Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 101-109).

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2006. / Härtill 5 uppsatser.

Disabled-2 (Dab2) is an adaptor protein that plays critical roles in various biological processes, including protein endocytosis, platelet activation and aggregation, tumor growth, and development. In platelets, Dab2 associates with membrane sulfatide at the platelet surface, modulating platelet inside-out and outside-in signaling pathways. A Dab2-derived peptide, named the sulfatide-binding peptide (SBP), is the minimal unit of Dab2 to exert its function as a negative regulator of platelet activation and aggregation. The work of this thesis refines the model of Dab2 SBP binding to sulfatide and provides structural and functional insights into the mechanism by which Dab2 SBP modulates platelet activation. Using molecular docking, lipid-protein overlay assay, nuclear magnetic resonance, and surface plasma resonance tools, this work identifies the critical residues within two major regions responsible for sulfatide interaction. First, docking a sulfatide to Dab2 SBP, a hydrophilic region, primarily mediated by Arg42, is thought to be responsible for the association with the sulfatide headgroup. We observed that Arg 42 could directly interact with sulfatide by forming hydrogen bonds with the OS atoms in the sulfatide head group. Further lipid-protein overlay assay and surface plasma resonance experiments confirmed that both the positive charge and stereochemistry of the side chain of Dab2 SBP Arg42 are required for the sulfatide binding. Moreover, Arg42 is found to be critical in the inhibition of P-selectin expression on activated platelets. The residues nearby Arg42 (i.e., Glu33, Ty38, and Lys 44) also contribute to sulfatide interaction. Second, the second polybasic motif located at the C-terminal -helix 2 is considered to interact with the acyl chain through hydrophobic interactions rather than direct binding to the charged sulfatide head group. Lysine residues in this region are suggested to exert a dual role in sulfatide association, that is, by favoring electrostatic interactions with the negatively-charged sulfatide and/or by employing their flexible hydrocarbon spacers for hydrophobic interactions with membrane lipids. Consistent with this suggestion, we found a hydrophobic patch in the wild type Dab2 SBP structure surrounded by Lys49, Lys51, and Lys53. Furthermore, the role of the second sulfatide binding motif in sulfatide binding is confirmed by mutagenesis analysis and lipid-protein overlay assays, highlighting the ability of molecular docking to accurately predict critical residues responsible for sulfatide binding. In summary, this work provides a detailed structural basis for Dab2 recognition by sulfatide through multiple biophysical methods. The corresponding biological implications in the inhibition of platelet activation are also evaluated by flow cytometry. By elucidating the underlying mechanisms of Dab2 mediating platelet activation through sulfatide binding, we provided structural and functional insights for designing a Dab2-derived peptide with altered sulfatide recognition features in platelets, which can be further employed in antiplatelet therapy. / Doctor of Philosophy / Platelets are blood cells that are fundamentally intended to help form clots to stop bleeding. They do so by being activated after getting signals from damaged blood vessels and reaching the injury site. Consequently, they form aggregates by attracting more platelets to clump on the clot. However, platelet activation induced by a tumor cell can, in turn, protect the tumor cell from immune system elimination and facilitates their growth and spread. This platelet-tumor complex formation suggests platelets as a therapeutic target for reducing tumor migration out of the bloodstream. Our study investigates the mechanism of a Disabled-2-derived peptide, named Dab2 SBP, which upon binding to a sulfatide lipid, can reduce the platelet activation extent, using molecular and cellular approaches. The results of this study may be instrumental in the generation of Dab2 SBP-derived peptides with altered sulfatide binding ability and selectivity, which may lead to a design of an antiplatelet drug that can limit the ability of tumor cells to invade other tissues.

No description available.

