Cytoskeletal reorganization in human blood platelets during spreading
Aishwarya Kishore Paknikar
Doctoral thesis
Date of Examination:
2017-01-19
Date of issue:
2017-06-21
Advisor:
Koester, Sarah Prof. Dr.
Referee:
Neher, Erwin Prof. Dr.
Referee:
Großhans, Jörg Prof. Dr.
Referee:
Tittmann, Kai Prof. Dr.
Referee:
Jakobs, Stefan Prof. Dr.
Referee:
Feussner, Ivo Prof. Dr.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-3E84-5
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Abstract
English
Anucleate, human blood platelets play an important role in haemostasis to prevent excessive blood loss from a vascular damage. Their actin and microtubule (MT) cytoskeleton is especially responsible for bringing about dramatic morphological and biochemical changes in them, which enables the platelets to change their shape, secrete granular contents, aggregate, adhere, spread, and retract in a platelet haemostatic plug, to seal the vascular breach. The platelets are thus extremely important cells from a medical point of view and the study of their cytoskeletal changes is essential. Because platelets do not have a nucleus, they cannot be transfected, microinjected or manipulated in any way. This has restricted the study of their cytoskeleton with classic post chemical fixation approaches. Two novel, cytoskeletal, fluorogenic probes, SiR-actin and SiR-tubulin have been recently introduced, which bind to filamentous actin (F-actin) and MTs respectively. These probes have now made it possible to monitor the F-actin and MT cytoskeleton in real-time, in cells, which are otherwise difficult to transfect. In this dissertation, these SiR-actin and SiR-tubulin probes are used to directly visualize the F-actin and MT changes occurring during platelet spreading in real-time, and the temporal dynamics of these two cytoskeletal components during platelet spreading are explored. The real-time F-actin dynamics in platelets point towards the existence of multiple time-scales of F-actin reorganization during their spreading. The real-time MT dynamics in platelets point towards their role in platelet exocytosis and show that myosin plays a major role in this process. Overall, this technique of labeling human platelets with the SiR-actin and SiR-tubulin probes, to monitor any cytoskeletal changes occurring in real-time during their spreading, will improve our understanding of the role of the platelet cytoskeleton in aiding platelet functions, better. This may perhaps help lead to the development of novel agents that can alleviate platelet-related disorders in which the cytoskeleton is impaired.
Keywords: platelets; cytoskeleton; spreading; real-time F-actin and microtubule
