Orientador: Flávio Henrique Caetano / Banca: Ana Maria Costa Leonardo / Banca: Maeli Dal Pai Silva / Banca: Maria Tercilia Vilela de Azeredo Oliveira / Banca: Carmem Silvia Fontanetti Christofoletti / Resumo: O presente estudo teve como objetivo analisar os leucócitos circulantes em microscopia de luz e eletrônica e sua distribuição sazonal, além de procurar estabelecer o período de renovação celular desses leucócitos, e principalmente de caracterizar os fagócitos mononucleares do sangue de tartaruga, e sua capacidade de fagocitose frente a material inerte. Neste trabalho utilizou-se seis tartarugas Phrynops hilarii, originárias de ilhas do estuário do rio Guaíba Porto Alegre (RS), que estavam ambientadas em nosso biotério. A coleta de sangue foi realizada em todos os períodos sazonais, por punção de vasos laterais do pescoço e coletados em tubos de ensaio heparinizados. Foram realizados esfregaços sanguíneos, corados com Leishmann e Giemsa, contando-se quinhentas células de cada animal e após a obtenção dos dados, foi aplicado o teste estatístico de Bonferroni. Para a análise autorradiográfica foi injetado 1000μCi / kg de thymidine-H A. Para microscopia eletrônica processamos a nata leucocitária obtida por meio de centrifugação do sangue, para a analise citoquímica incubamos com citidina-5'-monofosfato, betaglicerofosfato de sódio, Trimetafosfatase, para averiguar a resposta fagocitária utilizamos 0,01% de carvão coloidal.Os resultados mostram que os leucócitos de Phrynops hilarii tem descrição de leucócitos semelhantes às outras espécies, somente os basófilos e linfócitos não sofreram alterações em sua distribuição sazonal. Todos os leucócitos com exceção dos basófilos apresentaram renovação celular após sete dias. Caracterizamos monoblasto, promonócito, monócitos e macrófago no sangue circulante bem como a capacidade dos fagócitos mononucleares de fagocitar células mortas e materiais inerte. / Abstract: The aim of this study was to analyze the leukocytes in the blood using electronic and light microscopy and their seasonal distribution, also to characterize the leukocyte cells replacement and mainly to characterize the mononuclear phagocytes in the blood and their phagocytic capacity. In this study, it was used six turtles (Phrynops hilarii), caught at the Guaíba river estuary, Porto Alegre, Rio Grande do Sul, Brazil and lodged for 1 week at the Central Animal House, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Blood was obtained during the seasonal periods by puncturing the lateral vessels of the neck. The blood samples were stained by Leishmann and Giemsa, counting five hundred cells in each animal. After the obtained data, it was applied the Bonferroni test as statistical method. For the autoradiographic analysis, it was injected in the circulating blood 1000 μCi/kg of 3Hthymidine. For electronic microscopy, it was processed the leukocyte substrate by circulating blood centrifugation. For cytochemical analyses, blood smears were air dried, post-fixed in 4% formalin and submitted to the determination of the following enzyme activities: acid phosphatases (β-g1ycerophosphatase and citidine-5′-sodium monophosphatase), and trimetaphosphatase. The results showed that the leukocytes of Phrynops hilarii have the leukocytes description similar to the other species, only the basophiles and lymphocytes did not suffer alterations in their seasonal distribution. All the leukocytes, in exception of the basophiles showed cells replacement after seven days. It was characterized the monocytes and macrophages in the circulating blood as well as the phagocytes capacity. / Doutor

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-11Bitstream added on 2014-06-13T20:21:25Z : No. of bitstreams: 1 pitol_dl_dr_rcla.pdf: 2375764 bytes, checksum: 1ce362e81dd4a2ee6f04869e84243e98 (MD5) / O presente estudo teve como objetivo analisar os leucócitos circulantes em microscopia de luz e eletrônica e sua distribuição sazonal, além de procurar estabelecer o período de renovação celular desses leucócitos, e principalmente de caracterizar os fagócitos mononucleares do sangue de tartaruga, e sua capacidade de fagocitose frente a material inerte. Neste trabalho utilizou-se seis tartarugas Phrynops hilarii, originárias de ilhas do estuário do rio Guaíba Porto Alegre (RS), que estavam ambientadas em nosso biotério. A coleta de sangue foi