RESUMO
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
Although catheters with side holes allow high flow rate during hemodialysis, they also induce flow disturbances and create a critical hemodynamic environment that can favor fibrin deposition and thrombus formation. This study compared the blood flow and analyzed the influence of shear stress and shear rate in fibrin deposition and thrombus formation in nontunneled hemodialysis catheters with unobstructed side holes (unobstructed device) or with some side holes obstructed by blood thrombi (obstructed device). Computational fluid dynamics (CFD) was performed to simulate realistic blood flow under laminar and turbulent conditions. The results from the numerical simulations were compared with the fibrin distribution and thrombus architecture data obtained from scanning electron microscopy (SEM) and two photons laser scanning microscopy (TPLSM) on human thrombus formed in catheters removed from patients. CFD showed that regions of flow eddies and separation were mainly found in the venous holes region. TPLSM characterization of thrombi and fibrin structure in patient samples showed fibrin formations in accordance with simulated flux dynamics. Under laminar flow conditions, the wall shear stress close to border holes increased from 87.3±0.2 Pa in the unobstructed device to 176.2±0.5 Pa in the obstructed one. Under turbulent flow conditions, the shear stress increased by 47% when comparing the obstructed to the unobstructed catheter. The shear rates were generally higher than 5000/s and therefore sufficient to induce fibrin deposition. This findings were supported by SEM data documenting a preferential fibrin arrangement on side hole walls.
Assuntos
Cateterismo Venoso Central/efeitos adversos , Diálise Renal/efeitos adversos , Trombose/etiologia , Simulação por Computador , Fibrina/análise , Fibrina/metabolismo , Fibrina/ultraestrutura , Hemodinâmica , Humanos , Hidrodinâmica , Modelos Cardiovasculares , Estresse Mecânico , Trombose/metabolismo , Trombose/fisiopatologiaRESUMO
BACKGROUND: The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (µm2), the area of the fibrin fibers (µm2), and the width of the fibrin fibers (µm) were determined for the dog clots. The platelet area (µm2), the area of fibrin fibers (µm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. RESULTS: Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. CONCLUSIONS: The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.
Assuntos
Batroxobina/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/ultraestrutura , Gluconato de Cálcio/farmacologia , Fibrina/ultraestrutura , Fibrinolíticos/farmacologia , Animais , Batroxobina/administração & dosagem , Plaquetas/efeitos dos fármacos , Gluconato de Cálcio/administração & dosagem , Gatos/sangue , Cães/sangue , Quimioterapia Combinada , Espaço Extracelular/efeitos dos fármacos , Fibrina/efeitos dos fármacos , Fibrinolíticos/administração & dosagem , Masculino , Microscopia Eletrônica de Transmissão/veterinária , Trombose/veterináriaRESUMO
Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 µg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.
Assuntos
Anticoagulantes/metabolismo , Dermatan Sulfato/fisiologia , Fibrina/fisiologia , Fibrina/ultraestrutura , Animais , Anticoagulantes/fisiologia , Bovinos , Fibrina/metabolismo , Ligação Proteica/fisiologiaRESUMO
There is evidence that clot structure can be modulated by endothelial cells, wherein the fibrinogen αC domain plays a major role in the fibrin-cell interaction. The spatial distribution of fibrin fibers from fibrinogen Caracas V and Caracas I, with heterozygous mutation in the αC domain (Aα Ser432Cys and Aα Ser466stop, respectively) on human dermal microvasculature endothelial cells (HMEC-1), was studied by laser scanning confocal microscopy. In order to assess fibrin-cell interaction and the role of the αC domain, preliminary experiments were done with inert microspheres and RGD peptide included in the clotting reaction, and forming clots with fibrinogen fragment X (fibrinogen without αC domain). Groups of stressed fibers were observed near the cell surface and were related to fibrin-cell interactions, which were abolished by the RGD peptide, and by the absence of the αC domain. The fibrin network of fibrinogen Caracas V and Caracas I was very different from that of normal fibrinogen. In general, patient's clots were characterized by very thin, tightly packed fibrin fibers, with a substantially reduced network porosity. Near the cell's surface, both abnormal fibrinogens formed a very fine meshwork, with stressed fibers 'anchored' to the cell surface, a pattern that was lost far from the cell surface. The structure of normal and patient clots performed in the absence of cells resembled that observed far from the cell surface, concluding that Caracas V and Caracas I fibrin was modulated by the presence of endothelial cells.
