RESUMO
ABSTRACT: Nonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbß3-fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.
Assuntos
Células-Tronco Pluripotentes Induzidas , Trombose , Humanos , Megacariócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Plaquetas/metabolismo , Trombose/metabolismo , Fibrina/metabolismo , PlasmaRESUMO
This study aimed to perform histological, immunohistochemical, biomechanical, and wettability assessments of leukocyte- and platelet-rich fibrin (L-PRF) membranes obtained from the blood of healthy dogs. Ten client-owned Labrador Retriever dogs were enrolled. Blood samples were obtained from the external jugular vein using a vacuum tube without anticoagulant, which was immediately centrifuged at 400g for 12 min in a dedicated centrifuge. The L-PRF clot was removed from the tube, and the red clot was released from the buffy coat using a spatula. The membrane was produced using a PRF box. Histological examination identified the three portions of the L-PRF membranes. The first portion was composed mainly of red blood cells with the presence of a low number of leukocytes among them. The second portion was composed of white blood cells, mainly neutrophils. The third portion was composed of the fibrin network which was characterized by acidophilic staining. The immunohistochemical analysis showed that vascular endothelial growth factor and platelet-derived growth factor were expressed in all samples at different intensities, both in cellular components and fibrin mesh. The tensile test and wettability assessments were measured in membranes 30 min and 3 h after production. The 30 min L-PRF membranes supported twice the ultimate tensile strength compared to 3 h L-PRF membranes. The wettability of the 30 min sample membranes was statistically higher than the 3 h sample membranes. In conclusion, the centrifugation protocol allowed production of the L-PRF membrane using canine blood and this was confirmed by histological and immunohistochemical analysis. The mechanical resistance and wettability of the L-PRF membrane were significantly reduced over time.
Assuntos
Fibrina Rica em Plaquetas , Cães , Animais , Fibrina Rica em Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Molhabilidade , Leucócitos/metabolismo , Fibrina/metabolismoRESUMO
Rebalancing the hemostatic system by targeting endogenous anticoagulant pathways, like the protein C (PC) system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered in the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated PC (APC) to the hemostatic response, because PC activation occurs on the surface of endothelial cells. We used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, PC activation, and APC anticoagulant function. We found that inhibition of APC activity, in mice either harboring the factor V Leiden mutation or infused with an APC-blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with the role of APC as an anticoagulant. In contrast, inhibition of APC activity had no effect on hemostasis after penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of PC activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of APC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining PC activation during hemostasis and thrombosis.
Assuntos
Hemostáticos , Trombose , Animais , Anticoagulantes/farmacologia , Células Endoteliais/metabolismo , Fibrina/metabolismo , Hemostasia , Humanos , Camundongos , Proteína C/farmacologia , Trombina/metabolismo , Trombose/metabolismoRESUMO
Fibrinolysis occurs when plasminogen activators, such as tissue plasminogen activator (tPA), convert plasminogen to plasmin, which dissolves the fibrin clot. The proteolytic activity of tPA and plasmin is not restricted to fibrin degradation. In the extravascular space, these two proteases modify a variety of substrates other than fibrin, playing a crucial role in physiological and pathological tissue remodeling. In the brain, for example, tPA and plasmin mediate the conversion of brain-derived neurotrophic factor precursor (proBDNF) to mature brain-derived neurotrophic factor precursor (BDNF). Thus, the fibrinolytic system influences processes reported to be dysfunctional in depression, including neurogenesis, synaptic plasticity, and reward processing. The hypothesis that decreased fibrinolytic activity is an important element in the pathogenesis of depression is supported by the association between depression and increased levels of plasminogen activator inhibitor (PAI)-1, the main inhibitor of tPA. Also, various biochemical markers of depression induce PAI-1 synthesis, including hypercortisolism, hyperinsulinemia, hyperleptinemia, increased levels of cytokines, and hyperhomocysteinemia. Moreover, hypofibrinolysis provides a link between depression and emotional eating, binge eating, vegetarianism, and veganism. This paper discusses the role of reduced fibrinolytic activity in the bidirectional interplay between depression and its somatic manifestations and complications. It also reviews evidence that abnormal fibrinolysis links heterogeneous conditions associated with treatment-resistant depression. Understanding the role of hypofibrinolysis in depression may open new avenues for its treatment.
