scholarly journals Action of Extracellular Proteases of Aspergillus flavus and Aspergillus ochraceus Micromycetes on Plasma Hemostasis Proteins

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 782
Author(s):  
Alexander A. Osmolovskiy ◽  
Laura Schmidt ◽  
Anastasia V. Orekhova ◽  
Sergey K. Komarevtsev ◽  
Valeriana G. Kreyer ◽  
...  

In this study, we investigated the properties of proteolytic enzymes of two species of Aspergillus, Aspergillus flavus 1 (with a high degree of pathogenicity) and Aspergillus ochraceus L-1 (a conditional pathogen), and their effects on various components of the hemostasis system (in vitro) in the case of their penetration into the bloodstream. We showed that micromycete proteases were highly active in cleaving both globular (albuminolysis) and fibrillar (fibrin) proteins, and, to varying degrees, they could coagulate the plasma of humans and animals (due to proteolysis of factors of the blood coagulation cascade) but were not able to coagulate fibrinogen. The proteases of both Aspergillus fully hydrolyzed thrombi in 120–180 min. Micromycetes did not show hemolytic activity but were able to break down hemoglobin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3934-3934
Author(s):  
Christian J. Kastrup ◽  
Matthew K. Runyon ◽  
Feng Shen ◽  
Rustem F. Ismagilov

Abstract To investigate the biophysical mechanisms that regulate the spatial dynamics of blood coagulation, we have developed a set of microfluidic tools that allow analysis and perturbation of blood coagulation on the micrometer scale with precise control of fluid flow, geometry, and surface chemistry. Physiological coagulation occurs in a localized manner; specifically, coagulation is believed to occur exclusively at regions of substantial vascular damage and does not spread throughout the entire vascular system. In vitro analysis and characterization of these spatial dynamics requires the ability to reproduce and perturb this system, an ability that is not provided by the mixed reactor systems commonly used for in vitro studies of blood coagulation. We developed microfluidic devices with micrometer-scale channels and methods to coat these channels with various phospholipids, including components of the blood coagulation network such as thrombomodulin and tissue factor, to reproduce in vitro the geometry and surface chemistry of blood vessels in vitro. In a microfluidic device with channels coated with phospholipids and thrombomodulin, we demonstrated that clots propagate in a wave-like fashion with a constant velocity in the absence of flow. We also showed that propagation of coagulation from an occluded channel to a channel with flowing blood plasma can be regulated by the geometry of the junction and the shear rate in the channel with flowing plasma. We also developed microfluidic tools to probe the spatial dynamics of initiation of clotting by patterning surfaces with tissue factor reconstituted into phospholipids bilayers. When human plasma or whole blood was exposed to these surfaces in a microfluidic device, clotting occurred only on patches of tissue factor larger than a threshold size. This threshold patch size is controlled by the rate of activation of clotting factors at the patch and the rate of transport of activated factors off the patch. These results suggest a mechanism for how tissue factor can circulate in blood without causing clotting, and how small regions of vascular damage can exist without causing clotting. These results also suggest new biophysical mechanisms that may control interactions between the coagulation cascade and bacterial surfaces.


Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2133-2143 ◽  
Author(s):  
Roxane Darbousset ◽  
Grace M. Thomas ◽  
Soraya Mezouar ◽  
Corinne Frère ◽  
Rénaté Bonier ◽  
...  

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.


2020 ◽  
Vol 19 (1) ◽  
pp. 71-80
Author(s):  
Yu. A. Malinovskaya ◽  
E. I. Kovalenko ◽  
T. S. Kovshova ◽  
N. S. Osipova ◽  
O. O. Maksimenko ◽  
...  

Introduction. The use of polymeric biodegradable nanoparticles (NP) as drug delivery systems is a promising approach to overcome histohematomatic barriers. Thus, poloxamer 188-coated poly (lactide-co-glycolide) (PLGA) NP are able to overcome blood-brain barrier and to deliver therapeutic agents, in particular doxorubicin, into intracranial tumour upon intravenous administration. It is important to evaluate NP interaction with blood components in preclinical studies.The objective of the study was to investigate cytotoxicity and hemocompatibility of doxorubicin-loaded PLGA NP (Dox-PLGA NP), to essess NP uptake by glioblastoma cells.Materials and methods. The influence of NP on coagulation cascade was evaluated by prothrombin time measuring before and after plasma incubation with NP. To assess NP thrombogenicity the platelet activation level was determined by flow cytometry. The NP hemolytic activity (released hemoglobin concentration) was measured spectrophotometrically. NP cytotoxicity was determined by MTS assay. NP uptake by human glioblastoma cells was evaluated by flow cytometry.Results. Dox-PLGA NP did not influence blood coagulation time and thrombocyte activity at concentrations up to 100 mcg/mL: PT values were 12–15 s for all tested samples, and P-selectin expression level did not exceed 15 %. All samples were not hemolytic after 3 h of incubation. Cytotoxicity of doxorubicin released from PLGA NP on glioma U87MG cells was comparable to that of free doxorubicin. As shown by flow cytometry Dox-PLGA NP were efficiently internalized into the cells.Conclusion. The study of hemocompatibility confirmed the safety of Dox-PLGA NP: NP did not influence blood coagulation system and did not induce hemolysis. NP were efficiently internalized into the human glioblastoma cells and produced considerable antitumor effect in vitro.


