Dissertations / Theses on the topic 'Platelet dynamics'

This work describes the development and testing of a real-time three-dimensional computational fluid dynamics simulation of thrombosis and embolization to be used in the design of blood-contacting devices. Features of the model include the adhesion and aggregation of blood platelets on device material surfaces, shear and chemical activation of blood platelets, and embolization of platelet aggregates due to shear forces. As thrombus develops, blood is diverted from its regular flow field. If shear forces on a thrombus are sufficient to overcome the strength of adhesion, the thrombus is dislodged from the wall. Development of the model included preparing thrombosis and embolization routines to run in a parallel processing configuration, and estimating necessary parameters for the model including the adhesion strength of platelet conglomerations to the device surfaces and the criterion threshold for the coalescence of neighboring thrombi. Validation of the model shows that the effect of variations in geometry may be accurately predicted through computational simulation. This work is based on previous work by Paul Goodman, Daniel Lattin, Jeff Ashton, and Denzel Frost.

Extracorporeal Membrane Oxygenation (ECMO) is a life-saving therapy usedfor support in critical heart and/or lung failure. Patient’s blood is pumped viaan artificial lung for oxygenation outside of the body. The circuit is composedof a blood pump, cannulae for drainage and reinfusion, a membrane lung,tubing and connectors. Its use is associated with thromboembolic complicationsand hemolytic damage. Detailed numerical studies of two blood pumps anda lighthouse tip drainage cannula were undertaken to characterize the flowstructures in different scenarios and their link to platelet activation. The pumpsimulations were modelled according to manufacturer’s proclaimed use but alsoin off-design conditions with flow rates used in adult and neonatal patients.Lagrangian Particle Tracking (LPT) was used to simulate the injection ofparticles similar in size to platelets to compute platelet activation state (PAS).The results indicated that low flow rates impacted PAS similarly to high flowrates due to increased residence time leading to prolonged exposure to shearstress despite the fact that shear per se was lower at low flow rate. Regardingthe cannula, the results showed that a flow pattern similar to a jet in crossflowdeveloped at the side holes. A parameter study was conducted to quantifydrainage characteristics in terms of flow rate distribution across the holes wheninput variables of flow rate, modelled fluid, and hematocrit were altered. Thefindings showed, across all the cases, that the most proximal hole row drainedthe largest fraction of fluid. The effects due to the non-Newtonian nature ofblood were confined to regions far from the cannula holes and the flow structuresshowed very limited dependence on the hematocrit. A scaling law was found tobridge the global drainage performance of fluid between water and blood.

QC 210906

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.

The linear optical properties and exciton dynamics of different semiconductor nanostructures have been studied. A model to describe the absoprtion spectra of cadmium selenide (CdSe) nanoplatelets (NPLs) has been developed, which allows the extraction of parameters relating to excitons confined within the thickness of the NPLs. Giant oscillator strength transitions (GOST) have been observed in NPLs with a lifetime limited dephasing of the ground state excitons at low temperature of about 1 ps, using transient resonant four wave mixing in heterodyne detection. The observation of the GOST effect has been affirmed by a decrease in the low temperature dephasing time with increasing NPL area. In addition, in cadmium selenide/cadmium sulphide (CdSe/CdS) quantum dot in rod (QDR) samples, dephasing dynamics at low temperature have been described by a Gaussian distribution of decay rates. Density dynamics have shown a fast initial decay followed by a signal rise attributed to an interplay between acoustic phonons and fast population dynamics related to the QDR fine structure. Density dynamics at longer delays have been attributed to an ensemble including both excitons and trions. The low temperature dephasing has been attributed to relaxation of excitons from a bright state to a lower lying dark state. Finally, a homebuilt scanning probe microscopy instrument has been modified to allow simultaneous optical and scanning probe imaging for the study of correlation between charge state and optical properties of nanostructures.

