Kidney23.8%Haematological48.2%CNS/PNS9.5%Thrombotic events4.7%anti-dsDNA68.4%anti-SSA/anti-SSB47.6%anti-RNP19.0%anti-Sm33.0%Antiphospholipid antibodies14.2%Low C3/C457.1%Previous TherapyGlucocorticoid90.5%Hydroxychloroquine95.2%Methotrexate23.8%Mofetil Mycophenolate33.3%Ciclosporin28.5%Cyclophosphamide9.5%Azathioprine33.3%Rituximab14.3%Antiplatelet23.8%Anticoagulant therapy4.7%Disclosure of InterestsNone declared
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Aggarwal, Anu, Courtney L. Jennings, Emily Manning, and Scott J. Cameron. "Platelets at the Vessel Wall in Non-Thrombotic Disease." Circulation Research 132, no. 6 (March 17, 2023): 775–90. http://dx.doi.org/10.1161/circresaha.122.321566.
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Platelets are small, anucleate entities that bud from megakaryocytes in the bone marrow. Among circulating cells, platelets are the most abundant cell, traditionally involved in regulating the balance between thrombosis (the terminal event of platelet activation) and hemostasis (a protective response to tissue injury). Although platelets lack the precise cellular control offered by nucleate cells, they are in fact very dynamic cells, enriched in preformed RNA that allows them the capability of de novo protein synthesis which alters the platelet phenotype and responses in physiological and pathological events. Antiplatelet medications have significantly reduced the morbidity and mortality for patients afflicted with thrombotic diseases, including stroke and myocardial infarction. However, it has become apparent in the last few years that platelets play a critical role beyond thrombosis and hemostasis. For example, platelet-derived proteins by constitutive and regulated exocytosis can be found in the plasma and may educate distant tissue including blood vessels. First, platelets are enriched in inflammatory and anti-inflammatory molecules that may regulate vascular remodeling. Second, platelet-derived microparticles released into the circulation can be acquired by vascular endothelial cells through the process of endocytosis. Third, platelets are highly enriched in mitochondria that may contribute to the local reactive oxygen species pool and remodel phospholipids in the plasma membrane of blood vessels. Lastly, platelets are enriched in proteins and phosphoproteins which can be secreted independent of stimulation by surface receptor agonists in conditions of disturbed blood flow. This so-called biomechanical platelet activation occurs in regions of pathologically narrowed (atherosclerotic) or dilated (aneurysmal) vessels. Emerging evidence suggests platelets may regulate the process of angiogenesis and blood flow to tumors as well as education of distant organs for the purposes of allograft health following transplantation. This review will illustrate the potential of platelets to remodel blood vessels in various diseases with a focus on the aforementioned mechanisms.
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Welles, E. G., C. Bourne, J. W. Tyler, and M. K. Boudreaux. "Detection of Activated Feline Platelets in Platelet-rich Plasma by Use of Fluorescein-labeled Antibodies and Flow Cytometry." Veterinary Pathology 31, no. 5 (September 1994): 553–60. http://dx.doi.org/10.1177/030098589403100507.