realizada em todos os períodos sazonais, por punção de vasos laterais do pescoço e coletados em tubos de ensaio heparinizados. Foram realizados esfregaços sanguíneos, corados com Leishmann e Giemsa, contando-se quinhentas células de cada animal e após a obtenção dos dados, foi aplicado o teste estatístico de Bonferroni. Para a análise autorradiográfica foi injetado 1000μCi / kg de thymidine-H A. Para microscopia eletrônica processamos a nata leucocitária obtida por meio de centrifugação do sangue, para a analise citoquímica incubamos com citidina-5'-monofosfato, betaglicerofosfato de sódio, Trimetafosfatase, para averiguar a resposta fagocitária utilizamos 0,01% de carvão coloidal.Os resultados mostram que os leucócitos de Phrynops hilarii tem descrição de leucócitos semelhantes às outras espécies, somente os basófilos e linfócitos não sofreram alterações em sua distribuição sazonal. Todos os leucócitos com exceção dos basófilos apresentaram renovação celular após sete dias. Caracterizamos monoblasto, promonócito, monócitos e macrófago no sangue circulante bem como a capacidade dos fagócitos mononucleares de fagocitar células mortas e materiais inerte. / The aim of this study was to analyze the leukocytes in the blood using electronic and light microscopy and their seasonal distribution, also to characterize the leukocyte cells replacement and mainly to characterize the mononuclear phagocytes in the blood and their phagocytic capacity. In this study, it was used six turtles (Phrynops hilarii), caught at the Guaíba river estuary, Porto Alegre, Rio Grande do Sul, Brazil and lodged for 1 week at the Central Animal House, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Blood was obtained during the seasonal periods by puncturing the lateral vessels of the neck. The blood samples were stained by Leishmann and Giemsa, counting five hundred cells in each animal. After the obtained data, it was applied the Bonferroni test as statistical method. For the autoradiographic analysis, it was injected in the circulating blood 1000 μCi/kg of 3Hthymidine. For electronic microscopy, it was processed the leukocyte substrate by circulating blood centrifugation. For cytochemical analyses, blood smears were air dried, post-fixed in 4% formalin and submitted to the determination of the following enzyme activities: acid phosphatases (β-g1ycerophosphatase and citidine-5′-sodium monophosphatase), and trimetaphosphatase. The results showed that the leukocytes of Phrynops hilarii have the leukocytes description similar to the other species, only the basophiles and lymphocytes did not suffer alterations in their seasonal distribution. All the leukocytes, in exception of the basophiles showed cells replacement after seven days. It was characterized the monocytes and macrophages in the circulating blood as well as the phagocytes capacity.

Chelonia mydas, denominada tartaruga verde, é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e é considerada em perigo de extinção pela IUCN (2006). A fibropapilomatose, doença caracterizada por tumores cutâneos benignos (fibropapilomas), é uma das mais importantes ameaças à sobrevivência dessa espécie. Pesquisas sugerem o envolvimento de agentes infecciosos virais em associação com fatores ambientais e genéticos. Foram estudadas 47 tartarugas provenientes do litoral do estado de São Paulo, sendo 18 sem fibropapilomas e 29 acometidas. Dados de biometria das tartarugas, quantidade, localização e tamanho dos tumores foram anotados. O objetivo do trabalho foi avaliar a função dos leucócitos sangüíneos e o perfil hematológico das tartarugas acometidas ou não. As células receberam estímulo de Saccharomyces cerevisiae e Staphylococcus aureus para avaliação da fagocitose e miristato-acetato de forbol (PMA) para avaliação do burst oxidativo. Foram identificadas três populações celulares: heterófilos, monócitos e linfócitos. Os monócitos foram as células responsáveis pela fagocitose e pelo burst oxidativo. Animais com fibropapilomas apresentaram intensidade de burst oxidativo basal e induzido por PMA maior. No entanto, animais sem fibropapilomas apresentaram maior intensidade de fagocitose (estimulada por Saccharomyces cerevisiae). Foi realizado hemograma completo e a análise estatística demonstrou que houve diferença significativa (p<0,05), entre animais com e sem tumores, apenas para HCM (Hemoglobina