One of the most promising routes to create advanced materials is self-assembly. Self-assembly refers to the self-organisation of building blocks to form ordered structures. As the properties of the self-assembled materials will inherit the properties of the basic building blocks, it is then possible to engineer the properties of the materials by tailoring the properties of the building blocks. In order to create mesoscale materials, the self-assembly of molecular building blocks of different sizes and interactions is important. Mesoscopic materials can be obtained by using larger building blocks such as nano and colloidal particles. Colloidal particles are particularly attractive as building blocks because it is possible to design interparticle interactions by controlling both the chemistry of the particles' surface and the properties of the solvent in which the particles are immersed. The self-assembly of spherical colloidal particles has been widely reported in the literature. However, advances in experimental techniques to produce particles with different shapes and sizes have opened new opportunities to create more complex structures that cannot be formed using spherical particles. Indeed, the particles' shape and effective interactions between them dictate the spatial arrangement and micro-structure of the system, which can be engineered to produce functional materials for a wide range of applications. The driving forces determining the self-assembly of colloidal particles can be modified by the use of external influences such as geometrical confinement and electromagnetic forces. Geometrical confinement, for example, has been used to design quasi two-dimensional materials such as multi-layered structures of spheres, dimers, rods, spherical caps, and monolayers of platelets with various geometries and symmetries. In this dissertation, we present three computer simulations studies using Monte Carlo and Molecular Dynamics simulations determining the self-assembly of monolayer colloidal platelets with different shapes confined in two dimensions. These particles have been selected due to recent experiments in colloidal particles with similar shapes. All the particles' models are represented by planar polygons, and three different effects affecting their self-assembly have been analysed: (a) the curvature of the particles' vertices; (b) the curvature of the particles' edges; and finally (c) the addition of functional groups on the particles' surface. These studies aim to demonstrate that the subtle changes on the particle's shape can be used to engineer complex patterns for the fabrication of advanced materials. Monte Carlo simulations are performed to study the self-assembly of colloidal platelets with rounded corners with 4, 5, and 6-fold symmetries. Square platelets provide a rich phase behaviour that ranges between disorder-order and order-order phase transitions. Suprisingly, the disk-like shape of pentagons and hexagons prevents the total crystallisation of these systems, even at a high pressure state. A hysteresis gap is observed by the analysis of compression and expansion runs for the case of square platelets and the thermodynamic method known as direct coexistence method is used to be accurately determined the point of the order-order transition. Further, unexpected results are obtained by performing Molecular Dynamics simulations in systems with platelets with 3, 4, 5, and 6-fold symmetries when all the sides of each polygon are curved. Macroscopic chiral symmetry breaking is observed for platelets with 4 and 6-fold symmetries, and for the first time a rule is promoted to explain when these chiral structures can be formed driven only by packing effects. This unique rule is verified also for platelets with the same curved sides as previously when functional chains tethered to either vertices or sides. Indeed, square platelets with curved sides confined in two dimensions can form chiral structures at medium densities when flexible chains tethered to either vertices or sides. Triangular platelets with curved sides can form chiral structures only when the chains are tethered to the corners, since the chains experience an one-hand rotation to sterically protect one side. When the chains are symmetrically tethered to the sides, local chiral symmetry breaking is observed as both left-hand and right-hand sides on each vertex are sterically protected allowing the same probability for rotation either in clockwise or anticlockwise direction.

Les récepteurs P2Y12 constituent une cible majeure des médicaments antiplaquettaires dans le traitement préventif des évènements thromboemboliques chez les patients ayant un syndrome coronarien aigu. A ce titre, le ticagrelor, un antagoniste sélectif et réversible des récepteurs P2Y12, a une place de choix dans l’arsenal thérapeutique. L’objectif de ce travail est d’étudier les interactions entre le ticagrelor, l’ADP et les récepteurs P2Y12 et les lipides membranaires des plaquettes.Dans la première partie de ce travail, nos résultats ont montré que le ticagrelor et l’ADP modifient la composition, la distribution et la concentration des sphingomyélines contenues dans les microdomaines membranaires en lien avec l’activation ou l’inhibition des plaquettes.Dans la deuxième partie de ce travail, nous avons décrit, pour la première fois, l'interaction des récepteurs P2Y12 avec les deux métabolites du ticagrelor. En outre, nous avons montré des interactions similaires entre l’ADP et les antagonistes avec les récepteurs P2Y12 avec une différence au niveau de la poche de liaison indiquant le changement de la conformation des récepteurs.L’ensemble de nos données confortent le rôle du ticagrelor dans la réorganisation des lipides membranaires et suggèrent des interactions spécifiques et une modification de la conformation entre les récepteurs P2Y12, l’ADP, le ticagrelor et ses métabolites
P2Y12 receptors are a major target of antiplatelet drugs in preventing thromboembolic events in patients with acute coronary syndrome. As such, ticagrelor, a selective and reversible P2Y12 receptor antagonist, has a place of choice in the therapeutic management. The aim of this work is to study the interactions between ticagrelor, ADP and P2Y12 receptors and platelet membrane lipids.Our data support the role of ticagrelor in the reorganization of membrane lipids and suggest specific interactions and a modification of the conformation between P2Y12 receptors, ADP, ticagrelor and its metabolites.In the first part of this work, our results showed that ticagrelor and ADP modify the composition, distribution and concentration of sphingomyelins in membrane microdomains related to platelet activation or inhibition.In the second part of this work, we described, for the first time, the interaction of P2Y12 receptors with the two metabolites of ticagrelor. In addition, we showed similar interactions between ADP and P2Y12 receptor antagonists with a difference in the binding pocket indicating the change in receptor conformation

