Platelet activation and adhesion resulting in thrombus growth is essential for normal hemostasis, but can lead to irreversible, life-threatening vessel occlusion. In the current study, the contribution of platelet integrins, activation receptors and the contact system of blood coagulation in such pathological conditions was investigated in mice. / Plättchenaktivierung, -adhäsion und nachfolgende Thrombusbildung ist ein für die Hämostase essentieller Prozess, der jedoch zu irreversiblem lebensbedrohlichen Gefäßverschluss führen kann. In der vorliegenden Arbeit wurde die Rolle von Thrombozyten-Integrinen, aktivierenden Rezeptoren, sowie dem Kontaktsystem der Koagulation unter pathologischen Bedingungen im Maussystem untersucht.

by William, Wai-lun Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 85-95). / Chapter I. --- Introduction --- p.1 / Chapter II. --- Our Approach --- p.9 / Chapter III. --- Results and Discussion - Synthetic Strategy --- p.29 / Chapter IV. --- Results and Discussion - Pharmacological Activity --- p.44 / Chapter V. --- Conclusion --- p.49 / Chapter VI. --- Further Development --- p.53 / Chapter VII. --- Experimental Section --- p.55 / Chapter VIII. --- References --- p.85 / Chapter IX. --- Supplementary Materials --- p.96

Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury, but also contribute to arterial thrombosis, leading to myocardial infarction and stroke. Thrombus formation is the result of well-defined molecular events, including agonist-induced elevation of intracellular calcium ([Ca2+]i) and series of cytoskeletal rearrangements. With the help of genetically modified mice, the work presented in this thesis identified novel mechanisms underlying the process of platelet activation in hemostasis and thrombosis. Store-operated calcium entry (SOCE) through Orai1 was previously shown to be the main Ca2+ influx pathway in murine platelets. The residual Ca2+ entry in the Orai1 deficient platelets suggested a role for additional non-store-operated Ca2+ (non-SOC) and receptor operated Ca2+ entry (ROCE) in maintaining platelet calcium homeostasis. Canonical transient receptor potential channel 6 (TRPC6), which is expressed in both human and murine platelets, has been attributed to be involved in SOCE as well as in diacylglycerol (DAG)-triggered ROCE. In the first part of the study, the function of TRPC6 in platelet Ca2+ signaling and activation was analyzed by using the TRPC6 knockout mice. In vitro agonist induced Ca2+ responses and in vivo platelet function were unaltered in Trpc6-/- mice. However, Trpc6-/- mice displayed a completely abolished DAG mediated Ca2+-influx but a normal SOCE. These findings identified TRPC6 as the major DAG operated ROC channel in murine platelets, but DAG mediated ROCE has no major functional relevance for hemostasis and thrombosis. In the second part of the thesis, the involvement of the PDLIM family member CLP36 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. The GPVI/FcR-chain complex initiates platelet activation through a series of tyrosine phosphorylation events downstream of the FcR-chain-associated immunoreceptor tyrosine-based activation motif (ITAM). GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. The present study reports the adaptor protein CLP36 as a major inhibitor of GPVI-ITAM signaling in platelets. Platelets from mice expressing a truncated form of CLP36, (Clp36ΔLIM) and platelets from mice lacking the entire protein (Clp36-/-) displayed profound hyper-activation in response to GPVI-specific agonists, whereas GPCR signaling pathways remained unaffected. These alterations translated into accelerated thrombus formation and enhanced pro-coagulant activity of Clp36ΔLIM platelets and a pro-thrombotic phenotype in vivo. These studies revealed an unexpected inhibitory function of CLP36 in GPVI-ITAM signaling and established it as a key regulator of arterial thrombosis. / Die Aktivierung und die Aggregation von Thrombozyten (Blutplättchen) sind essentielle Prozesse, um Blutverluste nach Verletzungen zu begrenzen, sie spielen jedoch auch eine Rolle bei der arteriellen Thrombose, die zu Herzinfarkt und Schlaganfall führen kann. Die Thrombusbildung ist das Ergebnis wohldefinierter molekularer Vorgänge, die die Agonisten-induzierte Konzentrationserhöhung von intrazellulärem Kalzium ([Ca2+]i) und eine Reihe von Umlagerungen des Zytoskeletts mit einschließen. Die Ergebnisse dieser Arbeit, die mit Hilfe genetisch veränderter Mauslinien erzielt wurden, decken neue Mechanismen der Thrombozytenaktivierung in Thrombose und Hämostase auf. Es wurde bereits gezeigt, dass der durch Orai1 vermittelte Store-operated calcium entry (SOCE) den Haupteintrittsweg für Ca2+ in Mausthrombozyten darstellt. Der verbleibende Ca2+ Einstrom führte zur Annahme, dass zusätzlich non-store-operated Ca2+ (non-SOC) und receptor operated Ca2+ entry (ROCE) eine Rolle in der Aufrechterhaltung der Ca2+ Homöostase spielen. Dem Canonical transient receptor potential channel 6 (TRPC6), der in Thrombozyten des Menschen als auch der Maus exprimiert wird, wurde eine Rolle in dem SOCE und diacylglycerol (DAG)-vermitteltem ROCE zugeschrieben. Im ersten Teil dieser Arbeit wurde die Funktion von TRPC6 im Ca2+ Signaling und der Aktivierung von Thrombozyten mit Hilfe der TRPC6 defizienten Mauslinie untersucht. Die Funktion der Trpc6-/- Thrombozyten waren in vitro (z.B. Agonisten-induzierte Ca2+-Antworten) als auch in vivo unverändert. Jedoch zeigten Thrombozyten von Trpc6-/- Mäusen einen komplett fehlenden DAG vermittelten Kalziumeinstrom, aber normalen SOCE. Diese Ergebnisse identifizierten TRPC6 als den Haupt-DAG-aktivierten ROC Kanal in Mausthrombozyten. Jedoch hatte diese DAG vermittelte ROCE keine größere funktionelle Relevanz für Thrombose und Hämostase. Im zweiten Teil dieser Arbeit wurde die Rolle von CLP36, einem Mitglied der PDLIM Proteinfamilie, im Signalweg des Haupt-Kollagenrezeptors, Glykoprotein (GP) VI, auf Thrombozyten untersucht. Der GPVI/FcRKette Komplex initiiert die Thrombozytenaktivierung durch eine Reihe von Tyrosinphosphorylierungen, die dem FcR-Kette-assoziiertem immunoreceptor tyrosine based activation motif (ITAM) nachgeschaltet sind. GPVI-vermittelte Signale müssen sorgfältig reguliert sein, um eine unkontrollierte intravaskuläre Thrombozytenaktivierung zu verhindern. Jedoch sind die zugrunde liegenden Mechanismen nicht komplett verstanden. Die vorliegende Arbeit zeigt, dass das Adapterprotein CLP36 als ein wichtiger Inhibitor des GPVI-ITAM Signalwegs wirkt. Thrombozyten von Mäusen, welche eine trunkierte Form von CLP36 exprimieren, der die LIM-Domäne fehlt (Clp36ΔLIM), als auch von Mäusen, denen das komplette Protein fehlt (Clp36-/-), zeigten eine deutlich verstärkte Aktivierung als Antwort auf GPVI-spezifische Agonisten. Andere Signalwege aber waren nicht beeinflusst. Diese Veränderungen resultierten in einer schnelleren Thrombusbildung und erhöhten prokoagulatorischen Aktivität von Clp36ΔLIM Thrombozyten, welche sich letztendlich als prothrombotischer Phänotyp in vivo bemerkbar machten. Diese Ergebnisse deckten eine unerwartete inhibitorische Funktion von CLP36 im GPVI-ITAM Signalweg auf und etablierten CLP36 als einen wichtigen Regulator der arteriellen Thrombose.

Platelets play a pivotal role in hemostasis and thrombosis. It induces vascular retraction and clot formation through platelets activation and signal transmission, which promote ligands expressing on the surface of platelets, such as glycoproteins and P-selectin. Some surface glycoproteins gatherd to form complexes after activation. It bound to extracellular receptors such as collagen and thrombin to induce aggregation, which could also be induced by releasing agonists, such as arachidonic acid, adenosine diphosphate, epinephrine, serotonin and fibrinogen, to active the nearby platelets. The standard process of plateletapheresis in the Blood Center was to hold the platelets in still for one hour before stored on a vibrator. The process of holding platelets still for one hour before storage was omitted in some hospitals. It was not clear whether to omit the process has any effect on the quality of platelets. The expressions of P-selectin and vWF receptor, CD42b (Gp Ib£\) on platelets were analyzed by flow cytometry in this study. No significant differences (p= 0.77 and p= 0.62, respectively) were found. Similar results were obtained when functions of platelets were evaluated by agonists. It was concluded that leaving the platelets in room temperature for one hour to recover before keeping it on a vibrator would not enhance the functions of platelets aggregation significantly.