Assuntos
Coagulação Sanguínea , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Fibrina/ultraestrutura , Fibrinogênios Anormais/metabolismo , Trombose/sangue , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Western Blotting , Derme/citologia , Derme/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Fibrina/análise , Fibrinogênios Anormais/química , Fibrinogênios Anormais/genética , Heterozigoto , Humanos , Microscopia Eletrônica de Varredura , Mutação , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Trombose/patologia , VenezuelaRESUMO
This study used scanning electron microscopy (SEM) to evaluate the morphology and adhesion of blood components on root surfaces instrumented by curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser. One hundred samples from 25 teeth were divided into 5 groups: 1) Curettes; 2) Piezoelectric ultrasonic scaler; 3) Curettes plus piezoelectric ultrasonic scaler; 4) Er,Cr:YSGG laser; 5) Curettes plus Er,Cr:YSGG laser. Ten samples from each group were used for analysis of root morphology and the other 10 were used for analysis of adhesion of blood components on root surface. The results were analyzed statistically by the Kruskall-Wallis and Mann-Whitney tests with a significance level of 5%. The group treated with curettes showed smoother surfaces when compared to the groups were instrumented with piezoelectric ultrasonic scaler and the Er,Cr:YSGG laser. The surfaces instrumented with piezoelectric ultrasonic scaler and Er,Cr:YSGG laser, alone or in combination with hand scaling and root planing, did not differ significantly (p>0.05) among themselves. No statistically significant differences (p>0.05) among groups were found as to the adhesion of blood components on root surface. Ultrasonic instrumentation and Er,Cr:YSGG irradiation produced rougher root surfaces than the use of curettes, but there were no differences among treatments with respect to the adhesion of blood components.
Assuntos
Células Sanguíneas/citologia , Raspagem Dentária/instrumentação , Aplainamento Radicular/instrumentação , Raiz Dentária/ultraestrutura , Adesão Celular/fisiologia , Raspagem Dentária/métodos , Dentina/efeitos da radiação , Dentina/ultraestrutura , Fibrina/efeitos da radiação , Fibrina/ultraestrutura , Humanos , Lasers de Estado Sólido/uso terapêutico , Terapia com Luz de Baixa Intensidade/instrumentação , Microscopia Eletrônica de Varredura , Piezocirurgia/instrumentação , Aplainamento Radicular/métodos , Camada de Esfregaço , Curetagem Subgengival/instrumentação , Raiz Dentária/efeitos da radiaçãoRESUMO
This study used scanning electron microscopy (SEM) to evaluate the morphology and adhesion of blood components on root surfaces instrumented by curettes, piezoelectric ultrasonic scaler and Er,Cr:YSGG laser. One hundred samples from 25 teeth were divided into 5 groups: 1) Curettes; 2) Piezoelectric ultrasonic scaler; 3) Curettes plus piezoelectric ultrasonic scaler; 4) Er,Cr:YSGG laser; 5) Curettes plus Er,Cr:YSGG laser. Ten samples from each group were used for analysis of root morphology and the other 10 were used for analysis of adhesion of blood components on root surface. The results were analyzed statistically by the Kruskall-Wallis and Mann-Whitney tests with a significance level of 5 percent. The group treated with curettes showed smoother surfaces when compared to the groups were instrumented with piezoelectric ultrasonic scaler and the Er,Cr:YSGG laser. The surfaces instrumented with piezoelectric ultrasonic scaler and Er,Cr:YSGG laser, alone or in combination with hand scaling and root planing, did not differ significantly (p>0.05) among themselves. No statistically significant differences (p>0.05) among groups were found as to the adhesion of blood components on root surface. Ultrasonic instrumentation and Er,Cr:YSGG irradiation produced rougher root surfaces than the use of curettes, but there were no differences among treatments with respect to the adhesion of blood components.