Assuntos
Fibrinólise , Ativador de Plasminogênio Tecidual , Encéfalo/metabolismo , Depressão , Fibrina/metabolismo , Fibrinolisina/metabolismo , Fibrinólise/fisiologia , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
BACKGROUND: Platelet concentrates like leucocyte- and platelet-rich fibrin (L-PRF) have been widely evaluated in different oral surgical procedures to promote the healing process. However, liquid L-PRF products such as liquid fibrinogen have been poorly explored, especially in the biomimetic functionalization of dental implants. The aim of this in vitro study is to evaluate the interaction between 5 different dental implant surfaces and liquid fibrinogen. METHODS: Five commercially available dental implants with different surfaces (Osseospeed™, TiUnite™, SLActive®, Ossean®, and Plenum®) were immersed for 60 minutes in liquid fibrinogen obtained from healthy donors. After this period, the implants were removed and fixed for scanning electron microscopy (SEM). RESULTS: All dental implants were covered by a fibrin mesh. However, noticeable noncontact areas were observed for the Osseospeed™, TiUnite™, and SLActive® surfaces. On the other hand, Ossean® and Plenum® surfaces showed a dense and uniform layer of fibrin covering almost the entire implant surface. The Osseospeed™, TiUnite™, and SLActive® surfaces presented with lower blood cell numbers inside the fibrin mesh compared with the others. Moreover, at higher magnification, thicker fibrin fibers were observed in contact with Ossean® and Plenum® surfaces. The Plenum ®surface showed the thickest fibers which also inserted and interconnect to the microroughness. CONCLUSION: The initial contact between an implant surface and the fibrin network differs significantly among different implant brands. Further studies are necessary to explore the clinical impact of these observations in the osseointegration process of dental implants.
Assuntos
Fibrinogênio/metabolismo , Implantes Dentários , Fibrina/metabolismo , Humanos , Microscopia Eletrônica de Varredura , Projetos PilotoRESUMO
Leptospirosis is a neglected zoonosis, caused by pathogenic spirochetes bacteria of the genus Leptospira. The molecular mechanisms of leptospirosis infection are complex, and it is becoming clear that leptospires express several functionally redundant proteins to invade, disseminate, and escape the host's immune response. Here, we describe a novel leptospiral protein encoded by the gene LIC13086 as an outer membrane protein. The recombinant protein LIC13086 can interact with the extracellular matrix component laminin and bind plasminogen, thus possibly participating during the adhesion process and dissemination. Also, by interacting with fibrinogen and plasma fibronectin, the protein LIC13086 probably has an inhibitory effect in the fibrin clot formation during the infection process. The newly characterized protein can also bind molecules of the complement system and the regulator C4BP and, thus, might have a role in the evasion mechanism of Leptospira. Taken together, our results suggest that the protein LIC13086 may have a multifunctional role in leptospiral pathogenesis, participating in host invasion, dissemination, and immune evasion processes.
Assuntos
Leptospira interrogans , Leptospira , Leptospirose , Fibrina/metabolismo , Humanos , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Plasminogênio/metabolismo , Ligação ProteicaRESUMO
Blood Concentrates (BCs) are autologous non-transfusional therapeutical preparations with biological properties applied in tissue regeneration. These BCs differ in the preparation method, in fibrin network architecture, growth factors release as well as in platelet/cell content. Methodological changes result in distinct matrices that can compromise their clinical effectiveness. The present study evaluated the influence of different g-forces and types of tubes in the release of vascular endothelial growth factor (VEGF) from platelet-rich fibrin (PRF) as a function of time. The PRF-like samples were obtained with three g-forces (200, 400, and 800 x g) for 10 minutes in pure glass tubes or in polystyrene-clot activator tubes. Scanning and Transmission electron microscopy was used to morphometric analyzes of PRF's specimens and flow cytometry was used to quantify VEGF slow release until 7 days. Our results showed that platelets were intact and adhered to the fibrin network, emitting pseudopods and in degranulation. The fibrin network was rough and twisted with exosomic granulations impregnated on its surface. An increase in the concentration of VEGF in the PRF supernatant was observed until 7 days for all g forces (200, 400 or 800 xg), with the highest concentrations observed with 200 x g, in both tubes, glass or plastic. Morphological analyzes showed a reduction in the diameter of the PRF fibers after 7 days. Our results showed that g-force interferes with the shape of the fibrin network in the PRF, as well as affect the release of VEGF stored into platelets. This finding may be useful in applying PRF to skin lesions, in which the rapid release of growth factors can favor the tissue repair process. Our observations point to a greater clarification on the methodological variations related to obtaining PRF matrices, as they can generate products with different characteristics and degrees of effectiveness in specific applications.