Blutplättchen werden von Megakaryozyten im Knochenmark in einem Prozess produziert, an dem Aktin beteiligt ist. Aktin-Depolymerisierungsfaktor (ADF) und Cofilin sind Aktin-bindende Proteine, die als entscheidende Regulatoren im Aktinumsatz agieren, indem sie das Schneiden und Depolymerisieren von Filamenten unterstützen. Die Bedeutung von ADF/Cofilin und des Aktinumsatzes in der Bildung von Blutplättchen ist gegenwärtig nicht bekannt. In der vorliegenden Arbeit wurden Mäuse untersucht, die eine konstitutive ADF-Defizienz und/oder die eine konditionale n-Cofilin Defizienz (Cre/loxP) aufweisen. Um Cofilin nur in Megakaryozyten und Blutplättchen auszuschalten, wurden Cofilinfl/fl Mäuse mit PF4-Cre Mäusen verpaart. ADF- oder n-Cofilin-defiziente Mäuse hatten keinen oder nur einen geringen Phänotyp in Blutplättchen. Eine Defizienz von ADF und n-Cofilin führte hingegen zu einem beinahe kompletten Verlust der Blutplättchen, was mit Defekten in der Bildung von Plättchenzonen in Knochenmark-Megakaryozyten einherging. Weitere Untersuchungen an in vitro und ex vivo kultivierten Megakaryozyten zeigten eine Reduzierung der Bildung von Proplättchen und das Fehlen der typischen Verdickungen der Proplättchen. Diese Daten zeigen redundante aber essentielle Funktionen von ADF und n-Cofilin im terminalen Schritt der Plättchenbildung in vitro und in vivo, und belegen erstmals eine wichtige Rolle des Aktinumsatzes in diesem Prozess. Im zweiten Teil dieser Dissertation wurden die Mechanismen untersucht, die für die zelluläre Regulierung des Hauptkollagenrezeptors auf Blutplättchen, Glykoprotein VI (GPVI), verantwortlich sind. Nach einer Gefäßwandverletzung wird subendotheliales Kollagen freigelegt, wodurch GPVI die Aktivierung von Blutplättchen vermittelt, und damit zur Blutstillung (Hämostase), aber auch zum Verschluss eines verletzten Gefäßes beitragen kann, was letztendlich zu einem Myokardinfarkt oder einem Schlaganfall führen kann. Deshalb ist GPVI ein attraktives Zielprotein für eine anti-thrombotische Therapie, insbesondere weil frühere Studien gezeigt haben, dass anti-GPVI Antikörper eine irreversible Herunterregulierung des Rezeptors auf zirkulierenden Blutplättchen mittels Internalisierung und Abspaltung induzieren. Es wird vermutet, dass Metalloproteinasen der ADAM (a disintegrin and metalloproteinase domain) - Familie das Abspalten vermitteln, jedoch fehlt in vivo der Beweis dafür. Um die Mechanismen des Abspaltungsprozesses des GPVI Rezeptors in vivo besser verstehen zu können, wurden zwei Mauslinien, GPVI- und konditionale ADAM10-defiziente Mäuse, generiert und zusätzlich sogenannte „low TACE (TNFalpha converting enzyme)“ Mäuse analysiert. Es konnte gezeigt werden, dass GPVI in vitro von ADAM10 oder TACE in Abhängigkeit der Signalwege, die zum Abspalten des Rezeptors führen, geschnitten werden kann. Darüberhinaus wurde GPVI in vivo nach Antikörperverabreichung in ADAM10-defizienten Mäusen und „low TACE“ Mäusen herunterreguliert, was vermuten lässt, dass entweder beide Metalloproteinasen an diesem Prozess beteiligt sind oder noch eine zusätzliche Metalloproteinase für die GPVI Regulation in vivo verantwortlich ist
Platelets are produced by bone marrow megakaryocytes in a process involving actin dynamics. Actin-depolymerizing factor (ADF) and cofilin are actin-binding proteins that act as key regulators in actin turnover by promoting filament severing and depolymerization. The overall significance of ADF/cofilin function and actin turnover in platelet formation is presently unclear. In the first part of this thesis, platelet formation and function were studied in mice constitutively lacking ADF and/or mice with a conditional deficiency (Cre/loxP) in n-cofilin. To delete cofilin exclusively in megakaryocytes and platelets, cofilinfl/fl mice were crossed with PF4 (platelet factor 4)-Cre mice. While a single-deficiency in ADF or n-cofilin resulted in no or only a minor platelet formation defect, respectively, a double-deficiency in ADF and n-cofilin led to an almost complete loss of platelets. Bone marrow megakaryocytes of ADF/n-cofilin-deficient mice showed defective platelet zone formation. Interestingly, in vitro and ex vivo megakaryocyte differentiation revealed reduced proplatelet formation and absence of platelet-forming swellings. These data establish that ADF and n-cofilin have redundant but essential roles in the terminal step of platelet formation in vitro and in vivo. In the second part of the thesis, mechanisms underlying cellular regulation of the major platelet collagen receptor, glycoprotein VI (GPVI), were studied. GPVI mediates platelet activation on exposed subendothelial collagens at sites of vascular injury, and thereby contributes to normal hemostasis but also to occlusion of diseased vessels in the setting of myocardial infarction or stroke. Thus, GPVI is an attractive target for anti-thrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and ectodomain shedding. Metalloproteinases of the ADAM (a disintegrin and metalloproteinase domain) family are suspected to mediate this ectodomain shedding, but in vivo evidence for this is lacking. To study the mechanism of GPVI regulation in vivo, two mouse lines, Gp6 knock-out and Adam10fl/fl, PF4-Cre mice, were generated and in addition low TACE (TNFalpha converting enzyme) mice were analyzed. It was shown that GPVI can be cleaved in vitro by ADAM10 or TACE depending on the shedding-inducing signaling pathway. Moreover, GPVI was down-regulated in vivo upon antibody injection in ADAM10-deficient and low TACE mice suggesting that either both or an additional metalloproteinase is involved in GPVI regulation in vivo