Esse estudo utilizou microscopia eletrônica de varredura (MEV) para avaliar a morfologia e a adesão de elementos sanguíneos em superfícies radiculares instrumentadas com curetas, ultrassom piezoelétrico e laser de Er,Cr:YSGG. Foram utilizadas no presente estudo 100 amostras provenientes de 25 dentes que foram divididas em 5 grupos: 1) Raspagem manual com curetas; 2) Raspagem com ultrassom; 3) Associação instrumento manual e ultrassom; 4)Irradiação do laser de Er,Cr:YSGG;5)Associação raspagem manual com irradiação com laser de Er,Cr:YSGG. Dez amostras de cada grupo foram utilizadas para análise da morfologia e as outras 10 foram utilizadas para a análise de adesão de elementos sanguíneos. As eletromicrografias foram analisadas através dos escores de adesão de elementos sanguíneos e pelo índice de morfologia radicular e os resultados foram analisados estatisticamente através dos testes de Kruskall-Wallis e de Mann-Whitney com nível de significância de 5 por cento. O grupo que foi tratado com instrumentos manuais apresentou superfície mais lisa em relação aos grupos que foram instrumentados com ultrassom e com o laser de Er,Cr:YSGG. As superfícies instrumentadas com ultrassom e com o laser de Er,Cr:YSGG de forma isolada ou associada a raspagem manual não apresentaram diferenças estatísticas entre si (p>0,05). Não houve diferenças estatísticas entre os grupos em relação a adesão de elementos sanguíneos(p>0,05). A instrumentação ultrassônica e a irradiação com o laser de Er,Cr:YSGG produziram superfícies radiculares mais rugosas em relação a raspagem com curetas, porém não houve diferenças entre os tratamentos com relação à adesão de elementos sanguíneos.
Assuntos
Humanos , Células Sanguíneas/citologia , Raspagem Dentária/instrumentação , Aplainamento Radicular/instrumentação , Raiz Dentária/ultraestrutura , Adesão Celular/fisiologia , Raspagem Dentária/métodos , Dentina/efeitos da radiação , Dentina/ultraestrutura , Fibrina/efeitos da radiação , Fibrina/ultraestrutura , Terapia com Luz de Baixa Intensidade/instrumentação , Lasers de Estado Sólido/uso terapêutico , Microscopia Eletrônica de Varredura , Piezocirurgia/instrumentação , Aplainamento Radicular/métodos , Camada de Esfregaço , Curetagem Subgengival/instrumentação , Raiz Dentária/efeitos da radiaçãoRESUMO
Engineered nanoparticles are designed to perform specific functions and therefore have specific properties that could potentially be harmful. Nanoparticles such as titanium dioxide have the potential to become transparent and are therefore widely used in cosmetic products and sunscreen. Research on the toxicity of nanoparticles is of utmost importance and numerous in vitro studies have shown that some of these particles could have adverse health effects. The current study aimed to investigate the in vivo effects of two different titanium nanoparticles at two different concentrations after inhalation by experimental BALB/c mice. This was done to determine whether these particles will cause an inflammatory reaction, visible as alterations in platelet and fibrin ultrastructure. Mice were divided into five experimental groups comprising of a control group, high and low concentration groups exposed to the spherical-shaped particles, as well as high and low concentration groups exposed to the rod-shaped particles. The ultrastructure of the fibrin networks and platelet aggregates of these experimental groups were investigated and compared to that of controls. Results indicated that the fibrin networks of the exposed animals have a net-like covering over the major fibres, typical to that found in animals with inflammation. It can therefore be concluded that the nanoparticles used in this study may have the potential to cause an inflammatory reaction, affecting the haemostatic physiology.