Corpuscular Média) e os animais com fibropapilomatose apresentaram a menor média para esse parâmetro. A patogenia ainda é pouco elucidada, fato preocupante porque esta doença acomete muitas tartarugas jovens. Doenças relacionadas à poluição são um fator relevante para a redução populacional de animais e para o desequilíbrio dos ecossistemas. / Chelonia mydas, called green turtle, is a sea turtle that feeds and nests in brazilian coast and is considered endangered (Red List - IUCN, 2006). The fibropapillomatosis, disease characterized as benign cutaneous tumor (fibropapillomas), is a threat to Chelonia mydas. Studies suggest that fibropapillomatosis cause may be an association of virus, environmental and genetic factors. We studied 47 turtles from São Paulo north coast, twenty nine with fibropapillomas and 18 without this disease. The biometry of the turtles, quantity, localization and size of the tumors was registered. The goal of this work was to evaluate the leukocytes function and blood profile of the turtles with and without fibropapillomatosis. The cells were stimulated with Saccharomyces cerevisiae and Staphylococcus aureus to phagocytosis evaluation and phorbol miristate-acetate (PMA) to oxidative burst evaluation. Three populations cells were identified: heterophils, monocytes and lymphocytes. The monocytes were the responsible cells to phagocytosis and oxidative burst. The monocytes of the turtles with fibropapillomas displayed the higher oxidative burst fluorescence intensity (basal and stimulated).However, the monocytes of the turtles without fibropapillomas displayed the higher phagocytosis fluorescence intensity. The blood count and the statistics analysis were significant (p<0,05) only for MCH (Mean Corpuscular Hemoglobin) among turtles with and without fibropapillomas, the animals with fibropapillomatosis showed the minor average. The pathogenesis of fibropapillomatosis is still unclear and is a threat to green turtle survive because this disease affect young green turtles. Diseases related with pollution are a relevant factor for the reduction of animal population and ecosystem unbalance.

Os efeitos de EPA e DHA sobre a função de neutrófilos foram comparados. Para isso, foram realizados experimentos em neutrófilos isolados de ratos. Foram analisadas células com a membrana plasmática íntegra e fragmentação de DNA com o intuito de determinar as concentrações não tóxicas de EPA e DHA. Aumento da produção de peróxido de hidrogênio ocorreu a partir de concentrações menores de DHA (50 <font face=\"Symbol\">mM comparado com 100 <font face=\"Symbol\">mM de EPA). Já para a produção de ânion superóxido, EPA estimulou em doses menores (12.5 <font face=\"Symbol\">mM e o DHA em 100 <font face=\"Symbol\">mM). Ambos AGs aumentaram a síntese e liberação das citocinas CINC-2 e TNF-<font face=\"Symbol\">&#945 e não modificaram a produção de IL1-<font face=\"Symbol\">b e óxido nítrico após incubação das células por 18 horas. Somente DHA elevou a capacidade fagocitária e a atividade fungicida dos neutrófilos. EPA e DHA apresentaram efeitos distintos na produção de citocinas, fagocitose e atividade fungicida dos neutrófilos. Já na produção das EROS, EPA e DHA apresentaram efeitos similares, embora em concentrações diferentes. / The effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) on neutrophil function were compared. For this purpose, experiments were performed in isolated rat neutrophils. Cells with intact plasma membrane and DNA fragmentation were analyzed in order to determine the non-toxic concentrations of EPA and DHA. Production of H2O2 was increased in lower concentrations of DHA (50 <font face=\"Symbol\">mM compared to 100 <font face=\"Symbol\">mM of EPA). For production of O2 -, EPA stimulated at lower doses (12.5 <font face=\"Symbol\">mM compared to 100 <font face=\"Symbol\">mM of DHA). Both FAs increased synthesis and release of cytokines, CINC-2 and TNF-<font face=\"Symbol\">&#945, and did not change the production of IL-1<font face=\"Symbol\">b and nitric oxide after incubation of the cells for 18 hours. Only DHA increased the phagocytic capacity and fungicidal activity of neutrophils. These FAs showed distinct effects on cytokine production, phagocytosis capacity and fungicidal activity by neutrophils. For production of ROS, both EPA and DHA had similar actions, although at different concentrations.