Assuntos
Plaquetas/metabolismo , Fibrinólise , Fibrina Rica em Plaquetas/metabolismo , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Plaquetas/ultraestrutura , Centrifugação/efeitos adversos , Centrifugação/métodos , Feminino , Fibrina/metabolismo , Fibrina/ultraestrutura , Voluntários Saudáveis , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
Autologous fibrin has been widely used in surgical procedures for both soft and hard tissue repair. There are different protocols and devices to obtain this matrix, with varying centrifugal time, gravity force, speed, angle of the sample tube and spinning radius. The aim of this study was to compare three methods of obtaining autologous fibrin: L-PRF using the Intra-Spin L-PRF centrifuge (Dohan protocol), the advanced PRF (A-PRF) using the Intra-Spin L-PRF centrifuge and autologous leukocyte fibrin (ALF), using the Kasvi centrifuge. Venous blood was collected from 7 healthy volunteers, which were submitted to the 3 different methods of centrifugation. The membranes were tissue-processed and evaluated by immunohistochemistry for CD3, CD20, CD68 and CD138. For CD68+, a lower number of cells was immunolabelled in the L-PRF group when compared to the other groups (A-PRF and ALF). For CD3+, a lower number of immunolabellated cells was observed in the ALF group when compared to the remaining groups (p < 0.05). In the A-PRF group, the CD20+ cell count was lower than in the remaining groups. No difference was observed in CD138+ cell counts between the groups. The 3 protocols tested are suitable for obtaining autologous fibrin membranes.
Assuntos
Fibrina/metabolismo , Inflamação/patologia , Antígenos CD/metabolismo , Contagem de Células , Humanos , Leucócitos/metabolismo , Fibrina Rica em Plaquetas/metabolismoRESUMO
INTRODUCTION: Systemic hypercoagulation is often a severe complication of infective and inflammatory diseases, which overcome the hemostatic balance and lead to multiple thrombotic occlusions in the microvasculature and organ damage and is related to high mortality rates. SATI is a potent dual inhibitor of FXa and thrombin with antithrombotic efficacy in venous and arterial thrombosis models. In this study, the antithrombotic efficacy of SATI was investigated in a microthrombosis model in rats with an induced hypercoagulant state. MATERIALS AND METHODS: The hypercoagulant state was generated by infusion of TF in sixty rats (12 groups, consisting of 5 rats each). SATI was administered in two different doses by constant infusion and its antithrombotic efficacy was investigated using two different approaches: 1) measuring 125I-fibrin deposition in various organs and 2) continuous whole-body imaging of 111In-platelet biodistribution in anesthetized animals. RESULTS: After start of the TF infusion in rats with radioactively-labeled fibrinogen, the radioactivity was accumulated in liver, spleen, kidney, and mostly in the lung as a consequence of fibrin generation. SATI efficiently reduced the pulmonary deposition of fibrin in a dose- and time-dependent manner. In the SATI groups the splenic and renal radioactivity was enhanced at later time points probably as consequence of the clearance of 125I-fibrin(ogen). Imaging of rats that received 111In-platelets prior to systemic TF administration showed retention of the radioactivity mainly in the lungs in the control group. SATI efficiently blocked the platelet accumulation in the lungs and increased platelet recruitment by the spleen. CONCLUSIONS: SATI is a promising candidate for prevention of microcirculatory disturbances by inhibiting fibrin deposition and platelet accumulation in the lungs and thereby conferring organ protection. Both methods used in this study are suitable for investigating the antithrombotic efficacy of new drugs in microthrombosis. Continuous imaging of 111In-platelets allowed for follow-up of thrombus formation in living animals without the need for tissue harvesting.