2006 ◽  
Vol 30 (4) ◽  
pp. 731-738 ◽  
Author(s):  
Marcelo Cláudio Pereira ◽  
Georgia Rocha Vilela ◽  
Lívia Martinez Abreu Soares Costa ◽  
Reginaldo Ferreira da Silva ◽  
Anderson Felicori Fernandes ◽  
...  

Objetivou-se com este trabalho avaliar os efeitos inibitórios, "in vitro", de óleos essenciais dos condimentos, alecrim (Rosmarinus officinalis L.), cebola (Allium cepa L.), manjericão (Ocimum basilicum L.), menta (Mentha piperita L.) e orégano (Origanum vulgare L.), sobre o desenvolvimento de fungos. Os óleos foram extraídos pela técnica de arraste a vapor e testados nas concentrações de 500; 1000; 1500 e 2000 mg/mL-1. Como culturas de teste foram utilizados os fungos Fusarium sp.; Aspergillus ochraceus Wilhelm.; Aspergillus flavus Link e Aspergillus niger van Tieghem obtidos da micoteca do EcoCentro/EPAMIG em Lavras, MG. O óleo essencial do orégano inibiu o desenvolvimento dos fungos testados em todas as concentrações exceto o fungo A. niger que teve o seu desenvolvimento micelial inibido a partir da concentração de 1000 mg/mL-1,. Os óleos de alecrim, menta, cebola e manjericão tiveram um efeito pronunciado a partir da concentração de 1500 mg/mL-1.


2020 ◽  
Vol 10 (9) ◽  
pp. 397
Author(s):  
Kimihiko Takada ◽  
Mayuko Takano ◽  
Aiko Kunii ◽  
Kei Harayama ◽  
Akira Ito ◽  
...  

Background: Nobiletin is contained in Shiikuwasa fruit, a popular citrus fruit from Okinawa Prefecture in Japan. Nobiletin reportedly acts as a strong antioxidant, an anti-inflammatory agent, and an anti-cancer agent, and it suppresses the expression of TF which triggers blood coagulation. However, in vivo verification of in vitro reports is necessary. This study used a rat model of LPS-induced microthrombosis based on the in vivo studies as previously reported. Sustained intravenous injection of LPS changed all blood coagulation indicators in the direction of thrombus formation. The aim of this study was to determine if intake of nobiletin could suppress DIC-like symptoms.Methods: Experimental SD rats were fully anesthetized and fixed to an operating table. Either LPS alone or nobiletin (50 mg/kg) plus LPS was given to rats to investigate the repressive effects of nobiletin on the expression of blood coagulation factors.Results: After 4 h of LPS infusion (12.5 mg/kg/h, i.v.), PLT counts and Fbg levels in rat plasma decreased by 80% and 74%, respectively. PT and APTT were extended by 180% and 256%, respectively. TF activity and PAI-1 antigen levels were remarkably increased (54- and 86-fold, respectively vs. control). Pretreatment on nobiletin (50 mg/kg, p.o.) reduced or suppressed fluctuations in blood coagulation indices caused by LPS. TF activity was repressed almost completely by nobiletin pretreatment. After 4 h, PAI-1 antigen levels in nobiletin-treated animals were repressed 82.6% compared to LPS-treated rats. Nobiletin repressed LPS-induced changes in TF and PAI-1 more effectively than other parameters. Further, nobiletin repressed fibrin thrombi  formation in the renal glomeruli induced by LPS treatment.Conclusions: Nobiletin was found to reduce LPS-induced DIC-like symptoms in rats. In the fluctuations of blood indices related to the coagulation cascade, nobiletin suppressed the LPS-induced expression of PAI-1 and TF more effectively than other indices. The binding sites of transcription factors that are activated by LPS-induced signals reside in the promoter areas of TF and PAI-1 gene sequences. Thus, the suppression of TF and PAI-1 expression by nobiletin appears similar to mechanisms previously evaluated during in vitro experiments. Importantly, nobiletin repressed fibrin deposition in the renal glomeruli induced by LPS treatment and improved overall health. Nobiletin may function as an anti-thrombogenic agent when ingested daily. Keywords: nobiletin; LPS; DIC model; blood coagulation; anti-thrombogenic