Claritromicina e clofazimina são utilizadas no tratamento da hanseníase e em infecções causadas pelo complexo Mycobacterium avium, comuns em portadores do HIV. Devido à escassez de dados sobre a toxicidade de esquemas terapêuticos que associam estes fármacos, este estudo teve por objetivo avaliar os efeitos adversos desta terapia, em ratos machos Wistar, por meio da determinação de parâmetros hematológicos, hemostáticos e bioquímicos e correlação destes parâmetros com a dose e concentração plasmática dos medicamentos, em regime de doses únicas e múltiplas. Para tanto foram realizados: a) contagem global e específica de leucócitos (método manual) e ensaios de fagocitose e burst oxidativo de neutrófilos (citometria de fluxo); b) contagem de plaquetas (método manual), tempo de protrombina, tempo de tromboplastina parcial ativada, níveis plasmáticos dos fatores VII e X (método automatizado); c) níveis séricos de gama-glutamiltransferase (método cinéticocolorimétrico) e bilirrubinas total e direta (método colorimétrico); d) concentrações plasmáticas dos fármacos (Cromatografia Líquida de Alta Eficiência). Não houve diferenças entre as concentrações plasmáticas dos fármacos administrados em monoterapia ou politerapia. Entretanto, tanto clofazimina como claritromicina tiveram redução das concentrações plasmáticas em regime de doses múltiplas, quando comparadas à dose única. Houve aumento do número de leucócitos (dose múltipla) e de células polimorfonucleares (doses única e múltipla) nos grupos tratados com claritromicina em monoterapia ou associada à clofazimina, e redução das células mononucleares, em doses única e múltipla, nos mesmos grupos. Os fármacos parecem inverter a proporção entre células mono e polimorfonucleares. Observou-se aumento do burst oxidativo nos animais tratados com os fármacos tanto em monoterapia como em regime de politerapia. Entretanto, não houve diferença entre os tratamentos com os fármacos em relação ao controle DMSO, em dose única. Em doses múltiplas, os tratamentos com clofazimina e claritromicina em monoterapia ou politerapia estimularam o aumento do burst oxidativo (p < 0,0001) em relação ao controle DMSO. Não foram verificadas diferenças na fagocitose entre os grupos tratados e controle, tanto em dose única como em doses múltiplas. Tempo de protrombina e tempo de tromboplastina parcial ativada não foram alterados com o uso dos fármacos. Os fatores VII e X da coagulação tiveram aumento de suas atividades quando os ratos foram tratados em regime de dose múltipla com claritromicina, em regime de mono e politerapia. Houve perda de cerca de 8 % do peso de ratos tratados com clofazimina e 18 % daqueles tratados com claritromicina ou com a associação dos dois fármacos, no esquema de doses múltiplas, entretanto não houve diferença entre os grupos quando foram avaliados os níveis de gama-glutamiltransferase e bilirrubinas total e direta. Concluindo, clofazimina e claritromicina provocam alterações hematológicas, hemostáticas e bioquímicas e os resultados de concentração plasmática são valiosos para avaliação de efeitos adversos em estudos comparativos de monoterapia e politerapia entre os medicamentos. / Clarithromycin and clofazimine have been used to treat leprosy and infections caused by Mycobacterium avium complex in HIV patients. Because there are few data about the toxicity of treatment regimens involving these drugs, this study aimed to evaluate the adverse effects of this therapy in male Wistar rats through the determination of hematological, haemostatic and biochemical parameters and correlate them with the dose and plasma concentrations of drugs, under a single and multiple dose regimen. Evaluation was performed as follows: a) Global and specific count of leukocytes (manual method), phagocytosis and oxidative burst of neutrophils assays (flow cytometry), b) platelet count (manual method), prothrombin time, activated partial thromboplastin time, plasma levels of factors VII and X (automated method), c) Gamma-glutamyltransferase (kinetic-colorimetric method) and total and direct bilirubin serum levels (colorimetric method), d) plasma concentrations of drugs (High-Performance Liquid Chromatography). There were no differences between plasma concentrations of the drugs administered in monotherapy or polytherapy. However, the concentrations of both clofazimine and clarithromycin have decreased in plasma in multiple dose regimen compared to single dose. There was an increase in the number of leukocytes (multiple dose) and polymorphonuclear cells (single and multiple doses) in the groups treated with clarithromycin in monotherapy or in association with clofazimine, and a decrease in the number of mononuclear cells in single and multiple doses, in the same groups. Both drugs seemed to reverse the proportion between mononuclear and polymorphonuclear cells. The oxidative burst was observed in animals treated with drugs in polytherapy or in monotherapy, however there was no difference between the treatment with drugs and the control with DMSO in single dose. In multiple doses, treatment with clofazimine and clarithromycin in monotherapy or polytherapy stimulated the increase of oxidative burst (p <0.0001) compared to control. There were no differences in phagocytosis between the treated and control groups in single and multiple doses. Prothrombin time and activated partial thromboplastin time have not changed with the use drugs. In contrast, the activities of factors VII and X of coagulation have increased when rats were treated with multiple doses regimes with clarithromycin alone or in association with clofazimine. There was weight loss of 8% in rats treated with clofazimine and 18% in those treated with clarithromycin or with association of the drugs in the multiple doses regimen. However, there was no difference between the groups when gammaglutamyltransferase and total and direct bilirubin levels were analyzed. Therefore, clofazimine and clarithromycin induce hematological, hemostatic and biochemical changes and the results of plasma concentration is valuable for assessing adverse effects in comparative studies of monotherapy and polytherapy of these drugs.

