Selective activation of platelets by surfaces and soluble agonists

Resumen

Platelets are anuclear cell fragments circulating in blood whose primary function is to form blood clot at the site of injury and stop bleeding (hemostasis). They perform these functions by undergoing activation¿a process that involves changes in platelet shape and size, exposure of phospholipid phosphatidyl serine (PS) in the outer leaflet of plasma membrane, activation of GPIIb/IIIa receptor, membrane expression and secretion of cargo molecules stored in different intracellular granules (alpha, dense and lysosome). Platelets¿ key role in hemostasis is well established. Recent findings have furthermore implicated platelets in a variety of other processes, such as wound healing, angiogenesis, implant integration and rejection, tumor metastasis, as well as adaptive and innate immune responses. Such functional diversity implies that platelets exhibit a spectrum of different functional states that are induced in a stimulus-dependent manner. In other words, there are different kinds of activated platelets tailored to performing different functions. In this context, understanding how platelet activation is regulated, promises to revolutionize therapeutic approaches to cardiovascular disorders, cancers, treatment of recalcitrant wounds, and implant integration. In this work we focused on differences in the way platelets respond to different stimuli. By examining platelet activation at surfaces, we found that platelets adhering on glass and titania in the absence of calcium express different sets of activation markers. Glass-adhering platelets expressed CD62P (alpha granule marker), CD63 (dense granule iii marker), PS, and the active form of GPIIb/IIIa, while TiO2-adhering platelets expressed only CD63. In the presence of extracellular calcium, this difference between the two surfaces vanished. To investigate the mechanism underlying this phenomenon, we measured the dynamics of intracellular calcium in surface-adhering platelets. It was found to be different in platelets adhering on glass and on TiO2 in the absence of extracellular Ca2+. The differences correlated with the different platelets responses observed on these surfaces. These findings suggest that platelets can selectively undergo different sets of activation responses depending on the stimuli (TiO2 surface with or without surface bound calcium, glass or TiO2 surface in the absence of extracellular calcium), confirming the recent notion that platelets can tune their microenvironment in a stimuli-specific fashion. Studying platelet activation selectivity is challenging due to the lack of proper markers for distinguishing differently activated platelets. To circumvent this problem, we capitalized on the diversity and ubiquity of cell surface carbohydrates and the specificity of the lectin-carbohydrate interactions. Like all other cells, the surfaces of platelets are covered with various glycosylated proteins and lipids. We used fluorescently labeled lectins¿proteins that selectively bind certain mono- or oligosaccharides¿to investigate changes in platelet surface glycosylation upon agonist treatment. We tested a range of physiological and non-physiological agonists differing in terms of strength, receptors they act upon, and the corresponding intracellular signaling pathways. Each agonist caused a unique change in the platelet surface glycosylation pattern, eliciting a unique functional state, because cell surface carbohydrates serve specific functions in cell-cell and cell-matrix communications. Thus, for the first time, we were able to show that different agonists lead to different platelet functional states. The work presented in this Thesis provides insights into the platelet activation selectivity. It provides clear evidence that various platelet activation responses¿such as alpha and dense granule exocytosis, exposure of PS, activation of GPIIb/IIIa receptor and surface carbohydrate expression¿are stimuli-dependent. These findings have significant ramifications for understanding platelet signaling from the point of view of systems biology and considerable practical implications for the development of diagnostic approaches as well as personalized, therapeutic strategies