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1095-1095
Author(s):  
Mette Sondrup Andersen ◽  
Aage Kristian Olsen Alstrup ◽  
Julie Kirstine Andersen ◽  
Søren Risom Kristensen ◽  
Kåre Lehmann Nielsen

Abstract Abstract 1095 Heparin was discovered in 1916 and put into clinical trials in 1935. Despite advent of several anticoagulants during the last 75 years, heparin still remains the most widely used anticoagulant. None the less, several drawbacks of heparin exist i) it is difficult to determine the correct dosage, ii) heparins binds many different targets in humans, iii) side effects such as Heparin Induced Thrombocytopenia (HIT) is known (Hirsh J et al, Chest 2001). Consequently, intense emphasis have been put on finding new and improved inhibitory agents towards specific factors in the blood coagulation. Especially factor Xa (fXa) is considered an interesting target for inhibitors due to its central place in the coagulation cascade (Gross PL and Weitz, JI, Clinical Pharmacology and therapeutics 2009). Here we present a novel direct specific inhibitor of fXa, PifXa (protein inhibitor of coagulation factor Xa), which has been isolated from potato tubers. The inhibitor of the legume Kunitz type protein family was able to inhibit the activity of fXa using a mixed mode of inhibition with an apparent Ki of 2.5 nM, as determined using a low molecular weight substrate. Noteworthy, no inhibition of thrombin could be detected. Furthermore, the effect of the inhibitor could be detected using the activated partial thromboplastin time (aPTT) assay, which suggests that PifXa is not only capable of inhibiting free fXa but also complex/clot-bound fXa. Other known specific fXa inhibitors such as the pentasaccharide fondaparinux (Arixtra, GlaxoSmithKline) and low molecular weight heparin (LMWH) give rise to little or no effect in the aPTT assay. This observation has been attributed to the fact that these inhibitors only inhibit free fXa (Hirsh J et al, Chest 2001). PifXa was capable of significantly prolonging the tail bleeding time, but did not increase the bleeding amount significantly compared to the control in in vivo experiments conducted in rats. Hence, PifXa is highly specific towards the blood coagulation cascade, but do not interfere with the platelet plug formation in contrast to heparin, that can interfere with the thrombin induced platelet activation (Day, J et al, J of Cardiothoracic and Vascular Anesthesia 2004). Indeed, inhibition of activation of the platelets by PifXa could not be detected in in vitro experiments using platelet aggreometry. Furthermore, PifXa given in combination with the anti-platelet drug acetylsalicylic acid increased both the bleeding time and amount in the in vivo rat experiment significantly, demonstrating an additive effect of PifXa and the antiplatelet drug. The combined effect exceeded that of both heparin and fondaparinux. In contrast to other specific factor Xa inhibitors, the effect of PifXa, being a protein, can be fully reversed by addition of a specific polyclonal antibody. That this is in fact possible was demonstrated in vitro. The specificity of the inhibitor combined with the possibility to reverse the effect makes PifXa an interesting candidate drug during cardio pulmonary bypass where the general inconvenient requirement for IV administration of protein drugs is tolerable, a large dose of anticoagulants in a limited period of time is necessary, and thus administration of an antidote to reverse the effect at the end of the procedure is desired. Disclosures: Andersen: Aalborg University: Patents & Royalties. Nielsen:Aalborg University: Patents & Royalties.


