Thesis (M.S.)--Catholic University of America. / Includes bibliographical references.

Platelets play an essential role in haemostasis by adhering to the damaged vessel wall and forming a platelet plug to arrest bleeding. Although platelets are traditionally thought of as pro-coagulant, they possess the ability to harbour functional proteins that are key to fibrinolysis, the breakdown of the blood clot, on their surface. They are therefore substantially well equipped to regulate local fibrinolysis. This thesis aims to further define the role of platelets in fibrinolysis, in particular platelet-derived plasminogen activator inhibitor 1 (PAI-1) and plasminogen. PAI-1 is the principal physiological inhibitor of tissue-type plasminogen activator (tPA), and plasminogen is the zymogen for plasmin. In Chapter 3, we show that platelet-derived PAI-1 is released from platelet α-granules by an αIIbβ3 and fibrin dependent mechanism. We found that a significant portion of α-granular PAI1 is retained on the surface of highly activated PS-positive platelets, and activity analysis revealed the majority of PAI-1 on the platelet surface was in its active form. The functional role of platelet PAI-1 was investigated by analysis of tPA-mediated lysis of Chandler model thrombi. Our data revealed a striking dependence for platelet PAI-1 in stabilising platelet-rich thrombi against degradation. Chapter 4 characterises the expression of a novel transmembrane receptor, Plg-RKT, on the surface of human and mouse platelets. This revealed that plasminogen and Plg-RKT augment one another's binding to the platelet surface. Furthermore, analysis of plasminogen binding to the platelet surface revealed two distinct binding sites: 1) via Plg-RKT and 2) via a fibrin and αIIbβ3 dependent mechanism. Finally, Chapter 5 of this thesis discusses the optimisation of a system that monitors thrombus formation and fibrinolysis under flow. Use of this model will help to further elucidate the complex role that platelets play in controlling the balance between coagulation and fibrinolysis.

Thesis (Ph.D.)--Duquesne University, 2008. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 231-271) and index.

