Atherosclerosis is a major cause of mortality in industrialized nations. Atherosclerosis is characterized by plaque deposition which decreases the lumen diameter into a stenosis. The creation of a restriction increases shear rates pathologic levels exceeding 3,500/s. Following plaque cap rupture, thrombus may form from the accumulation of millions of platelets, occluding the vessel, leading to heart attack and stroke. Studies of high shear thrombosis show that platelet activation, GPIIb/IIIa and vWF are involved. However, some recent studies also suggest that high shear aggregation is not dependent on activation or GPIIb/IIIa. Several antiplatelet pharmaceuticals against activation and GPIIb/IIIa have been proposed, but their efficacy in patients remains mixed. The overall objective of this project is to determine the factors necessary for thrombosis to occlusion in very high shear regions seen in diseased arteries. Our central hypotheses are that platelet activation and the subsequent conformational change in GPIIb/IIIa are necessary for thrombosis, and that higher concentrations of vWF in the plasma will increase thrombosis. To this end, we developed a new high shear hemodynamic model utilizing 30mLs of whole blood and quantified thrombus thickness, volume accumulation and accumulation rates. We demonstrate that thrombosis to occlusion stems from a second phase of Rapid Platelet Accumulation (RPA). Thrombus accumulation is completely prevented by PGE1 inhibition of platelet activation. Similarly, GPIIb/IIIa blockade via abciximab prevented significant thrombus deposition and RPA. We also found that increasing plasma vWF levels in high shear regions increased thrombus thickness and suggestively increased RPA rates. The results clarify the need for activation of mural platelets for long term thrombus accumulation without the activation of circulating platelets.

Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 1977 / Includes bibliographical references. / by Frederick H. Silver. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Mechanical Engineering

Understanding the differential bonding mechanics underlying bleeding disorders is of crucial importance to human health. In this research insight is provided into how four of these bleeding disorders (each with somewhat similar clinical characteristics), work at the molecular bond level. The bleeding diseases studied here can result from defects in the platelet glycoprotein (GP) Ibα the von Willebrand factor (vWF) molecule, or the ADAMTS-13 enzyme. Types 2B and 2M von Willebrand Disease (VWD) result in excess bleeding, yet type 2B has increased binding affinity between platelet GPIbα and vWF, while type 2M has decreased binding affinity between these two molecules. Platelet type VWD (pt-VWD) causes mutations in the GPIbα molecule and has similar characteristics to type 2B VWD. Further, in thrombotic thrombocytopenic purpura, bleeding results when there is a lack of active ADAMTS-13 enzyme. Each disease results in patient bleeding, but due to different mechanisms. This dissertation will explore the bonding mechanics between GPIbα and vWF and how they are altered in each disease state. To observe the GPIbα-vWF bonding mechanics, rolling velocities, transient tethering lifetimes, and tether frequency were determined using a parallel plate flow chamber. Data from these experiments suggest that wt-wt interactions are force dependent and have biphasic catch-slip bonding behavior. The data show that the shear stress at which the maximum mean stop time occurs differs between gain-of-function and loss-of-function mutations. Using similar methods, we study the changes resulting from pt-VWD mutations in GPIbα, and find that the catch bond seen for wt-wt interactions is lost for these mutations. Further, the data suggest that interactions with gain-of-function GPIbα mutations may be transport rather than force dependent. Finally, how the GPIbα-vWF tether bond changes for thrombotic thrombocytopenic purpura was also investigated to show that the bond lifetime in the absence of the enzyme is increased presenting a possible rationale for why bleeding occurs in this disease. Overall, the data show how the bonding mechanics of the GPIbα-vWF tether bond differ in four bleeding diseases. Further, these observations offer potential explanations for how these changes in the bonding mechanism may play a role in the observed patient bleeding.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disease that affects mostly women in reproductive age and sometimes cause permanent damage. The development of cardiovascular disease, especially atherosclerosis, is the leading cause of death in SLE. However, its etiology and relationship with the development of atherosclerosis remain unknown. Several studies have shown that platelets have relevant properties to thrombogenesis, such as the release of ADP, a molecule capable of inducing platelet aggregation. Adenosine, derived from the hydrolysis of ATP and ADP, in turn has antiagreggant properties. The control of extracellular levels theses molecules and subsequent purinergic signaling induced by them is carried out by enzymes NTPDase, E-NPP, 5'-nucleotidase and ADA. Therefore, the objective of this study was to evaluate the activity of these ectonucleotidases in platelets and platelet aggregation profile in patients with LES. An increase in the activity of the enzymes NTPDase, E-NPP, 5'-nucleotidase and ADA was observed in platelets of patients with lupus compared with control subjects. No difference on platelet aggregation of patients with lupus was observed when compared to control. The increase in activity of E-NTPDase, E-NPP and 5'-nucleotidase seems to be a compensatory organic response against the pathological condition, to generate higher concentration of adenosine. But the ADA activity also is increased in platelets of patients with SLE and may decrease the concentration of adenosine, which favors prothrombotics process. Thus, the results suggest that the ectoenzymes may be involved in the modulation of atherosclerotic processes that occur in SLE. / O lúpus Eritematoso Sistêmico é uma doença crônica, inflamatória e sistêmica, que afeta principalmente mulheres em idade reprodutiva e desencadeia danos permanentes na maioria dos casos. O desenvolvimento de doenças cardiovasculares, especialmente aterosclerose, é a principal causa de morte em pacientes lúpicos. Entretanto sua etiologia e relação com o desenvolvimento de aterosclerose permanecem desconhecidos. Vários estudos demonstraram que as plaquetas apresentam propriedades relevantes na trombogênese, como a liberação de ADP, uma molécula capaz de induzir a agregação plaquetária. A adenosina, proveniente da hidrólise do ATP e ADP, por sua vez apresenta propriedades antiagregantes. O controle dos níveis extracelulares destas moléculas e a consequente sinalização purinérgica por elas induzidas é realizado pelas enzimas NTPDase, E-NPP, 5'-nucleotidase e ADA. Sendo assim, o objetivo deste trabalho foi avaliar a atividade destas ectonucleotidases em plaquetas bem como o perfil da agregação plaquetária em pacientes lúpicos. Um aumento na atividade das enzimas NTPDase, E-NPP, 5'-nucleotidase e ADA foi observado em plaquetas de pacientes com LES, quando comparado com sujeitos controles. Nenhuma diferença no perfil de agregação plaquetária de pacientes lúpicas foi observada, quando comparado ao controle. O aumento na atividade da NTPDase, da E-NPP e da 5'-nucleotidase parece ser uma resposta compensatória orgânica frente ao estado patológico para gerar maior concentração de adenosina. Porém a atividade da ADA também encontra-se aumentada em plaquetas de pacientes com diagnóstico de LES, podendo diminuir a concentração de adenosina, o que favorece estágios prótrombóticos. Desse modo, os resultados encontrados sugerem que as ectoenzimas podem estar envolvidas na modulação dos processos ateroscleróticos que ocorrem no LES.