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Platelets contribute to prethrombotic or thrombotic states; however, accepted evaluation methods (i.e., in vitro testing by use of an aggregometer) of platelet function in cats can be difficult because of the large volume of blood required from which platelets are isolated and the potential for platelet activation due to difficult venipunctures in sometimes uncooperative or excited animals. The activation problem also contributes to errors in platelet counts. Platelets from four domestic short haired cats (two males, two females, 2–3 years old) minimally restrained without sedation or anesthesia were evaluated. Blood (5 ml) was collected by jugular venipuncture directly into syringes containing 3.8% trisodium citrate (nine parts blood to one part anticoagulant) plus prostaglandin E1 (3 μM; 0.25, 0.5, 1, or 2 μl/500 μl citrate) or 3.8% trisodium citrate alone. Prostaglandin E1, which is a stable metabolite of arachidonic acid with platelet inhibitory properties similar to those of prostaglandin I2 was added to the anticoagulant to prevent activation of platelets during the collection process. Feline platelets exposed to prostaglandin E1 became immediately and persistently nonreactive to agonists, which negated their use in functional studies (aggregation, 14C-serotonin release, binding of fluorescein-conjugated antifibrinogen) but improved platelet counting accuracy. Detection of in vivo activation of platelets in prethrombotic and thrombotic states in humans has been done by identification of activation-dependent molecules on platelet surfaces by use of specific antibody recognition and detection by flow cytometric analysis. Many activation-dependent platelet surface receptor changes are species specific; however, fibrinogen appears to be conserved across species. Results of flow cytometric analysis by forward and side angle light scatter easily identified feline platelets in 10 μl of platelet-rich plasma but were less definitive in identification of platelets in whole blood. Feline platelets in platelet-rich plasma activated by ADP (5 or 50 μM), which allowed binding of fibrinogen to exposed fibrinogen receptors, were labeled with fluorescein-conjugated anti-human fibrinogen. Labeled platelets were identified as a separate population by flow cytometric staining pattern and fluorescence intensity from nonlabeled platelets or nonspecifically-labeled resting platelets. This technique should allow detection of activated feline platelets from extremely small quantities of blood, which could facilitate repetitive and serial platelet testing in diseases thought to be accompanied, caused, or augmented by thrombotic conditions.
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De Ceunynck, Karen E. P., Christian G. Peters, Sharjeel A. Chaudhry, Abhishek Jain, Sarah J. Higgins, Omozuanvbo Aisiku, Jennifer Fitch-Tewfik, et al. "A Chemical APC Mimetic Protects Endothelium from Thromboinflammatory Injury." Blood 128, no. 22 (December 2, 2016): 3835. http://dx.doi.org/10.1182/blood.v128.22.3835.3835.
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Abstract Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in animal models of inflammation and APC has been used clinically in sepsis and wound healing. Clinical use of APC in sepsis, however, was terminated as it was compromised by APC's anticoagulant activity, which is associated with bleeding and limits its dosing in patients. We used a small molecule approach to circumvent this problem. With support from the Molecular Libraries Program, we screened 302,457 compounds to identify small molecules that modulated PAR1-mediated platelet activation. One class of PAR1-targeted compounds, which we termed parmodulins, was found to act at the cytosolic face of PAR1, at the G-protein binding sites. When evaluated in endothelial cell cultures,parmodulin 1 (PM1) and parmodulin 2 (PM2) inhibited apoptosis induced by thrombin, TNF-α, or staurosporine, in a manner similar to APC. PAR1 knockdown using siRNA abolished these protective effects demonstrating that parmodulins elicit a cytoprotective pathway by acting through PAR1. To assess the mechanism of action of parmodulin cytoprotection, we first evaluated proximal signaling mechanisms. Parmodulins stimulated phosphorylation of PI3-kinase and Akt in endothelium. Inhibition of Gβγ blocked parmodulin-induced phosphorylation of Akt, indicating that parmodulins act at the cytosolic face of PAR1 by releasing Gβγ. Transcriptional profiling of over 30,000 genes and specific evaluation of NF-kB transcriptional activation showed that exposure to PM2 blocked TNF-α-induced transcriptional activation. In addition to interfering with inflammatory signaling, parmodulins also stimulated the upregulation of cytoprotective proteins such as stanniocalcin-1. Since our premise was that parmodulins