Overexpression of the enzyme iNOS induces apoptotic cellular death by increasing indices of pro-inflammation and oxidative stress. Aerobic physical activity has been known to have anti- inflammatory benefits and reduce oxidative stress. Purpose: Therefore, this study aimed to examine the impact of aerobic fitness on LPS-induced iNOS mRNA expression and the relationship of this expression with indices of oxidative stress, pro-inflammation and apoptosis in isolated leukocytes. Methods: Whole blood samples from aerobically fit and unfit males were stimulated with and without LPS. Thereafter, iNOS mRNA expression and MDA, TNF-α and p53 concentrations were analyzed. Results: iNOS mRNA expression levels following LPS stimulation were not increased in both groups, and correlational analyses were not consistent with mechanistic predictions. Discussion: Numerous factors including timing of sample quantification, the high level of health of the subject population, and alternative intracellular mechanisms impacting biomarkers analyzed, may have influenced leukocyte iNOS mRNA expression levels.

<p>This thesis describes a new <i>in vitro</i> slide chamber model that makes it possible to conduct studies of molecular and cellular interactions between whole blood and biomaterials. The model proved to be a suitable tool for detection of cell and platelet binding to a biomaterial surface. It was possible to monitor activation of the blood cascade systems and cells in the fluid phase and detect surface-bound molecules.</p><p>One finding was that thrombin generation is primarily triggered by FXII on a biomaterial surface since corn trypsin inhibitor, inhibited thrombin generation in blood.</p><p>Another finding was that thrombin generation was dependent on variety types of blood cells, since thrombin generation was almost negligible in platelet-rich plasma. When various preparations of blood cells were used to reconstitute platelet-rich and platelet-poor plasma, erythrocytes were shown to be the most efficient cell type in triggering thrombin generation. Inhibition of platelet aggregation with aspirin and Ro44-9883 was associated with a decrease in thrombin generation, confirming that platelet activation is necessary for normal coagulation activation. These findings suggest that the central events consist of an initial low-grade generation of thrombin that involves erythrocytes and possibly leukocytes which leads to activation of platelets; and a second platelet-dependent amplification loop that produces most of the thrombin.</p><p>Titanium exposed to whole blood produced high amounts of thrombin. Stainless steel and PVC, generated lower amounts. This indicates that titanium might be less suitable as a biomaterial in devices that are in direct contact with blood for prolonged time. Considering the superior osteointegrating properties of titanium and titanium's response to blood, a correlation between high thrombogenicity and good osteointegration seems to exist.</p><p>Compstatin, that binds to complement component C3, effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/C3 fragments to the surface. Our results suggest that a biomaterial is able to activate complement through both the classical and alternative pathways and that the classical pathway alone is able to maintain a substantial bioincompatibility reaction. The results show that complement activation is a prerequisite for activation and binding of PMNs to the surface in the <i>in vitro</i> model.</p>

The ability of human polymorphonuclear leukocytes (PMNL; neutrophils), to sense and move to sites of infection is essential for our defense against pathogens. Cell motility is critically dependent on a dynamic remodeling of morphology. The morphological polarization toward chemoattractants, such as N-formyl-Met-Leu-Phe (fMLF), is associated with temporary extension and stabilization of lamellipodia in the direction of movement. The underlying mechanisms of cell motility are, however, still not entirely elucidated. It is therefore an urgent task to extend the present experimental evidence to give solid basis for a comprehensive model. Here it is shown that nitric oxide (NO) stimulates the morphological response of neutrophils, most likely due to transient increases in [Ca2+]i, following addition of NO-donors. This will, hypothetically, activate gelsolin and other actin filament severing proteins, leading to a subsequent decrease in filamentous actin. The incapability to efficiently turnover the actin filament network then blocks all motile activity. It is also shown that N-formyl peptide receptors on polarized neutrophils accumulate non-uniformly towards regions involved in motility. It is suggested that neutrophils use the asymmetric receptor distribution for directional sensing and sustained migration. A model for lamellipodium extension, where water fluxes play a pivotal role is presented. It is suggested that water fluxes through water-selective aquaporin (AQP) channels, contribute to the propulsive force for formation of various membrane protrusions and, thus, cell motility. It is well known that small G proteins of the Rho family GTPases play important roles in the intracellular signaling underlying cell motility. In morphologically polarized neutrophils it is shown that Cdc42, Rac2 and RhoA display spatially distinct distributions, which allows for sequential chemoattractant stimulation of neutrophil motility. The specific localizations of Rac2, Cdc42 and RhoA relative to each other and filamentous actin and fMLF receptors support the hypothesized order of activation and regulation of neutrophil cell motility. In conclusion, the detailed analysis of motility-related issues presented here provide new data allowing further refinement of previous models of neutrophil motility.

