Introdução: O consumo habitual de refeições ricas em gordura tem se mostrado indutor de doenças cardiovasculares (DCV), afetando o equilíbrio entre os sistemas de coagulação e fibrinólise e também induzindo o aumento de marcadores inflamatórios. Por outro lado, o exercício físico tem sido indicado como intervenção por atenuar o incremento da inflamação e equilibrar os sistemas hemostáticos em indivíduos que consomem uma refeição hiperlipídica (RH). Objetivo: Verificar o efeito subagudo de duas sessões com intensidades diferentes de exercício aeróbio na curva lipêmica, inflamação, hemostasia em sujeitos jovens saudáveis submetidos à refeição hiperlipídica. Metodologia: Onze sujeitos eutróficos do sexo masculino, fisicamente ativos, com idade média de 23 ± 3 anos participaram do estudo que foi composto por três protocolos com dois dias consecutivos cada. No dia 1 os sujeitos realizavam um dos três protocolos que era realizado de forma randomizada, os protocolos eram divididos em: exercício de baixa intensidade (BI), exercício de moderada intensidade (MI) e repouso (Con). No dia dois 12h após a realização do exercício prévio os sujeitos consumiam uma RH (15% proteínas, 35% carboidratos e 50% lipídeos). Foram realizadas coletas de sangue para analise de triglicerídeos (TG), Colesterol total (CT), lipoproteínas de alta densidade (HDL), lipoproteínas de baixa densidade (HDL) e Glicose, no basal (BS) e a cada hora de 1 à 5h após a RH. As coletas sanguíneas para análise de Ativador de plasminogênio (tPA), Inibidor do ativador de plasminogênio do tipo 1 (PAI-1), Fator de necrose tumoral alfa (TNFα), Interleucina 6 (IL-6) e Interleucina 10 (IL-10) foram realizadas no momento basal, 1h, 3h e 5h após a RH. Resultados: Os protocolos BI e MI apresentaram menor área abaixo da curva (AUC) de TG em relação ao Con (P<0,05). Houve diferença significativa no PAI-1 em relação ao BI quando comparado ao MI e Con e de tPA do protocolo BI em relação ao Con no momento 1h pós refeição (P<0,05). No FVII, os protocolos MI e BI foram significativamente menores que o Con no momento 1h pós RH (p<0,05). Houve diferença significativa em TNFα entre os protocolos MI e Con no momento 1h pós RH (P<0,05) e foram encontradas diferenças em IL-10 nos protocolos MI e Con nos momentos 1h e entre os protocolos MI e BI nos momentos 1h, 3h e 5h pós RH (P<0,05). Houve diferença em IL-6 em todos os momentos de todos os protocolos em relação ao momento basal (BS). Conclusão: A RH aumenta o estado inflamatório e desregula o equilíbrio entre coagulação e fibrinólise, o protocolo BI e MI atenuam a curva de TG em relação ao Con, o protocolo MI melhorou o estado inflamatório diminuindo TNFα e incrementado IL-10 e o protocolo BI melhorou a relação entre coagulação e fibrinólise atenuando a diminuição de tPA e diminuindo o incremento de PAI-1 e ambos os protocolos MI e BI não incrementaram FVII 1h após RH. / Background: Regular consumption of high-fat meals has been considered to play a role in the development of cardiovascular diseases. The increase of postprandial lipemia after a high-fat meal consumption can imbalance the relationship between coagulation and fibrinolysis and, by consequence, enhance an inflammatory response. Conversely, exercise has been considered an important intervention, once it may attenuate inflammatory responses and counterbalance hemostatic systems during the postprandial period. Purpose: Verify the subacute effect of two exercise bouts performed at different intensities on postprandial lipemia, inflammation and hemostasis after the consumption of a high-fat meal. Methods: Eleven healthy and physically active male subjects with average age of 23 ± 3 years completed 2-day trials in three conditions: Control, low-intensity exercise (LI) and moderate-intensity exercise (MI). Subjects performed an exercise bout (LI or MI) or no exercise (Control) on the evening of day 1. On the morning of day 2, a high-fat meal was provided (15 % of protein, 35 % of carbohydrates and 50 % of lipids). Blood was sampled at fasting (0 h) and every hour from 1 to 5 h for triglycerides (TG), total cholesterol, HDL, LDL and glucose. For plasminogen activator inhibitor-1 (PAI-1), plasminogen activator (tPA), tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6) and interleukin-10 (IL- 10), blood was sampled at 0, 1, 3 and 5 h. Results: TG area under the curve (AUC) was lower in LI and MI than Control (P<0.05). For PAI-1, there was a difference from LI to MI and Control at 1 h (P<0.05). For tPA, there was a difference from LI to Control at 1 h (P<0.05). For FVII the protocols MI and BI there was difference from Con in at 1h. For TNFα, there was a difference from MI to Control at 1 h (P<0.05). IL-10 concentration was different from MI to Control at 1 h and from MI to LI at 1, 3 and 5 h (P<0.05). Fasting IL-6 concentrations were different between all conditions (P<0.05). Conclusion: The consumption of a high-fat meal increases the inflammatory process and deregulates the balance between coagulation and fibrinolysis. Exercise, independent of the intensity, can reduce TG AUC compared to Control. MI can reduce TNFα and increases IL-10, while LI regulates coagulation and fibrinolysis balance, which can be explained by the increase in tPA and increase in PAI-1.