could achieve cytoprotective effects without anticoagulant effects, we compared dose curves of APC versus PM2 in both apoptosis assays and standard clotting assays. APC prolonged the aPTT at concentrations lower than those required to achieve cytoprotection of the endothelium. The low APC concentration used in our study was similar to plasma concentrations measured in clinical studies. Hence, these data were consistent with the fact that clinical bleeding was observed at doses of APC used for sepsis. In contrast, despite inhibiting apoptosis as effectively as APC, PM2 had no effect on plasma aPTT at any concentration. Nonetheless, PM2 was able to inhibit LPS- and TNF-α-induced thrombin generation and FXa activation on endothelium owing to its cytoprotective effect. PM2 also prevented TNF-α-induced accumulation of platelets on endothelium in bioengineered microvessels. These data demonstrate that PM2 can reduce inflammation-induced endothelial pro-thrombotic phenotype even without directly inhibiting coagulation factors. To assess the endothelial protective effects of PM2 in vivo we evaluated leukocyte rolling in cremaster venules of mice. Infusion of PM2 significantly reduced surgery-induced leukocyte rolling flux compared to vehicle-treated mice. As selectins are critically involved in leukocyte rolling we monitored soluble E-selectin levels in LPS-induced inflammation. Treatment of mice with PM2 significantly reduced the LPS-induced release of soluble E-selectin. Previously, we demonstrated that PM2 blocks platelet accumulation in a mouse laser injury model of thrombus formation. We here show that infusion of PM2 also significantly reduces fibrin accumulation to 25% of control (p<0.001) at the site of laser injury. Together our data show that PM2 exerts endothelial-mediated anti-inflammatory, anti-coagulant and anti-thrombotic effects in vitro and in vivo. These results demonstrate that modulating PAR1 at the cytosolic face could represent an alternative approach to APC in the treatment of thromboinflammatory disorders like sepsis. Disclosures No relevant conflicts of interest to declare.
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Bertani, Fabio, Dalila Di Francesco, Maria Dolores Corrado, Maria Talmon, Luigia Grazia Fresu, and Francesca Boccafoschi. "Paracrine Shear-Stress-Dependent Signaling from Endothelial Cells Affects Downstream Endothelial Function and Inflammation." International Journal of Molecular Sciences 22, no. 24 (December 10, 2021): 13300. http://dx.doi.org/10.3390/ijms222413300.
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Cardiovascular diseases (CVDs), mainly ischemic heart disease (IHD) and stroke, are the leading cause of global mortality and major contributors to disability worldwide. Despite their heterogeneity, almost all CVDs share a common feature: the endothelial dysfunction. This is defined as a loss of functionality in terms of anti-inflammatory, anti-thrombotic and vasodilatory abilities of endothelial cells (ECs). Endothelial function is greatly ensured by the mechanotransduction of shear forces, namely, endothelial wall shear stress (WSS). Low WSS is associated with endothelial dysfunction, representing the primary cause of atherosclerotic plaque formation and an important factor in plaque progression and remodeling. In this work, the role of factors released by ECs subjected to different magnitudes of shear stress driving the functionality of downstream endothelium has been evaluated. By means of a microfluidic system, HUVEC monolayers have been subjected to shear stress and the conditioned media collected to be used for the subsequent static culture. The results demonstrate that conditioned media retrieved from low shear stress experimental conditions (LSS-CM) induce the downregulation of endothelial nitric oxide synthase (eNOS) expression while upregulating peripheral blood mononuclear cell (PBMC) adhesion by means of higher levels of adhesion molecules such as E-selectin and ICAM-1. Moreover, LSS-CM demonstrated a significant angiogenic ability comparable to the inflammatory control media (TNFα-CM); thus, it is likely related to tissue suffering. We can therefore suggest that ECs stimulated at low shear stress (LSS) magnitudes are possibly involved in the paracrine induction of peripheral endothelial dysfunction, opening interesting insights into the pathogenetic mechanisms of coronary microvascular dysfunction.
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Totzeck, Matthias, Raluca-Ileana Mincu, Simone Mrotzek, Dirk Schadendorf, and Tienush Rassaf. "Cardiovascular diseases in patients receiving small molecules with anti-vascular endothelial growth factor activity: A meta-analysis of approximately 29,000 cancer patients." European Journal of Preventive Cardiology 25, no. 5 (January 29, 2018): 482–94. http://dx.doi.org/10.1177/2047487318755193.