Las nanopartículas han sido diseñadas para realizar funciones específicas, y por lo tanto, tienen propiedades específicas que podrían ser perjudiciales. Las nanopartículas, como el dióxido de titanio tienen el potencial de llegar a ser transparentes, pudiendo ser ampliamente utilizadas en productos cosméticos y protectores solares. La investigación sobre la toxicidad de las nanopartículas es de suma importancia y numerosos estudios in vitro han demostrado que algunas de estas partículas, podrían tener efectos adversos para la salud. El presente estudio tuvo como objetivo investigar los efectos in vivo de dos nanopartículas de titanio diferentes en dos concentraciones después de la inhalación experimental de ratones BALB/c. Esto se realizó para determinar si las partículas provocan una reacción inflamatoria, visible como alteraciones en la ultraestructura de plaquetas y fibrina. Los ratones se dividieron en cinco grupos experimentales, que comprende un grupo control y grupos expuestos a nanopartículas de forma esférica de alta y baja concentración, así como grupos expuestos a nanopartículas en forma de barra de alta y baja concentración. Fueron investigadas la ultraestructura de las redes de fibrina y agregados plaquetarios de estos grupos experimentales y se comparó con la de los controles. Los resultados indicaron que en los animales expuestos se observó una red de fibrina que recubría las fibras más grandes, típicas de las que se encuentran en los animales con inflamación. Por lo tanto, puede concluirse que las nanopartículas utilizadas en este estudio pueden tener el potencial de causar una reacción inflamatoria, afectando a la fisiología hemostática.
Assuntos
Animais , Camundongos , Fibrina/ultraestrutura , Nanopartículas/toxicidade , Plaquetas/ultraestrutura , Titânio/toxicidade , Modelos Animais de Doenças , Fibrina , Exposição por Inalação , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Plaquetas , Testes de Toxicidade , Titânio/administração & dosagemRESUMO
OBJECTIVE: To compare the adhesion and maturation of blood components on chemically conditioned root surfaces. METHOD AND MATERIALS: Clinical root samples of human teeth were obtained (n = 150) and manually scaled. Five groups of 30 samples were treated as follows: (1) saline solution irrigation (control); (2) 24% EDTA gel; (3) 25% citric acid solution; (4) tetracycline solution (50 mg/mL); and (5) 30% sodium citrate solution. After these treatments, 15 samples of each group received a blood drop and were analyzed by SEM. The remaining 15 had their surface morphology evaluated for collagen fibrils exposure by SEM. Photomicrographs were analyzed according to the score of adhesion of blood components. Kruskal-Wallis and Dunn multiple comparison tests were employed. RESULTS: The control group was characterized by the absence of blood elements on the surface. The best result was observed in the citric acid group, which had a dense fibrin network with blood elements adhered. The EDTA group showed a moderate fibrin network formation. In contrast, a scarce fibrin network and a few cells were present in the tetracycline samples, and an absence of blood elements was found on sodium citrate specimens. The citric acid group was statistically different from the control group (P < .01). No differences were found among the control, EDTA, tetracycline, and sodium citrate groups (P > .05). CONCLUSION: Under these experimental conditions, citric acid is indicated to stabilize clots on the root surface, which act as a scaffold for connective tissue cell development.