碩士
國立成功大學
醫學工程研究所
83
An in vitro flow system is usually used to investigate the distribution features of adhered platelets under different flow conditions. The four controlled conditions are: (1) fibrin-coated surface, shear rate 445/s; (2) fibrincoated surface, shear rate 70/s; (3) fibrinogen-coated surface, shear rate 445/s; (4) fibrinogen-coated surface, shear rate 70/s. 　　The purpose of this study is to apply image processing technique for quantitative analysis of the platelets images. First of all, the identification of platelets is realized by sequential procedures, including automatic thresholding, unit-platelet models computation and recognition of platelets' centers by comparing correlation coefficients. Based upon the measures of the number of identified platelets and the mass center of those platelets distribution, the accuracy of the platelets identification is more than 92% in average and is also better than the previous method. 　　Next, the distribution features of adhered platelets are characterized using the statistical analysis and the fractal analysis. Fractal dimension is a new parameter for quantifying the degree of clustering. From the results of the simulation study, two viewpoints could be derived. (1) Under the same number of the attached platelets, the more uniform distribution would have the larger fractal dimensions, and the more clustering distribution would have the smaller fractal dimensions. (2) For the higher of the number of attached platelets, the fractal dimension will be larger. Finally, the unit-platelet tracking process is also implemented and demonstrated in this study. 　　A simple GUI (Graphic User Interface) is also added for rendering the physiologists a user friendly man-machine interface.

The objective of this work was to further our understanding of the influence hemodynamic forces have on the cardiovascular system. Specifically, the effect of shear stress on platelet aggregability under two pathophysiological conditions was studied. Elevated shear stress levels in stenosed vessels induce platelet aggregation. Increased plasma catecholamine concentrations have also been implicated in the onset of acute coronary ischemic syndromes. This first study was designed to examine the synergistic platelet activation by the interaction of shear stress and epinephrine. Platelets suspensions sheared at subthreshold levels in a cone and plate viscometer showed little or no aggregation unless pretreated with subthreshold concentrations of epinephrine. Monoclonal antibody blockade of glycoproteins (Gp) Ib and IIb/IIIa showed that the synergistic platelet aggregation required functional Gp IIb/IIIa but could partially bypass Gp Ib-von Willebrand factor (vWF) interaction. Binding studies indicated that fibrinogen is required for platelet priming, but a functional role for vWF could not be ruled out. Synergistic platelet aggregation was also observed in whole blood following exercise to elevate endogenous levels of catecholamines in blood donors. Vascular smooth muscle cells (SMCs) are exposed to fluid dynamic shear stress at sites of vessel wall damage. This second study was designed to examine the effects of shear stress on the production of platelet inhibitory nitric oxide (NO) and carbon monoxide (CO) from SMCs. A modified cone and plate viscometer was used to subject monolayers of SMCs to arterial levels of shear stress. Shear stress was observed to induce mRNA and protein production of inducible heme oxygenase (HO-1), which generates CO, but not to affect inducible NO synthase, in a shear stress and time dependent manner. Washed platelets, used to detect the production of CO from SMCs, contained elevated levels of cGMP and demonstrated an inhibited aggregation response to ADP following coincubation with sheared SMCs. The elevated intra-platelet cGMP levels and inhibited aggregation response could only be blocked by incubating the SMCs with the HO-1 inhibitor tin protoporphyrin. These results indicate that shear stress may play a variety of roles with regard to platelet aggregability under pathological conditions, helping to control blood fluidity.

Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.