Type 2 diabetes is caused by defect pancreatic islet β-cells together with peripheral insulin resistance. The disease is often accompanied by obesity with associated low-grade visceral adipose tissue inflammation, which contributes to insulin resistance. As a consequence of, and a possible compensation for the increased insulin demand, blood flow to the pancreatic islets is increased in animal models of diabetes. This increased blood perfusion might with time affect the vascular network as well as β-cells within the islets. This thesis investigates the role of changes of blood perfusion in pancreatic islets and adipose tissues, as well as the recruitment to and composition of leukocyte subpopulations in insulin-sensitive tissues in experimental models of diabetes. Blood flow measurements in islets and adipose tissues of rats and mice were performed using the microsphere technique, while leukocyte recruitment was studied in the mouse cremaster muscle using intravital microscopy. Increased islet blood flow was observed in the GK rat model of type 2 diabetes, which was decreased by acute as well as continuous 2-week inhibition of β3-adrenoceptors without affecting plasma insulin concentrations. Increased inflammatory leukocyte recruitment was observed in both alloxan-induced and high-fat diet-induced diabetes. However, an impaired bacterial clearance was observed in diabetic mice, which was due to impaired phagocytosis. A gender difference was detected in mice fed a high-fat diet, since obese female mice did not show increased levels of pro-inflammatory circulatory markers or inflammatory leukocytes in the adipose tissue. The main effector cell in the adipose tissue inflammation in high-fat-fed male mice seemed to be the pro-inflammatory macrophage. The Treg population in adipose tissue was increased in female mice, but remained unchanged in male mice on high-fat diet. In conclusion, increased islet blood flow in type 2 diabetes could be reversed by β3-adrenoceptor inhibition, which may maintain islet function. The diabetes-associated hyperglycemia activated leukocytes but impaired their phagocytic ability. High-fat-fed female mice showed less peripheral inflammation due to a smaller number of recruited inflammatory macrophages and a high-fat diet-induced Treg population in intra-abdominal adipose tissues.

This thesis describes a new in vitro slide chamber model that makes it possible to conduct studies of molecular and cellular interactions between whole blood and biomaterials. The model proved to be a suitable tool for detection of cell and platelet binding to a biomaterial surface. It was possible to monitor activation of the blood cascade systems and cells in the fluid phase and detect surface-bound molecules. One finding was that thrombin generation is primarily triggered by FXII on a biomaterial surface since corn trypsin inhibitor, inhibited thrombin generation in blood. Another finding was that thrombin generation was dependent on variety types of blood cells, since thrombin generation was almost negligible in platelet-rich plasma. When various preparations of blood cells were used to reconstitute platelet-rich and platelet-poor plasma, erythrocytes were shown to be the most efficient cell type in triggering thrombin generation. Inhibition of platelet aggregation with aspirin and Ro44-9883 was associated with a decrease in thrombin generation, confirming that platelet activation is necessary for normal coagulation activation. These findings suggest that the central events consist of an initial low-grade generation of thrombin that involves erythrocytes and possibly leukocytes which leads to activation of platelets; and a second platelet-dependent amplification loop that produces most of the thrombin. Titanium exposed to whole blood produced high amounts of thrombin. Stainless steel and PVC, generated lower amounts. This indicates that titanium might be less suitable as a biomaterial in devices that are in direct contact with blood for prolonged time. Considering the superior osteointegrating properties of titanium and titanium's response to blood, a correlation between high thrombogenicity and good osteointegration seems to exist. Compstatin, that binds to complement component C3, effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/C3 fragments to the surface. Our results suggest that a biomaterial is able to activate complement through both the classical and alternative pathways and that the classical pathway alone is able to maintain a substantial bioincompatibility reaction. The results show that complement activation is a prerequisite for activation and binding of PMNs to the surface in the in vitro model.