Assuntos
Condicionamento Ácido do Dente/métodos , Coagulação Sanguínea/efeitos dos fármacos , Quelantes/uso terapêutico , Dentina/efeitos dos fármacos , Raiz Dentária/efeitos dos fármacos , Células Sanguíneas/ultraestrutura , Adesão Celular/efeitos dos fármacos , Citratos/uso terapêutico , Ácido Cítrico/uso terapêutico , Colágeno/ultraestrutura , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/ultraestrutura , Raspagem Dentária , Dentina/ultraestrutura , Ácido Edético/uso terapêutico , Fibrina/efeitos dos fármacos , Fibrina/ultraestrutura , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Aplainamento Radicular , Método Simples-Cego , Camada de Esfregaço , Cloreto de Sódio , Citrato de Sódio , Tetraciclina/uso terapêutico , Raiz Dentária/ultraestruturaRESUMO
Modul8® is a composite mixture of natural products that are known to be an immunomodulator. In the current study the effect of this immunomodulator is tested on an experimental asthmatic BALB/c mouse model to investigate its properties on the white blood cell count in the blood and bronchial lavage of the animals since white blood cells play a fundamental role in the inflammatory process involved in asthma. As it is known that platelets also play an important role in the immune system, the ultrastructure of platelets and fibrin networks were also investigated by scanning electron microscopy. The animals were sensitised, nebulized and treated over a period of 43 days until termination. Results from the blood smears as well as the bronchial lavage smears revealed significantly higher eosinophil counts in the asthmatic group compared to the control and treated groups. Changes in the ultrastructure of the platelets and fibrin networks could also be observed, with the Modul8® -treated group appearing similar to that of the control group where thick major and thin minor fibres could clearly be distinguished and a tight mass of platelet aggregate could be observed. Whereas the fibrin networks from the asthmatic animals appeared flimsy with a tight mass of thin fibres covering the thick major fibres. The asthmatic platelet aggregates appeared granular without the tight round appearance of the control platelet aggregates. It is therefore concluded that Modul8® positively influences the white blood cell counts by altering the asthmatic profile to look similar to that of the control. Also, it seems as if Modul8® has a stabilizing effect on the platelets and fibrin networks. From these results it can be suggested that Modul8® might successfully be used in the treatment of inflammatory conditions such as asthma.
Modul8® es una mezcla compuesta de productos naturales que es conocida por ser un inmunomodulador. En el presente estudio, el efecto de este inmunomodulador se prueba de forma experimental en el modelo de ratón asmáticos BALB/c, para investigar sus propiedades sobre el conteo de glóbulos blancos en la sangre y lavado bronquial de los animales, ya que los glóbulos blancos desempeñan un papel fundamental en el proceso de respuesta inflamatoria implicado en el asma. Como es sabido, también las plaquetas desempeñan un papel importante en el sistema inmunológico, así, la ultraestructura de las plaquetas y las redes de fibrina también fueron investigadas por microscopía electrónica de barrido. Los animales fueron sensibilizados, nebulizados y tratados durante un período de 43 días hasta el término. Los resultados de los frotis de sangre, así como los de lavado bronquial revelaron un número significativamente mayor de eosinófilos en el grupo de asmáticos en comparación con el control y grupos tratados. Cambios en la ultraestructura de las plaquetas y redes de fibrina también pueden ser observados, donde el grupo tratado con la Modul8® aparece similar a el grupo control, donde los fibras de mayor grosor y menor grosor pueden ser claramente distinguidas y además, puede ser observada una apretada masa de plaquetas aglutinadas. Considerando las redes de la fibrina en animales asmáticos parecen endebles con una apretada masa de fibras de menor grosor que cubren las fibras de mayor grosor. Los agregados de plaquetas en asmáticos aparecen granulares sin el aspecto apretado del agregado plaquetario que rodea al grupo control. Por tanto, se concluye que Modul8® positivamente influye en el conteo de glóbulos blancos mediante la alteración del perfil de asmáticos a un aspecto similar al del control. Además, parece como si Modul8® tuviera un efecto estabilizador en las plaquetas y las redes de fibrina. De estos resultados se puede sugerir que Modul8® puede ser utilizado...