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Background Targeted therapy with tyrosine kinase inhibitors with anti-vascular endothelial growth factor activity improves survival of cancer patients. Cardiovascular complications are critical and it is unknown whether these require specific treatment strategies. We aimed to clarify the associated risk of cardiovascular adverse events in patients treated with tyrosine kinase inhibitors. Design The design of this study was a meta-analysis of randomised controlled trials. Methods We searched PubMed, Cochrane, EMBASE and Web of Science databases for randomised controlled trials published until January 2017 that assessed patients with different types of cancer treated with or without tyrosine kinase inhibitors in addition to standard chemotherapy. Results A total of 29,252 patients from 71 randomised controlled trials were included. Tyrosine kinase inhibitor treatment was associated with a higher cardiac ischaemia relative risk (relative risk = 1.69; 95% confidence interval: 1.12–2.57; p = 0.01), with the highest risks observed for sorafenib and patients with renal cancer. Risk of thrombocytopaenia (relative risk = 2.2; 95% confidence interval: 1.73–2.79; p < 0.001) was highest for regorafenib and patients with breast cancer. Left ventricular systolic dysfunction was increased after tyrosine kinase inhibitor therapy (relative risk = 2.53; 95% confidence interval:1.79 – 3.57; p < 0.001), with the highest risks reported for sunitinib and hepatocellular cancer. QT corrected interval prolongation (relative risk = 6.25; 95% confidence interval: 3.44–11.38; p < 0.001) and arterial hypertension (relative risk = 3.78; 95% confidence interval: 3.15-4.54; p < 0.001) were reported. The relative risks of arterial adverse events, cerebral ischaemia, venous adverse events and pulmonary embolism were similar across groups. Conclusion Tyrosine kinase inhibitors increase the risk of severe cardiovascular and particularly thrombotic adverse events. Specific treatment regimens when prescribing tyrosine kinase inhibitor therapies appear desirable.
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Sullenger, Bruce A. "Aptamers as Rapid Onset and Rapidly Reversible Antithrombotic Agents." Blood 134, Supplement_1 (November 13, 2019): SCI—16—SCI—16. http://dx.doi.org/10.1182/blood-2019-121056.
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Clinical evaluation of our factor IXa RNA aptamer in two thousand patients undergoing percutaneous coronary intervention has demonstrated that aptamers can rapidly and potently inhibit their target proteins in patients and that antidote molecules can rapidly and precisely control such activity in the minute time frame. These observations suggest that aptamers represent useful molecules to tightly control biochemical processes in humans in real time. To begin to explore this potential further, we have started to evaluate the utility of antidote-mediated control of aptamers for a variety of other therapeutic and diagnostic applications. We will describe our recent progress developing a rapidly controllable factor Xa anticoagulant aptamer to effectively yet reversibly control blood coagulation during cardiopulmonary bypass surgery and a rapidly reversible VWF aptamer for improving the treatment of thrombotic stroke. We have observed that aptamers, which target exosites on coagulation factors, can complement active site inhibitors to yield potent anticoagulant regiments (Gunaratne et al., 2018) that can support circulation of blood through extracorporeal oxygenator circuits. Moreover we have observed that an aptamer targeting the A1 domain of VWF can serve as a rapid onset and rapidly reversible antithrombotic agent. This aptamer prevents platelet recruitment and can induce recanalization of occluded arteries while a matched antidote oligonucleotide can rapidly reverse such anti-platelet activity and thereby limit bleeding following vascular injury (Nimjee et al., 2019). Collectively these clinical and preclinical studies lead us to believe that rapidly controllable aptamers represent valuable therapeutic agents that will provide physicians the ability to monitor and precisely control blood coagulation, as well as other biological pathways, in real time in response to individual patients' needs. GunaratneR, KumarS, FrederiksenJW, et al. Multimodal, antidote-controllable anticoagulation for cardiopulmonary bypass using aptamer-drug pairs. Nature Biotechnology 2018;36:606-613. NimjeeSM, Dornbos, IIID, Pitoc GA, et al. Preclinical Development of a VWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels. Molecular Therapy. 2019;27:1228-1241. Disclosures Sullenger: Basking Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties.