Assuntos
Inibidores do Fator Xa , Trombina/antagonistas & inibidores , Animais , Plaquetas/metabolismo , Fator Xa/farmacologia , Fibrina/metabolismo , Microcirculação , Ratos , Distribuição TecidualRESUMO
The aim of this study was the preparation and histological evaluation of Leukocyte- and Thrombocyte-Rich Fibrin (L-TRF) membranes obtained from the blood of four bird species. Forty adult healthy birds were divided into four groups of equal size: G1 - macaws, G2 - domestic chickens, G3 - parrots, G4 - toco toucans. A total of 0.5 mL of blood was collected from each bird, put into a glass tube without anticoagulant and centrifuged at 3000 rpm for 10 min. L-TRF membranes produced after compression of the clot were processed for histological analysis. The ratio of thrombocytes/area was not significantly different among Groups G2, G3 and G4, but a significant difference was found between Groups G1 and G2 with the highest thrombocyte concentration/area in G1. The groups did not differ statistically in the number of leukocytes/area. The fibrin-to-cells ratio did not vary statistically among Groups G1, G2 and G3, but this ratio was significantly higher in Group G4 than in the other groups. The thrombocyte-to-leukocyte ratio was the highest in Group G1, but it did not differ among Groups G2, G3 and G4. In conclusion, the centrifugation protocol allowed the production of L-TRF membranes in the four bird species studied. Histologically, cell ratios were analogous in domestic chickens and parrots, and macaws had the highest ratio of thrombocytes.
Assuntos
Plaquetas/metabolismo , Fibrina/metabolismo , Leucócitos/metabolismo , Membranas/metabolismo , Animais , Aves , Galinhas , Papagaios , Fibrina Rica em Plaquetas/metabolismoRESUMO
A fibrinolytic enzyme was produced by microalga Chlorella vulgaris cultivated in autotrophic and mixotrophic conditions added corn steep liquor, purified by a single chromatographic step, then biochemical characterization and in vitro thrombolytic activity was performed. Maximum cell concentration (1637.45⯱â¯15â¯mgâ¯L-1) and productivity (181.93â¯mgâ¯L-1â¯day-1) was obtained in mixotrophic culture using 1% corn steep liquor. Enzyme-extracted microalgal biomass was purified by acetone precipitation and DEAE Sephadex anion exchange chromatography up to 2 fold with recovery of 4.0%. After purification, fibrinolytic activity was 1834.6â¯Uâ¯mg-1 and 226.86â¯mm2 by spectrophotometry and fibrin plate assays, respectively. SDS-PAGE results exhibited a protein band of about 45â¯kDa and fibrinolytic band was detected by fibrin zymography. Enzyme activity was enhanced in the presence of Fe2+ and inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), which suggest it to be a metal-dependent serine protease. The extract also showed a red blood cell lysis <4% and in vitro thrombolytic activity of 25.6% in 90â¯min of reaction. These results indicate that the fibrinolytic enzyme from C. vulgaris may have potential applications in the prevention and treatment of thrombosis.