Statins are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol. The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases. Prenyl groups provide a membrane anchor that is essential for the correct membrane localisation and function of these proteins. Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins. Two such proteins are Rab27b and Rap1, small GTPase proteins that are involved in the secretion of platelet granule and integrin activation. We hypothesise that statins can impair prenylation of Rab27b and Rap1a in platelets and thereby attenuate platelet function. The specific aims of the project were to analyse the impact of statins on the prenylation status of Rab27b and Rap1a in platelets. As Rab27b and Rap1a are known to be involved in secretion of platelet granules a secondary aim was to analyse the downstream effects of statins on this process following activation. Finally, we assessed the impact of treatment of platelets with statins on thrombus formation, stability and resistance to fibrinolysis. Platelets incubated with statins overnight were separated into cytosolic (aqueous) and membrane (detergent) components and visualised by Western blot. An accumulation of Rab27b and Rap1a was observed in the cytosolic compartments of statins treated platelets compared to untreated platelets, thus indicating indirect evidence that statins attenuate prenylation of Rab27b and Rap1a in platelets. The most effective statin in attenuating prenylation of Rab27b and Rap1a was atorvastatin (ATV). The inhibitory effect of statins on prenylation was recovered by GGPP, indicating that the mechanism of inhibition involved the mevalonate pathway. Release of ADP from platelet dense granules was significantly impeded following overnight treatment with ATV. In line with the inhibition of prenylation of Rab27b and Rap1a by ATV, addition of GGPP rescued the release of ADP from platelet dense granules. This suggests that attenuation of dense granules release by ATV occurs via interference in the mevalonate pathway and the inhibition of Rab27b prenylation. Furthermore, ATV significantly attenuates α-granules release in thrombin stimulated platelets, which was visualised as impaired accumulation of endogenous P-selectin, PAI-1 and fibrinogen on the activated membrane. Changes in the activation of α₁₁bβ₃ integrin on the stimulated platelet surface, observed as defective binding of exogenous fibrinogen and PAC-1, were also evident following treatment of platelets with ATV. In addition, ATV treatment of platelets reduced binding of CD41a, indicating that the copy number and activation of α₁₁bβ₃ integrin on stimulated platelets was significantly reduced. Statins were also found to significantly inhibit thrombin-induced platelet aggregation following incubation of platelets overnight with therapeutic concentrations of statins. Surprisingly GGPP did not rescue platelet aggregation indicating that different mechanisms are involved in inhibition of platelet responses by statins. Incubation of whole blood with ATV overnight significantly altered several haemostatic parameters. Using thromboelastography we demonstrated a delay in the coagulation time and clot formation time. Maximum clot firmness was also significantly reduced in the presence of statins compared to the control. The effect on clot firmness generally arises from platelet dysfunction and/or a change in fibrinogen concentration and function; the latter was ruled out using a Fibtem test, which shows no difference between treated and untreated whole blood. Similarly, formation of platelet-rich plasma clots was significantly delayed following pre-treatment with ATV overnight. These clots also exhibited lower maximal absorbances, which could represent differences in the fibrin network structure. In line with the reduction in fibrinogen binding defective clot retraction was also observed in platelet-rich plasma pre-treated with ATV overnight. Similar clot retraction results were observed with tirofiban and CytoD, suggesting that the inhibitory effect of ATV may involve modulation of α₁₁bβ₃ integrin activation. Platelet-rich plasma clots formed post-treatment with statins were visualised by confocal microscopy and revealed significant alterations in clot structure; observed as thinner fibrin fibres and fewer platelet aggregates. Additionally, we demonstrated that statins modulate clot stability and shorten time to lysis. Clots formed from platelet rich plasma that was subjected to incubation with ATV overnight revealed faster lysis by tPA compared to the absence of statin. These findings are also in agreement with the lysis of Chandler model thrombi formed from overnight incubated whole blood with ATV, which demonstrated faster lysis rate mediated by tPA. Furthermore, statins were shown to change the clot thrombodynamics as assessed by HemaCore analyser, which shows that stains implicate both clot growth in response to TF-coated comb and spontaneous clot lysis by tPA. In conclusion, statins directly inhibit Rab27b and Rap1a prenylation in platelets and down-regulated dense granules release. Inhibition of Rab27b and Rap1a prenylation, and dense granules release was recovered by GGPP, indicating that these effects are mediated through the mevalonate pathway. Impairment of platelet aggregation by statins resulted via multiple mechanisms as GGPP did not recovered the inhibition of aggregation by ATV. Statins also modulate fibrinogen binding, α-granules release, clot retraction and clot formation and stability in vitro. Together these results suggest that statins may directly attenuate the platelet response in vivo. The pleotropic effect of statins on platelets may contribute to the protective function of these class of drugs in cardiovascular diseases.

Thesis (M.S.)--University of Michigan, 1995. / Thesis date on spine.

Thesis (M.S.)--University of Michigan, 1995. / Thesis date on spine.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