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Pankratenko, T. E., O. V. Moskalets, and T. Yu Abaseeva. "SOLUBLE ADHESION MOLECULES IN CHILDREN WITH HEMOLYTIC UREMIC SYNDROME." Medical Immunology (Russia) 20, no. 6 (December 15, 2018): 871–76. http://dx.doi.org/10.15789/1563-0625-2018-6-871-876.
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Atypical hemolytic uremic syndrom (aHUS) is a rare severe life-threatening form of thrombotic microangiopathy. aHUS is thought to be primarily mediated by dysfunctional complement regulation, due to mutations or genetic rearrangement of the complement components, or regulatory factors, as well as autoantibody production to the complement factors. These alterations promote uncontrolled complement activation by the alternative pathway on the surface of endothelial cells, followed by endothelial dysfunction, microthrombosis and organ damage, especially, renal pathology. Many studies showed that the biomarkers of endothelial activation/dysfunction including intercellular adhesion molecule type 1 (ICAM-1) and vascular cell adhesion molecule type 1 (VCAM-1) were associated with severe disease and could predict complications and clinical outcomes in different disorders. Increase of these biomarkers was observed in aHUS as well. Until recently, aHUS resulted in unfavorable outcomes, with high death rates in acute phase, and up to 2/3 patients progressed to the end-stage renal failure. Understanding the role of complement dysregulation in aHUS pathogenesis has led to major changes in therapeutic approaches. Eculizumab (a humanized anti-C5 monoclonal antibody) inhibits the terminal complement pathway. This drug has revolutionized treatment and improved prognosis in aHUS. Those patients who received eculizumab have shown sharp decreae in the C3 levels. However, the questions concerning duration of targeted therapy inremission still remains unclear.The aim of the present study was to evaluate serum С3, sICAM-1, sVCAM-1 levels in the children with aHUS remission supported by eculizumab maintenance treatment, or without it. Serum C3, sICAM-1 and sVCAM-1 levels were determined in 25 children with aHUS (14 treated with eculizumab and 15, without eculizumab). A control group included 17 children with a history of typical HUS. Serum levels of C3, sICAM-1 and sVCAM-1 were within normal age ranges in all the groups. The children treated with eculizumab showed decreased C3 levels (99±20 mg/l vs 112±15 mg/l, and 123±40 mg/l, respectively), and increased sICAM-1 levels (483±103 ng/ ml vs 343±50 ng/ml and 401±91 ng/ml, respectively) compared to other groups (p < 0.05). No differences in sVCAM-1 levels were revealed in the groups. Hence, no signs of subclinical complement activation or endothelial disfunction were revealed in the group free of eculizumab therapy. Normal C3, sICAM-1 and sVCAM-1 levels in blood indicate that normal endothelial state could be restored in aHUS, and this condition is maintained after discontinuation of the targeted therapy. Our results suggest that C3, sICAM-1 and sVCAM-1 monitoring may be useful for further management of these patients and for prediction of relapses.
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Patiño-Trives, A. M., C. Perez-Sanchez, A. Ibañez-Costa, P. S. Laura, M. Luque-Tévar, I. Arias de la Rosa, M. C. Ábalos-Aguilera, et al. "OP0038 SPLICEOSOME ALTERATIONS IN LEUCOCYTES FROM APS, SLE AND SLE+APS PATIENTS ARE CLOSELY RELATED TO THEIR MAIN CLINICAL FEATURES." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 20.2–20. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2485.