According to the World Health Organization (WHO), Cardiovascular Disease (CVD) is the leading cause of death in the world. Biomechanics and fluid dynamics of blood flow play an important role in CVD mediation. Shear stress plays a major role in platelet-substrate interactions and thrombus formation and growth in blood flow, where under both pathological and physiological conditions platelet adhesion and accumulation occur. In this study, a three-dimensional dynamic model of platelet-rich thrombus growth in stenosed vessels using computational fluid dynamics (CFD) methods is introduced. Platelet adhesion, aggregation and activation kinetics are modeled by solving mass transport equations for blood components involved in thrombosis. The model was first verified under three different shear conditions and at two heparin levels. Three-dimensional simulations were then carried out to evaluate the performance of the model for severely damaged (stripped) aortas with mild and severe stenosis degrees. For these cases, linear shear-dependent functions were developed for platelet-surface and platelet-platelet adhesion rates. It was confirmed that the platelet adhesion rate is not only a function of Reynolds number (or wall shear rate) but also the stenosis severity of the vessel. General correlations for adhesion rates of platelets as functions of stenosis and Reynolds number were obtained based on these cases. The model was applied to different experimental systems and shown to agree well with measured platelet deposition. Then, the Arbitrary Lagrangian Eulerian (ALE) formulation was used to model dynamic growth by including geometry change in the simulation procedure. The wall boundaries were discretely moved based on the amount of platelet deposition that occurs on the vessel wall. To emulate the dynamic behavior of platelet adhesion kinetics during thrombus growth, the validated model for platelet adhesion, which calculates platelet-surface adhesion rates as a function of stenosis severity and Reynolds number, was applied to the model. The model successfully predicts the nonlinear growth of thrombi in the stenosed area. These simulations provide a useful guide to understand the effect of growing thrombus on platelet deposition rate, platelet activation kinetics and occurrence of thromboembolism (TE) in highly stenosed arteries. / Ph. D. / Continuous platelet deposition in coronary arteries creates a narrow necking area at some susceptible regions such as bifurcations, stented arteries and ruptured vessel walls. These narrow regions, known as stenoses, are the number one cause of heart attacks. In this work, a predictive model of platelet deposition (i.e. thrombosis) is developed based on previous experimental and clinical data on human blood. Fluid shear stresses play a major role in platelet-substrate interactions and thrombus formation and growth in blood flow, where under both pathological and physiological conditions platelet adhesion and accumulation occur. In addition to simulating the blood flow patterns in arteries with computational fluid dynamics (CFD), the model is able to reliably predict the amount of platelets deposited in injured areas, the severity of the blockage in the blood flow, and the time for occlusion. The results of our model are much more accurate than previous models and are validated by comparing them to clinical data for thrombus formation in stenosed arteries. Thus, the project contributes towards better diagnosis and treatment of vascular disease with implications on the monitoring and management of cardiovascular diseases and providing useful guidelines to design improved devices such as left ventricular assist devices, mechanical heart valves, stents, etc.

Oxidative damage has been suggested to play a key role in the pathogenesis of atherosclerosis and other cardiovascular disease. Increased free radical production induced by oxidative stress can oxidise low density lipoproteins, activates platelets, induces endothelial dysfunction and disturbs glucose transport by consuming endogenous antioxidants. Using a combination, of in vitro and in vivo experimental models, the primary aims of the studies undertaken for this thesis were to examine whether different antioxidants could negate risk factors leading to thrombosis, atherosclerosis and other cardiovascular diseases. The studies utilised the mechanisms involved in platelet activity and glucose uptake by skeletal muscle myotubes. The first study determined if olive leaf extract would attenuate platelet activity in healthy human subjects. Blood samples (n=11) were treated with five different concentrations of extract of Olea europaea L. leaves ranging from 5.4£gg/mL to 54£gg/mL. A significant reduction in platelet activity (pless than0.001) and ATP release from platelets (p=0.02) was observed with 54£gg/mL olive leaf extract. The next crossover study compared the effect of exercise and antioxidant supplementation on platelet function between trained and sedentary individuals. An acute bout of 1 hour exercise (sub maximal cycling at 70% of VO2max) was used to induce oxidative stress in 8 trained and 8 sedentary male subjects, before and after one week supplementation with 236 mg/day of cocoa polyphenols. Baseline platelet count and ATP release increased significantly (pless than0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher than in the sedentary (235¡Ó37 vs. 208¡Ó34 x109/L, p less than 0.05), whereas platelet activation in trained subjects was lower than sedentary individuals (51¡Ó6 vs. 59¡Ó5%, p less than0.05). Seven days of cocoa polyphenol supplementation did not change platelet activity compared to the placebo group. The third study determined the effect of 5 weeks of either 100mg/day £^-Tocopherol (n=14), 200mg/d £^-Tocopherol (n=13) or placebo (n=12) on platelet function, lipid profile and the inflammatory marker C-reactive protein. Blood £^-tocopherol concentrations increased significantly (pless than0.05) relative to dose. Both doses attenuated platelet activation (pless than0.05). LDL cholesterol, platelet aggregation and mean platelet volume were decreased by 100mg/d £^-tocopherol (all pless than0.05). The final study determined the effect of glucose oxidase induced oxidative stress and £^-tocopherol treatment on glucose transport and insulin signalling in cultured rat L6 muscle cells. One hour treatment with 100mU/mL glucose oxidase significantly decreased glucose uptake both with and without 100nM insulin stimulation (pless than0.05). Pre-treatment with 100ƒÝM and 200ƒÝM £^-tocopherol partially protected cells from the effect of glucose oxidase, whereas 200ƒÝM £^-tocopherol restored both basal and insulin stimulated glucose transport to control levels. Glucose oxidase-induced oxidative stress did not impair basal or insulin stimulated phosphorylation of Akt or AS160, but 200ƒÝM £^-tocopherol improved insulin-stimulated phosphorylation of these proteins. In summary, the results from the studies undertaken for this thesis provide evidence that antioxidant supplementation maintains normal platelet function, exerts a positive effect on blood lipid profile and improves glucose uptake in normal healthy asymptomatic population as well as under conditions of induced oxidative stress. Antioxidants including foods rich in cocoa, olive and gamma tocopherol have the potential to combat oxidative stress induced risk factors leading to cardiovascular diseases.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2005. / Härtill 5 uppsatser.