ADAMTS13 is a metalloprotease that regulates the length, and thus, the platelet-capturing capacity of von Willebrand factor. The regulation of ADAMTS13 activity remains poorly understood. Numerous circulating proteases cleave ADAMTS13 in vitro, impairing its activity, but the physiological significance of this mechanism remains unknown. Two commonly cleaved regions within ADAMTS13 were identified and mutants were developed: two with one of each region mutated (T4L and T8L mutants), one with both regions mutated (T4L/T8L or “double” mutant), and one with an additional elastase site mutated (T4L/T8L + I380G). This work characterizes the mutants’ resistance to proteolysis and compares the activity of the double mutant to wild-type ADAMTS13 (WT). Each mutant and WT was incubated with purified coagulation and neutrophil proteases, activated neutrophils, or added to plasma before initiating coagulation with or without tissue plasminogen activator. Cleavage patterns were visualized with western blot. FRETS-VWF73 and microfluidic flow assays were used to compare WT and mutant activity. Coagulation proteases cleave both predicted sites within WT, and the double mutant exhibits near complete resistance to cleavage over 3 hours. Resistance to degradation by neutrophil proteases is prolonged in the double mutant, but additional cleavage sites are present. Elastase cleavage is prevented in the T4L/T8L + I380G mutant. In plasma, WT is degraded upon initiating coagulation and subsequent fibrinolysis, which is prevented in the double mutant. WT is also degraded in the presence of activated neutrophils, and the double and T4L/T8L + I380G mutants exhibit improved but incomplete resistance. Finally, the mutants exhibit similar activity to WT using FRETS-VWF73 and the microfluidic assay. This work validates the location of two protease-sensitive regions within ADAMTS13 and confirms the resistance of the double mutant to coagulation proteases in vitro. Future work will complete the activity analysis, and compare the mutants’ therapeutic efficacy to WT in vivo. / Thesis / Master of Science (MSc) / Current drugs used to dissolve blood clots can cause major bleeding. Therefore, safer treatments need to be developed. An important step in the clotting pathway is platelet accumulation in the injured vessel. Platelets stick to string-like protein, von Willebrand Factor (VWF), and ADAMTS13 is a protein that regulates this by cutting VWF strings. ADAMTS13 shows promise as a treatment for clots without causing bleeding, but it is unclear how its activity is controlled. ADAMTS13 can be degraded by other proteins, however the importance of this process in the body is unknown. This work characterizes a degradation-resistant ADAMTS13 mutant, which may be used to study whether ADAMTS13 degradation reduces its therapeutic effectiveness. The mutant has normal VWF-cutting activity, is resistant to degradation by clotting proteins, and is partially resistant to proteins released by neutrophils, an important immune cell in clotting. Future studies will investigate its effectiveness at treating clots in animals.

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c-Cbl protein functions as an E3 ligase and scaffolding protein, where three residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses upon integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet spreading, suggesting a differential role of these tyrosine residues. The physiological role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-CblYF/YF knock-in mice. c-Cbl KO and c-Cbl YF/YF platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl YF/YF platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet spreading and clot retraction / Physiology

Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a large homopolymeric glycoprotein that is synthesized and stored by platelets and endothelial cells until activation-induced release. MMRN1 is able to support platelet adhesion through mechanisms involving von Willebrand factor (VWF) and glycoprotein (GP)Ibα, and β3 integrins on activated platelets, and it enhances platelet adhesion to fibrillar collagen, potentially by binding to putative MMRN1-specific GPAGPOGPX (where O is hydroxyproline and X is valine or glutamine) motifs in fibrillar collagens. Using mice with and without selective Mmrn1 deficiency, the goals of this thesis were: 1) further characterize the ability of Mmrn1 to enhance platelet adhesion to collagen, 2) explore the role of fluid shear stress in the ability of Mmrn1 to enhance platelet adhesion, and 3) test the specificity of the GPAGPOGPX motif for Mmrn1 and the ability of GPAGPOGPX to support or enhance platelet adhesion. Mmrn1-deficient (Mmrn1-/-) mouse platelets showed impaired aggregate formation on fibrillar collagen surfaces under high (1500 s-1) and low (300 s-1) shear flow compared to wild-type (Mmrn1+/+) mouse platelets, which was due to reduced initial adhesion and a slower rate of platelet accumulation onto collagen surfaces. Similarly, Mmrn1-/- platelets formed smaller aggregates on immobilized recombinant (r)Vwf surfaces compared to wild-type platelets, and Mmrn1-/- platelets had impaired adhesion and aggregate formation on immobilized murine fibrinogen, but not fibrin, when platelets were pre-activated to release Mmrn1. Type I fibrillar collagen was found to contain a variant of the GPAGPOGPX motif (GPAGPOGPI), and GPAGPOGPX motifs supported adhesion of wild-type, but not Mmrn1-/-, platelets. When presented with the VWF-binding GPRGQOGVMGFO motif and the integrin α2β1-binding GFOGER motif present in fibrillar collagens, the GPAGPOGPX motifs synergistically enhanced platelet adhesion. These findings expand upon the known adhesive functions of platelet multimerin 1 and update knowledge of the motifs that support platelet adhesion to fibrillar collagens. / Dissertation / Doctor of Philosophy (Medical Science)