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Background:To date, although multiple molecular approaches have illustrated the various aspects of Primary Antiphospholipid Syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome plus lupus (APS plus SLE), no study has so far fully characterized the potential role of posttranscriptional regulatory mechanisms such as the alternative splicing.Objectives:To identify shared and differential changes in the splicing machinery of immune cells from APS, SLE and APS plus SLE patients, and their involvement in the activity and clinical profile of these autoimmune disorders.Methods:Monocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS plus SLE) and 50 healthy donors (HD) were purified by immunomagnetic selection. Then, selected elements of the splicing machinery were evaluated using a microfluidic qPCR array (Fluidigm). In parallel, extensive clinical/serological evaluation was performed, comprising disease activity, thrombosis and renal involvement, along with autoantibodies, acute phase reactants, complement and inflammatory molecules. Molecular clustering analyses and correlation/association studies were developed.Results:Patients with primary APS, SLE and APS plus SLE displayed significant and specific alterations in the splicing machinery components in comparison with HD, that were further specific for each leukocyte subset. Besides, these alterations were associated with distinctive clinical features.Hence, in APS, clustering analysis allowed to identify two sets of patients representing different molecular profile groups with respect to the expression levels of splicing machinery components. Principal component analyses confirmed a clear separation between patients. Clinically, cluster 1 characterized patients with higher thrombotic episodes and recurrences than cluster 2 and displayed a higher adjusted global APS score (aGAPSS). Accordingly, these patients showed higher levels of inflammatory mediators than cluster 2.Similarly, in patients with APS plus SLE, clustering analysis allowed to identify two sets of patients showing differential expression of splicing machinery components. Clinical and laboratory profiles showed that cluster 2 characterized patients that had suffered more thrombotic recurrences, most of them displaying an aGAPSS over 12 points and expressing higher levels of inflammatory mediators than cluster 1. The incidence of lupus nephropathy was similarly represented in both clusters.Lastly, in SLE patients, molecular clustering analysis identified two sets of patients showing distinctive clinical features. One cluster characterized most of the patients positive for anti-dsDNA antibodies, further suffering lupus nephropathy, and a high proportion of them also presenting atheroma plaques and high levels of inflammatory mediators.Correlation studies further demonstrated that several deranged splicing machinery components in immune cells (i.e. SF3B1tv1, PTBP1, PRP8 and RBM17) were linked to the autoimmune profile of the three autoimmune diseases, albeit in a specific way on each disorder. Accordingly, in vitro treatment of HD lymphocytes with aPL-IgG or anti-dsDNA-IgG changed the expression of spliceosome components also found altered in vivo in the three autoimmune diseases. Finally, the induced over/downregulated expression of selected spliceosome components in leukocytes modulated the expression of inflammatory cytokines, changed the procoagulant/adhesion activities of monocytes and regulated NETosis in neutrophils.Conclusion:1) The splicing machinery, profoundly altered in leukocytes from APS, APS plus SLE and SLE patients, is closely related to the activity of these diseases, their autoimmune and inflammatory profiles. 2) The analysis of the splicing machinery allows the segregation of APS, APS plus SLE and SLE, with specific components explaining the CV risk and renal involvement in these highly related autoimmune disorders.Acknowledgements:Funded by ISCIII, PI18/00837 and RIER RD16/0012/0015 co-funded with FEDERDisclosure of Interests:None declared
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Rehani, Teena, Adria Jonas, and Arne Slungaard. "The Major Phagocyte Peroxidase-Derived Oxidant HOSCN Stimulates Gene-Specific p38 MAPK- Dependent, NF-κb-Mediated Activation of Tissue Factor, VCAM-1 and ICAM-1 Expression In Endothelium." Blood 116, no. 21 (November 19, 2010): 1481. http://dx.doi.org/10.1182/blood.v116.21.1481.1481.