Assuntos
Humanos , Recém-Nascido , Lactente , Asma/diagnóstico , Asma/sangue , Asma/veterinária , Fatores Imunológicos/análise , Fatores Imunológicos/farmacologia , Fatores Imunológicos , Fibrina/ultraestrutura , Plaquetas/ultraestrutura , Camundongos Endogâmicos BALB C/anatomia & histologia , Camundongos Endogâmicos BALB C/sangueRESUMO
The purpose of the present study was to compare the platelet and fibrin network ultrastructure of humans to eight different animal species in order to determine the differences between human and animal platelet and fibrin morphology, and to determine whether the animals studied differ in their platelet and fibrin morphology, and whether these differences can be observed by scanning electron microscopy. Platelets and fibrin networks play an important role both in the coagulation process as well as physiologically in allergic processes and immunological mechanisms. The thickness of human fibrin networks were compared to mouse (Mus musculus), equine (Equus caballus), vervet monkey (Chlorocebus aethiops previously Cercopithecus aethiops), oryx (Oryx gazella), ovine (Ovis aries), penguin (Spheniscus demersus), rabbit (Oryctolagus cuniculus) and sea turtle (Caretta caretta). Fibers were measured and divided into thin (minor) fibers, intermediate fibers and thick (major) fibers. The results obtained indicated that for each of the three fibrin classes, the size ranges of the monkey, oryx and equine were not significantly different to one another, and the human, penguin, oryx and ovine not significantly different to one other. From these results it can be concluded that mammals and aves possess a distinct tri-modal fibrin fiber distribution, different from that of the studied reptilian species where the sea turtle possesses a distinct bimodal fibrin fiber distribution and it can be suggested that the utilization of mammalian and avian models, in terms of fibrin fiber distribution patterns, might be a suitable alternative for ultrastructural studies.
El propósito del presente estudio fue comparar la ultraestructura de plaquetas y las redes de fibrina de los seres humanos y de ocho diferentes especies de animales, con el fin de determinar las diferencias morfológicas de estas estructuras y si las diferencias pueden ser observadas por microscopía electrónica de barrido. Las plaquetas y las redes de fibrina desempeñan un papel importante tanto en el proceso de coagulación como, fisiológicamente en procesos alérgicos y mecanismos inmunológicos. Elgrosor de las redes de fibrina humana fue comparado con las del ratón (Mus musculus), equino (Equus caballus), mono vervet (Chlorocebus aethiops, anteriormente Cercopithecus aethiops, antílope Africano (Oryx gazella), ovino (Ovis aries), pingüino (Spheniscus demersus), conejo (Oryctolagus cuniculus) y tortuga marina (Caretta caretta). Las fibras fueron medidas y agrupadas en fibras delgadas (menor), fibras intermedias y fibras gruesas (grandes). Los resultados obtenidos indicaron que para cada una de las tres clases de fibrina, los rangos de su tamaño en el mono, antílope africano y en equino no fueron significativamente diferentes entre sí, mientras que en humano, pingüino, antílope africano y ovino no fueron significativamente diferentes entre éstos. De estos resultados se pudo concluir que mamíferos y aves poseen una distribución tri-modal de fibras de fibrina, distinta a la de las especies de reptiles estudiadas, donde la tortuga de mar posee una distribución bimodal de fibras de fibrina. Se puede sugerir que la utilización de los modelos mamíferos y aviar, en términos de patrones de distribución de fibras de fibrina, pueden ser una alternativa adecuada para los estudios ultraestructurales.