2021 ◽  
Author(s):  
A. Slimani ◽  
B.N. Nahal ◽  
D. Seddiki ◽  
M.S. Belghit

Mold growth is among the major causes of health impairment of cereals, in particular durum wheat (Triticum durum) for the synthesis of mycotoxins such as aflatoxins B1 and ochratoxin A (OTA), originally from poisoning in the consumer. In this context, the objectives of this work is the search and characterization of fungal strains Producers mycotoxins such as Aspergillus, Penicillium in semolina and their derivatives (traditional and industrial couscous) and detect and quantify total aflatoxins, aflatoxins B1 and ochratoxin mycotoxicologique to assess the risk associated with the consumption of these foods. In this regard, our work focuses on mycological and mycotoxicologique study of semolina and couscous deemed most commercialized in the town of Bechar-Algeria after a socio-economic survey. The mycological study testifies the high degree of pollution of our samples by Aspergillus, Penicillium. The expertise of genera reveals the high degree of invasion of our samples by Aspergillus, Penicillium. The examination of fungal procession characterizing our samples shows a very high index of distribution, or of fidelity of Penicillium 43.75% of our sample and 28.38% Aspergillus. The presence of these species is evidence that our samples have been abused, but especially poorly stored; should be noted the involvement of the genera Alternaria 7.10%, Fusarium 13.70%. Thin-layer chromatographic (TLC) analysis revealed that 50% of Aspergillus flavus-parasiticus strains were aflatoxin G-producing and aflatoxin B-free in our samples. Of the Aspergillus ochraceus strains 50% were OTA producers. The presumption of toxicity of the various samples appeared positive on TLC. The test of Elisa has confirmed the presence of the OTA in our samples, the analysis of its results shows that the majority of the rates of OTA taken on our analyzed samples follow the European standard, these rates are between 1.01 and 1.9, except for one sample (couscous) which has shown a rate much higher than the standard recommended by the regulation (> 100 ppb), the samples of semolina had a rate of OTA lower than the beginning of the detection (1 ppb). The results of the presence of AFB spread out between 4.93 ppb and > 40 ppb. The antifungal activity of the resin of Boswellia carterii was tested on the following strains: Aspergillus niger, Aspergillus flavus, Penicillium expansum. And kneaded according to the technique of diagonal growth on intermediate solid medium (PDA). The results showed that the yield of the aqueous extract varied between 96.2 and 99.8%. The results of the extracts also showed activity against the fungi studied 48.6% and 96.2%.


1969 ◽  
Vol 7 (12) ◽  
pp. 46-48

Aprotinin (Trasylol - FBA), a polypeptide extracted from bovine lung, inhibits various proteolytic enzymes in vitro, including trypsin and plasmin, and in higher concentration inhibits blood coagulation.1 2 In 1965 we concluded that aprotinin was of uncertain value in acute pancreatitis.3 More recent work with the drug suggests that it should be reassessed, both as an inhibitor of trypsin in acute pancreatitis, and as an inhibitor of the fibrinolytic enzyme plasmin and of blood coagulation in the defibrination syndrome, the haemorrhagic state characterised by depletion of fibrinogen and other clotting factors.


2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Yiming Wang ◽  
Dan Zhang ◽  
Xiaohong Xu ◽  
Xi Lei ◽  
Yiping Wang ◽  
...  

Magnesium lithospermate B (MLB) is one of the major components of Salvia miltiorrhiza root (Danshen). Danshen extracts have been used to control cardiovascular disease for centuries. In 2005, intravenous injection of Danshen depside salt was approved in China for treatment of chronic angina. Although clinical observations have suggested that Danshen extracts inhibited thrombosis, the exact mechanism has not been adequately explored. Using an in vitro whole blood clotting assay, we observed that MLB (250 μM) significantly reduced clot size. Both the clot wet and dry weights were decreased following treatment (108.3 mg vs. 63.5 mg, and 32.8 mg vs. 18.5 mg, p<0.05, respectively). Using thromboelastography, we found that MLB markedly decreased the mechanical strength of the clot and modestly delayed initiation of coagulation in cell-free blood plasma prepared by centrifugation (10,000 хg, 10 min). Under confocal microscopy, we further observed that MLB significantly reduced the density of the fibrin network formed in plasma following thrombin treatment, suggesting that MLB targets coagulation factors to inhibit coagulation. Recent network pharmacology analyses predict that MLB may interact with VWF, factor XIII (FXIII), or thrombin in the coagulation cascade. We found that MLB did not inhibit VWF-dependent platelet agglutination induced by botrocetin. ELISA revealed that MLB also did not significantly alter the binding of activated FXIII (FXIIIa) to fibrinogen. However, when native FXIII from blood plasma was used for the same assay, MLB significantly reduced the binding of FXIIIa to fibrinogen. Since generation of FXIIIa from FXIII is thrombin-dependent, these data suggest that MLB inhibits thrombin activity or thrombin generation. Indeed, we found that MLB markedly inhibited thrombin-induced gel-filtered human and mouse platelet aggregation. These data demonstrated a novel role for MLB in the inhibition of blood coagulation and platelet aggregation, likely through direct inhibition of thrombin function, although we cannot exclude its additional anti-thrombotic activities. Thus, purified MLB may represent an efficient, low-cost agent for treatment of artery and deep vein thrombosis.


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