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Abstract Abstract 1481 Thiocyanate (SCN-) is, unexpectedly, the principal physiologic substrate for eosinophil peroxidase (EPO) and a major (i.e., accounting for 50% of H2O2 consumed) substrate for myeloperoxidase (MPO). The product of these reactions is HOSCN, a weak, exclusively sulfhydryl-reactive oxidant that we have previously shown to be a uniquely potent (up to 100-fold) oxidant transcriptional inducer of human umbilical vein endothelial cell (HUVEC) tissue factor (TF), ICAM-1 and VCAM-1 expression via a mechanism dependent upon NF-κB p65/p50 activation. We hypothesized that oxidative activation of p38 MAPK is a necessary and proximal step in the activation of NF-κB p65/p50 at the TF, VCAM-1 and ICAM-1 promoters. To test this we utilized the p38 MAPK-specific pyridinyl imidazole inhibitor SB 203580 (SB). In HUVEC monolayers exposed 4h to 150 μM HOSCN in M199 medium containing 10% FCS, SB strongly inhibited (>90%, ED50 300 nM) TF activity, decreased VCAM-1 expression assessed by western blot by > 50% but had no discernible effect upon ICAM-1 expression. qRT-PCR analysis confirmed that the effects of SB on these three molecules was transcriptionally mediated. Immunoprecipitation of HOSCN-treated HUVEC whole cell extracts with a polyclonal anti-phospho-p38 MAPK reagent showed rapid (within 1 min) phosphorylation of p38 MAPK that lasted >30 min with consequent kinase activity documented by detection of the downstream target product phospho HSP27. Confocal immunofluorescence confirmed the rapid and durable induction of phospho p38 MAPK from undetectable to abundant, primarily cytoplasmic, but also some nuclear localization. We further hypothesized that the disparate effects of SB on these three molecules reflects differences in the sequences of their NF-κB-binding sites that affect their ability to bind the p65 NF-kB subunit. Chromatin immunoprecipitation (ChIP) analysis of HUVEC treated for 3h with HOSCN using an anti-p65 antibody revealed that SB inhibited by 75% the 10-fold increase in binding of p65 to the authentic endogenous VCAM-1 and NF-κB binding loci but had no discernible effect on p65 binding to the ICAM-1 locus. An identical pattern was seen in HUVEC exposed 30 min to the prototypical inflammatory cytokine TNF. To test the physiologic significance of these findings we assessed the effect of SB upon PMN and eosinophil binding to HOSCN-exposed HUVEC monolayers treated as described above. SB pretreatment of HUVEC blocked by > 80% the 3–8-fold increase in binding of PMN and eosinophils that occurs in HOSCN-exposed monolayers. Blocking antibodies to ICAM-1 and VCAM-1 demonstrate that both of the adhesion molecules contribute to HOSCN-induced PMN and eosinophil adhesion. We conclude that HOSCN generated by PMN and eosinophils attached or subjacent to vascular endothelium has the capacity to rapidly activate endothelial p38 MAPK-dependent activation of p65 binding to the TF and VCAM-1, but not ICAM-1, NF-κB binding loci; and these differences manifest in physiologically relevant transcriptional regulation of protein expression. Therefore, p38 MAPK inhibitors may have anti-thrombotic and anti-inflammatory potential, the latter particularly in states, such as allergic inflammation, that are most dependent on VCAM-1. Disclosures: No relevant conflicts of interest to declare.
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Mcewan, P. A., Robert K. Andrews, and Jonas Emsley. "Glycoprotein Ibalpha Inhibitor Complex Crystal Structure at High Resolution." Blood 114, no. 22 (November 20, 2009): 774. http://dx.doi.org/10.1182/blood.v114.22.774.774.