Orientador: Gilberto de Nucci / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T10:02:10Z (GMT). No. of bitstreams: 1 AbibJunior_Eduardo_D.pdf: 639040 bytes, checksum: e96fa4a810e2980947816b23f3c078a1 (MD5) Previous issue date: 2005 / Resumo: Objetivo: Esta tese tem por objetivos: avaliar a agregação plaquetária e o comportamento da pressão arterial em três momentos (sem alho; alho dose única (3,5 g) e alho dose diária (3,5 g) duas vezes ao dia por 4 dias) em voluntários sadios; Analisar a resposta de agregação plaquetária in vitro adicionando extrato de alho diluído em PRP e por ultimo correlacionar os dados obtidos da analise da agregação com os parâmetros TxB2, GMPc entre in vivo e in vivo. Para Analise em in vivo foram selecionados dezoito (18) voluntários do sexo masculino, entre 18 a 45 anos, saudáveis, para estudo não randomizado, aberto e divididos em tres grupos (Grupo Sem alho; Grupo Com Alho Único e Grupo Alho Diário). Amostras de sangue dos voluntários foram coletadas de acordo com horários pré-estabelecidos. Após execução da agregação plaquetária, Pressão arterial através da MAPA e quantificação dos níveis de TXB2, foram realizadas análises estatisticas. Para analise in vitro foram selecionados 5 voluntários sadios, de ambos os sexos, isentos de qualquer medicação uma semana antes coleta. O sangue foi coletado e o PRP foi separado e adicionado extrato de alho em volume determinado. Após execução da agregação plaquetária e quantificação dos níveis de TXB2, foram tb realizadas análises estatisticas. Tendo estes dados tanto in vivo quanto in vivo procedeu-se a analise comparativa entre eles. Resultados : Na analise in vivo, tanto a agregação plaquetária quanto a inibição da formação de TxB2 não se observou diferença entre os outros grupos independente do agonista utilizado. Na analise in vitro, os resultados sugeriram que o extrato de alho, em quantidades pequenas, inibi a agregação plaquetária Os resultados se confirmaram com o TXB2, pois quantidades de extrato que foram capazes de inibir a agregação plaquetária induzida por todos agonistas, inclusive àquela induzida por AA, não causou diminuição significativa da síntese de TXA2 induzida por AA. Houve variação significativa da PA sistólica e FC com administração diária de alho fresco comparada ao sem alho e alho único. Conclusão: Concluímos que outros mecanismos podem estar envolvidos na inibição da agregação plaquetária que não da inibição da ciclooxigenase plaquetária quando utilizado o extrato de alho. Não há uma inibição da agregação plaquetária através da ação sobre a ciclooxigenase quando observado em voluntários que ingeriram alho fresco. A administração de alho in natura, pequenas quantidades (3,5g de dente de alho = 16 mg alicina/g de alho) pode contribuir para promover alterações no comportamento hemodinâmico como observado através da MAPA em voluntários sadios / Abstract: Objective: This thesis has as objectives: to evaluate the platelet aggregation and the behavior of blood pressure in three moments (control; garlic single dose (3,5 g) and garlic daily dose (3,5 g) twice a day for 4 days) in healthy volunteers; To analyze the in vitro platelet aggregation answer adding garlic extract diluted in PRP and the last to correlate the obtained data from the aggregation analysis with the TxB2, GMPc parameters between in vivo and in vivo. For the in vivo Analysis eighteen (18) healthy volunteers of the masculine gender between 18 and 45 years old were selected, for an open, non-randomized study and divided into three groups (Control; Group With Single Garlic and Group Daily Garlic). Samples of the volunteers' blood were collected according to the pre-established schedules. After execution of the platelet aggregation, blood Pressure through AMBP and quantification of TXB2 levels , statistical analyses were accomplished. For in vitro analysis 5 healthy volunteers of both genders were selected, free of any medication one week before collection. The blood was collected and the PRP was separated and added garlic extract in determined volume. After execution of the platelet aggregation and quantification of TXB2 levels, statistical analyses were also accomplished. Having these in vivo data as well in in vivo the comparative analysis between them was preceeded. Results: There was significant variation of the systolic BP and HR with daily administration of fresh garlic compared to control and single garlic. Regarding the platelet aggregation it was observed difference between the daily garlic group and the other two groups (P <0.005) when used agonist arachidonic acid. In the in vitro analysis, the results suggested that the garlic extract, in small amounts, can inhibit the platelet aggregation without affecting in a significant way the activity of ciclooxygenase. The results were confirmed with the TXB2, for amounts of extract that were capable to inhibit the platelet aggregation induced by all agonists, including that one induced by AA, didn't cause significant decrease of TXA2 synthesis induced by AA. Conclusion: We concluded that other mechanisms can be involved in the inhibition of the platelet aggregation other than the inhibition of the platelet ciclooxygenase when used the garlic extract. There is not an inhibition of the platelet aggregation through the action on the ciclooxygenase when observed in volunteers that ingested fresh garlic. The administration of garlic in natura, small quantities (3,5g garlic glove = 16 mg allicim/g garlic) can contribute to promote alterations in the hemodynamic behavior as observed through the AMBP in healthy volunteers / Doutorado / Clinica Medica / Doutor em Clínica Médica