Assuntos
Chlorella vulgaris/enzimologia , Fibrina/metabolismo , Fibrinolíticos , Proteínas de Plantas , Cromatografia por Troca Iônica/métodos , Eritrócitos/efeitos dos fármacos , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologiaRESUMO
Pathogenic Leptopira is the etiological agent of leptospirosis, the most widespread zoonotic infection in the world. The disease represents a major public health problem, especially in tropical countries. The present work focused on two hypothetical proteins of unknown function, encoded by the genes LIC13059 and LIC10879, and predicted to be surface-exposed proteins. The genes were cloned and the proteins expressed using E. coli as a host system. We report that the recombinant proteins interacted with extracellular matrix (ECM) laminin, in a dose-dependent fashion and are novel potential adhesins. The recombinant proteins were called Lsa25.6 (rLIC13059) and Lsa16 (rLIC10879), for Leptospiral surface adhesins, followed by the respective molecular masses. The proteins attached to plasminogen (PLG), generating plasmin, in the presence of PLG-activator uPA. Both proteins bind to fibrinogen (Fg), but only Lsa25.6 inhibited fibrin clotting by thrombin-catalyzed reaction. Moreover, Lsa16 interacts with the mammalian cell receptor E-cadherin, and could contribute to bacterial attachment to epithelial cells. The proteins were recognized by confirmed leptospirosis serum samples, suggesting that they are expressed during infection. The corresponding leptospiral proteins are surface exposed based on proteinase K accessibility assay, being LIC10879 most probably exposed in its dimer form. The data of this study extend the spectrum of surface-exposed proteins of L. interrogans and indicate a possible role of the originally annotated hypothetical proteins in infection processes.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Coagulação Sanguínea , Leptospira interrogans/metabolismo , Leptospirose/microbiologia , Adesinas Bacterianas/genética , Animais , Caderinas/metabolismo , Clonagem Molecular , Simulação por Computador , Feminino , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Laminina/metabolismo , Leptospira interrogans/genética , Leptospirose/sangue , Camundongos , Camundongos Endogâmicos BALB C , Plasminogênio/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin ß1. Ablation of integrin ß1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin ß1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin ß1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function.
Assuntos
Integrina beta1/metabolismo , Integrina beta1/fisiologia , Células-Tronco Mesenquimais/fisiologia , Adipócitos/citologia , Adipogenia , Tecido Adiposo/citologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Fibrina/metabolismo , Fibrina/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Células Mieloides , Células-Tronco/citologiaRESUMO
Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.
Assuntos
Bothrops/sangue , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Animais , Caseínas/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Hemorragia/tratamento farmacológico , Metaloendopeptidases/metabolismo , Camundongos , Inibidores de Proteases/sangue , Inibidores de Proteases/química , Proteólise/efeitos dos fármacosRESUMO
The influence of a static magnetic field (SMF) on crude enzyme extracts with proteolytic activity is described and discussed. Proteolytic enzymes, which hydrolyze peptide bonds, and fibrinolytic enzymes, which dissolve fibrin clots, have industrial relevance, and applicability dependent on improvements of productivity and activity. We investigated whether a moderate SMF affects proteolysis in different in vitro tests: general proteolysis of azocasein substrate, and static and dynamic fibrinolytic processes (to compare fibrin gel configuration under exposure). Crude enzyme extracts, obtained from solid state fermentation of Mucor subtilissimus UCP (Universidade Católica de Pernambuco, Recife, Brazil) 1262, were used to carry out assays under slightly heterogeneous fields: a varied vertical SMF (for tests in Eppendorf tubes, from 0.100 to 0.170 T) and a varied horizontal SMF (for tests in Petri dishes, from 0.01 to 0.122 T), generated by two permanent magnets (NdFeB alloy). Results showed significant differences (P < 0.05) in static fibrinolysis assays after 24 h of exposure. The mean diameter of halos of fibrin degradation in the treated group increased by 21% compared to the control group; and the pixel number count of fibrin consumption (in a computational analysis of the area of each halo) enhanced by 30% with exposure. However, in dynamic fibrinolysis assays, no effects of SMF were observed. These results suggest a response of fibrin monomers to the SMF as a possible cause of the observed effects. Bioelectromagnetics. 38:109-120, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Fibrinólise , Campos Magnéticos , Mucor/metabolismo , Peptídeo Hidrolases/metabolismo , Biocatálise , Caseínas/metabolismo , Fibrina/química , Fibrina/metabolismo , Cinética , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
The water-soluble sulfated xylogalactoarabinans from green seaweed Cladophora falklandica are constituted by a backbone of 4-linked ß-l-arabinopyranose units partially sulfated mainly on C3 and also on C2. Besides, partial glycosylation mostly on C2 with single stubs of ß-d-xylopyranose, or single stubs of ß-d-galactofuranose or short chains comprising (1â5)- and/or (1â6)-linkages, was also found. These compounds showed anticoagulant activity, although much lower than that of heparin. The effect of a purified fraction (F1) on the fibrin network was studied in detail. It modifies the kinetics of fibrin formation, suggesting an impaired polymerization process. Scanning electron microscopy showed a laxer conformation, with larger interstitial pores than the control. Accordingly, this network was lysed more easily. These fibrin properties would reduce the time of permanence of the clot in the blood vessel, inducing a lesser thrombogenic state. One of the possible mechanisms of its anticoagulant effect is direct thrombin inhibition.