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Abstract Abstract 774 Introduction: The platelet Glycoprotein Ib/V/IX (GpIb/V/IX) complex is considered a major target for anticoagulant therapy. The primary function of the receptor is to mediate platelet adhesion to von Willebrand factor (VWF) bound to damaged sub-endothelium. This represents the first critical step for platelet adhesion under conditions of high fluid shear stress. GpIb/V/IX is implicated in a number of thrombotic pathological processes such as stroke or myocardial infarction and the bleeding disorders Bernard-Soulier syndrome, platelet type von Willebrand disease (Pt-VWD) and thrombotic thrombocytopenic purpura. We have successfully determined the structure of the GpIbalpha N-terminal domain in complex with a potent (sub nM) 11meric peptide inhibitor (OS1) of the interaction with VWF. Methods: We have determined the crystal structure to 1.8Å resolution using molecular replacement. Results. The peptide sequence CTERMALHNLC was readily identifiable bound to GpIbalpha between the extended regulatory (R) loop and the concave surface of the leucine rich repeats. The peptide adopts one and a half turns of an alpha-helix and contacts three subsites (S1, S2 and S3). S1 and S2 reside within the leucine rich repeats and S3 has a unique feature as this subsite involves contact with the regulatory R-loop stabilizing it in a well defined conformation with helical character. This loop alters conformation between an extended beta-hairpin in the VWF-A1 bound structure and a more compact largely disordered structure in the unliganded structure. In this regard, the Pt-VWD mutations of GpIbalpha, G233V and M239V, which reside in the R-loop act by inducing a beta-conformation and thus result in a high affinity form of the receptor. Conclusions: These studies provide a strategy for targeting the GpIbalpha-VWF interaction using small molecules or alpha-helical peptides exploiting the GpIbalpha subsites described here and acting allosterically to stabilise a low affinity conformation of the receptor with an alpha helical R-loop. Ligand mimetic peptide complex crystal structures for the platelet receptors integrin aIIbb3 with RGD, and alpha2beta1 with a collagen peptide have been described and the former are currently in therapeutic use for treatment of thromboemboletic disorders. Targeting the GpIbalpha-VWF interaction may provide anti-thrombotic drugs which affect platelet adhesion under high shear stress without compromising normal processes of platelet adhesion and aggregation which may be required for normal hemostasis to function. Targeting protein-protein interactions is considered one of the great contemporary challenges in drug discovery. The understanding of how the S1S2S3 subsites provide very effective inhibition of a large protein-protein interaction has wide applicability. LRR proteins are an extended family mediating protein-protein interactions involved in a variety of disease processes such as sepsis, asthma, immunodeficiencies, atherosclerosis, alzheimers (leucine rich repeat kinase) and leukaemia (leucine rich repeat phosphatase). The structural fit of the helical curvature of the peptide with the arc of the leucine rich repeats may provide a basis for further development of alpha-helical peptide mimetics targeting other members of the LRR family which utilize the concave face. Disclosures: No relevant conflicts of interest to declare.
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Sakai, Kazuya, Masataka Kuwana, Hidenori Tanaka, Kazuyoshi Hosomichi, Atsushi Hasegawa, Hiroki Uyama, Kenji Nishio, et al. "HLA loci predisposing to immune TTP in Japanese: potential role of the shared ADAMTS13 peptide bound to different HLA-DR." Blood 135, no. 26 (June 25, 2020): 2413–19. http://dx.doi.org/10.1182/blood.2020005395.
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Abstract Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model.
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Ahishev, D. M. "SYNTHESIS OF CALIX[4]ARENES WITH FIXED CONFORMATION AS POTENTIAL INHIBITORS OF FIBRIN POLYMERIZATION." Biotechnologia Acta 16, no. 2 (April 28, 2023): 7–10. http://dx.doi.org/10.15407/biotech16.02.007.
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Aim. The purpose of the present study was to develop a method for the fixation of ‘fixed’ conformation for estimation of the impact of calix[4]arene structure on the efficacy of its anticoagulant activity. This was achieved by substitution of the lower rim of C-145 analogue. Methods.Calix[4]arene C-145F (compound 6) was obtained in 4 steps starting with Duff reaction. Calix[4]arene methylene-bis-phosphonic ester 3 was prepared via addition of diisopropylphosphite in presence of metallic sodium to the parent calix[4]arene aldehyde 2. Further steps included Mitsunobu reaction, that afforded dipropoxycalix[4]arene 5 with rather good yields (80%), following the hydrolysis step that resulted in compound 6 in almost quantitative yield. Modeling of 3D-structure of calix[4]arene C-145 and its analogue C-145F was performed in Maestro, Schrodinger software. Results. Using a 2D NMR-NOESY spectroscopy, we can observe a distinct cross-peak between an aromatic singlet with a chemical shift on 7.72 ppm and protons of isopropyl group with a chemical shift on 1.62 ppm, which are moved in the strong field. Conclusions. The easy method of the fixation of conus conformation of calix[4]arene cup will be useful for synthesis of novel functionally active compounds. We believe that further development and study of different calix[4]arenes will help scientists to obtain bioactive molecules that could be prospective anti-thrombotic drugs.