Assuntos
Humanos , Animais , Fibrina/análise , Fibrina/provisão & distribuição , Fibrina/ultraestrutura , Plaquetas/citologia , Plaquetas/ultraestrutura , Mamíferos/anatomia & histologia , Mamíferos/sangue , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Varredura/veterinária , Répteis/anatomia & histologia , Répteis/sangueRESUMO
We have studied some biophysical properties of the fibrin network during the normal state of pregnancy and in patients with recurrent miscarriage (RM), in the first trimester of pregnancy. The fibrin polymerization process, followed by turbidity, showed that the rate of fibrin monomer assembly and the final turbidity was increased in the pregnant group (normal and with history of RM) compared to non-pregnant women (normal and RM), which is consistent with the increased fibrinogen concentration during pregnancy. No changes were observed in the Darcy constant (Ks) of RM clots, pregnant or not; however, in pregnant control subjects the Ks increased (p = 0.03). The fibrin lysis rate was increased in pregnant women compared to non-pregnant, being faster in women with RM. The rheological properties of the fibrin network in the non-pregnant group (control and RM patients) were similar; in the pregnant state, the fibrin network of the control group was 1.3 times stiffer compared to the control non-pregnant women, and almost unchanged in RM patients. In this study we have found changes in the clot structure that seem to be related to normal pregnancy and an increased rate of the fibrin lysis process in the RM patients, which may have clinical relevance.
Assuntos
Aborto Habitual/etiologia , Fibrina/metabolismo , Fibrinólise , Complicações Hematológicas na Gravidez/sangue , Primeiro Trimestre da Gravidez , Aborto Habitual/sangue , Testes de Coagulação Sanguínea , Feminino , Fibrina/ultraestrutura , Hemostasia , Humanos , Microscopia Eletrônica de Varredura , Nefelometria e Turbidimetria , Gravidez , Tromboelastografia , Fatores de Tempo , VenezuelaRESUMO
To elucidate some of the links between homocysteine and vascular disease, we have evaluated the effect of the amino acid on the formation (by kinetics studies), structure (by electron microscopy) and lysis of the fibrin network, using tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). We have studied whether homocysteine could alter the activity of the components involved in fibrinolysis (by amidolytic and thrombolytic methods). The results showed that homocysteine-associated networks were more compact and branched than controls (52 +/- 6 vs 44 +/- 5 fibers/field, P = 0.008), and were formed by shorter and thicker fibers. This clot proved to be more resistant to fibrinolysis with u-PA than control [lysis time 50%: 257 +/- 16 (homocysteine) vs 187 +/- 6 min (control); P < 0.004], but there were no differences with t-PA. Homocysteine did not affect the biological activities of plasmin, or plasminogen activation by t-PA and u-PA. Defective fibrinolysis with u-PA was therefore associated with homocysteine-fibrin structural alterations rather than the homocysteine effect on the biological activities of the fibrinolytic components evaluated. Results suggest that hyperhomocysteinemic patients could produce tight clots, were more resistant to lysis, and generated a procoagulant environment in situ. We believe that our findings may contribute to understanding the mechanisms involved in the homocysteine harmful effect.
Assuntos
Fibrina/ultraestrutura , Fibrinólise/fisiologia , Homocisteína/metabolismo , Plasma/metabolismo , Coagulação Sanguínea/fisiologia , Fibrina/química , Fibrina/metabolismo , Fibrinolisina/metabolismo , Humanos , Microscopia Eletrônica , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Inativadores de Plasminogênio/metabolismoRESUMO
On the basis of growing clinical evidence, it is well known that elevated plasma homocysteine (Hcy) levels are associated with higher risk of venous and arterial thrombosis. Several experimental studies have been carried out in order to elucidate the mechanisms involved that still remain unclear. The aim of our study was to evaluate the homocysteine effects on formation and structure of plasmatic fibrin network. We also assayed homocystine and cysteine to determinate possible participation of thiol group in the tested activity. Aliquots of a pool of plasma incubated separately with sulfur compounds were clotting with thrombin. Fibringeneration and fibrin networks were evaluated by kinetic studies and scanning electronic microscopy, respectively. No significant differences were observed on fibrin generation of the substances assayed in comparison to control. The scanning electronic microscopy showed that Hcy-associated networks were different from control, with shorter, thicker and more branched fibers, resulting in a more compact structure and probably more resistant to fibrinolysis. The thiol group would be involved in this effect. Our findings would be a new contribution to elucidate the mechanisms involved in harmful effects associated to hyperhomocysteinemia.