Assuntos
Anticoagulantes/farmacologia , Clorófitas/química , Fibrina/metabolismo , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Anticoagulantes/química , Configuração de Carboidratos , Avaliação Pré-Clínica de Medicamentos/métodos , Fibrinólise/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Varredura , Coelhos , Alga Marinha/química , Sulfatos/química , Xilose/análogos & derivados , Xilose/químicaRESUMO
UNLABELLED: Background. The regenerative capacity of the liver is critical for proper responses to injury. Fibrin extracellular matrix (ECM) deposition is a common response to insult and contributes to inflammatory liver injury. However, the role of this matrix in hepatic regeneration has not been determined. OBJECTIVE: The purpose of this study was first to determine the role of fibrin ECM in hepatic regeneration followed by the role of the fibrin-binding αvß3 integrin in mediating this effect. MATERIAL AND METHODS: C57Bl/6J (WT) or PAI-1 knockout (KO) mice underwent 70% partial hepatectomy (PHx); plasma and histologic indices of regeneration were determined, as well as expression of key genes involved in hepatic regeneration. RESULTS: PHx promoted transient fibrin deposition by activating coagulation and concomitantly decreasing fibrinolysis. Inhibiting fibrin deposition, either by blocking thrombin (hirudin) in WT mice or by knocking out PAI-1, was associated with a decrease in hepatocyte proliferation after PHx. This strongly suggested a role for fibrin ECM in liver regeneration. To investigate if αvß3 integrin mediates this action, we tested the effects of the anti-αvß3 cyclic peptide RGDfV in animals after PHx. As was observed with inhibition of fibrin deposition, competitive inhibition of αvß3 integrin delayed regeneration after PHx, while not affecting fibrin deposition. These effects of RGDfV correlated with impaired angiogénesis and STAT3 signaling, as well as transient endothelial dysfunction. In conclusion, these data suggest that αvß3 integrin plays an important role in coordinating hepatocyte division during liver regeneration after PHx via crosstalk with fibrin ECM.
Assuntos
Proliferação de Células , Fibrina/metabolismo , Hepatectomia/métodos , Hepatócitos/metabolismo , Integrina alfaVbeta3/metabolismo , Regeneração Hepática , Fígado/metabolismo , Fígado/cirurgia , Transdução de Sinais , Animais , Coagulação Sanguínea , Proliferação de Células/efeitos dos fármacos , Fibrinólise , Genótipo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Integrina alfaVbeta3/antagonistas & inibidores , Fígado/efeitos dos fármacos , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos Cíclicos/farmacologia , Fenótipo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
PURPOSE: To determine the effectiveness of low-intensity therapeutic ultrasound (LITUS) on wound healing in rats with third-degree burns. METHODS: Twenty rats were divided into the Control Group that comprised four rats without third-degree burns that did not undergo LITUS, the Burned Group (BG), comprising eight rats with third-degree burns that did not undergo LITUS, and the Burned with Treatment Group (BTG), comprising eight rats with third-degree burns that were administered LITUS. LITUS began 24 h after injury and involved daily applications for 8 min at 0.1 W/cm2 for 14 days. RESULTS: The BTG lost less weight than the BG (Q=2.75; p<0.05). No visible differences were apparent among the groups' lesions on day 4. By the end of treatment, wound healing was more evident in the BTG. No statistically significant differences were found between the BG and the BTG in relation to the parameters measured using the histological changes in burn wound healing scoring system. CONCLUSION: The LITUS protocol applied to the animals with third-degree burns accelerated the formation of fibrin-leukocyte crusts and significantly reduced weight loss. However, burn wound healing was not accelerated.
Assuntos
Queimaduras/terapia , Tecido Conjuntivo/lesões , Ondas Ultrassônicas , Cicatrização , Animais , Queimaduras/classificação , Queimaduras/patologia , Fibrina/metabolismo , Temperatura Alta/efeitos adversos , Masculino , Ratos Wistar , Reepitelização , Redução de PesoRESUMO
Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbß3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.