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

Investigations on platelet-derived growth factor (PDGF) and serotonin (5-HT), molecules stored in platelet granules, imply their potential effects in regulating megakaryopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. In addition, numerous reports indicate that melatonin, a derivative from serotonin effectively enhances platelet counts in patients with thrombocytopenia. However, their exact roles on human megakaryocytes and the underlying mechanisms remain unknown. Present studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in an irradiated-mouse model. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow aspirate. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. Our findings demonstrated that PDGF activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors (PDGFRs) with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockade of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia. We explored how serotonin regulated megakaryopoiesis and proplatelet formation. Our results indicated that serotonin (5-HT) significantly promoted CFU-MK formation and reduced apoptosis on megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryopoiesis through stimulating the release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO. Furthermore, thousands of membrane-derived microparticles (MPs) arose from MSCs and the TPO RNA/proteins contained within MPs were also considerably increased under serotonin treatment. In summary, our findings demonstrated an important role serotonin played on megakaryopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs in both dissociative and MP-encapsulated form, which indirectly promoted megakaryopoiesis. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Polymorphisms of platelet glycoproteins (GPs) are frequently targets for anti-platelet antibodies. At least 19 antigenic polymorphisms have been identified on platelet GPs. Antibodies against the HPA-lb polymorphism (a Leu to Pro switch at amino acid residue 33 of the IIIa sub-unit of GP IIb/Illa) have been attributed to as much as 90% of all cases of neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura in caucasians. The HPA-lb polymorphism has also been equivocally associated with coronary artery disease, particularly early onset (<60 years of age) myocardial infarction. Current technology for identifying individuals with the HPA-lb phenotype is limited to the labor-intensive, highly technical and expensive process of DNA amplification by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) analysis.This study proposes an alternative method for phenotyping individuals for the HPA-1 polymorphism using the Biocytex Platelet HPA-1 kit. The kit identifies the HPA-1 polymorphism utilizing two monoclonal CD61 (platelet glycoprotein IIb/IIIa) antibodies, one of which has a lowered affinity for GP llb/Illa possessing the HPA-lb polymorphism. Fluorescent labeling of bound antibody allows for flow cytometric quantitation of antibody binding capacity (ABC) for both monoclonal antibodies, and ratios derived from the ABC can be used to phenotype previously unknown samples.The Biocytex HPA-1 kit identified 73 of 74 (98.6%) individuals possessing the HPA1 a/HPA-1 a phenotype, 22 of 22 (100%) HPA-1 a/HPA-1 b individuals and 4 of 4 (100%) HPAIb/HPA-Ib individuals. All HPA-lb phenotypes were confirmed by PCR/RFLP. Total accuracy of the test system was 99%. / Department of Biology

The binding between the 45 kDa N-terminal domain of the a subunit of the GPIb-IX-V complex (GPIbαN) on the platelet membrane and the A1 domain of von Willebrand factor (VWF-A1), a multimeric protein circulating in the plasma, plays a key role in platelet adhesion and thrombus initiation at sites of cut-injury and atherosclerotic plaque rupture where blood vessels are subjected to high haemodynamic shear. A fundamental yet unresolved issue is how haemodynamic force upregulates this interaction (binding kinetics) and how a mechanical stimulus is translated into a biochemical signal (mechanotransduction). In order to address above issues, we setup a new biomembrane force probe (BFP) with the drifting reduction, temperature control and concurrent fluorescence imaging. My research findings are summarized into three aims: 1. VWF regions surrounding A1 hinder A1-GPIbα interaction at zero force, which is relieved by increasing force that stabilizes the interaction, giving rise to a VWF-GPIbα catch bond. 2. Three transport-related physical factors: receptor-ligand separation distance, Brownian motion and diffusivity govern the VWF-GPIbα association. 3. Mechanical force and structural variation regulate platelet signaling via the engagement duration of GPIbα mechanosensor. My thesis study advances our understanding of the biophysical and structural basis of how the VWF activation, its interaction with GPIbα and signal transduction are regulated by force when platelets' haemostatic functions are most needed.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

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