Assuntos
Fibrina/efeitos dos fármacos , Homocisteína/farmacologia , Compostos de Sulfidrila/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fibrina/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Varredura , Trombina/farmacologia , Trombose/etiologiaRESUMO
Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.
Assuntos
Transtornos de Proteínas de Coagulação/fisiopatologia , Fibrinogênios Anormais/metabolismo , Albuminas/metabolismo , Substituição de Aminoácidos , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/genética , Transtornos de Proteínas de Coagulação/sangue , Transtornos de Proteínas de Coagulação/genética , Dextranos/farmacologia , Fibrina/genética , Fibrina/metabolismo , Fibrina/ultraestrutura , Fibrinogênios Anormais/genética , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Humanos , Microscopia Confocal , Mutação , Trombofilia/sangue , Trombofilia/genéticaRESUMO
A new dysfibrinogenemia associated with thrombophilia has been identified in a Venezuelan kindred. Thrombin and Reptilase times were prolonged and the accelerating capacity of the patient's fibrin on the t-PA-induced plasminogen activation was decreased. In addition the affinity of fibrinogen for plasminogen was diminished. Permeability and electron microscopy studies revealed that the abnormal clot was made up of thin and densely packed fibres giving rise to a reduced fibrin gel porosity. This was confirmed by turbidity studies showing a decreased fibre mass/length ratio. Affected members were heterozygous for an Aalpha 532 Ser-->Cys mutation as demonstrated by genetic analyses. This abnormal fibrinogen has been designated as Fibrinogen Caracas V. The family study showed a convincing association between the mutation and thrombotic manifestations. The thrombotic tendency may be ascribed to lack of accelerating capacity of fibrin to induce fibrinolysis caused by an abnormal clot structure with thin fibres and reduced porosity.
Assuntos
Fibrinogênios Anormais/genética , Trombose/etiologia , Adolescente , Adulto , Substituição de Aminoácidos , Coagulação Sanguínea/genética , Testes de Coagulação Sanguínea/métodos , Análise Mutacional de DNA , Saúde da Família , Feminino , Fibrina/farmacologia , Fibrina/ultraestrutura , Fibrinogênios Anormais/metabolismo , Fibrinogênios Anormais/ultraestrutura , Heterozigoto , Humanos , Radioisótopos do Iodo , Cinética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Mutação/genética , Nefelometria e Turbidimetria , Linhagem , Plasminogênio/efeitos dos fármacos , Plasminogênio/metabolismo , Plasminogênio/normas , Recidiva , Análise de Sequência de DNA , Trombofilia/etiologia , Trombofilia/genética , Trombose/genética , Ativador de Plasminogênio Tecidual/farmacologiaRESUMO
Fibrinogen Caracas I is a dysfibrinogenemia with a mild bleeding diathesis and a defective wound healing. We have characterized this abnormal fibrinogen using transmission electron microscopy (TEM) in combination with turbidity and permeation studies. Turbidometric and permeability analysis showed that the abnormal fibrin had a significantly decreased mass:length ratio and fiber diameter. In addition, the permeability studies of plasma fibrin clots showed that the gel porosity of the abnormal fibrinogen was reduced. Images of the abnormal fibrin structure obtained using TEM showed that the fibers were thinner, much less branched and less ordered than normal fibers. Diminished fibrin fiber diameter and reduced fibrin gel porosity have been taken as hallmarks of thrombophilic dysfibrinogenemias. The results of the present study show that these features are not necessarily predictive of thrombophilia. Further studies performed on a larger number of dysfibrinogenemias need to be conducted in order to establish the implications of these parameters on the clinical outcome.