Mechanisms regulating human platelet cytoskeleton dynamics and shape change

Abstract

Platelets are small blood cells derived from megakaryocytes that play essential roles in primary haemostasis and regulate vascular integrity. Circulating quiescent platelets have a characteristic flat discoid shape maintained by a circumferential marginal band structure composed of bundled microtubules. Injury to blood vessels activates platelets, causing the marginal bands to elongate and coil, resulting in a dramatic discoid-to-sphere cell shape change. Confocal imaging demonstrated that microtubule motor proteins kinesin and dynein might drive the marginal band coiling process. However, the mechanisms of marginal band regulating platelet shape change are still unclear. Recent studies suggest that marginal band dynamic is controlled by a platelet-specific tubulin code, which pathogenic tubulin mutations can lead to platelet dysfunction and bleeding disorders. My thesis aimed to understand the molecular mechanisms and determine the cytoskeletal components that regulate platelet shape change.

First, I developed and optimised a protocol to isolate tubulin and microtubule-associated proteins (MAPs) from cultured HeLa cells. I then used this protocol to purify microtubules and MAPs using human platelets isolated from blood. Using mass spectrometry, I identified RMDN-3, KIF13A, and MAP1B as candidate MAPs for further characterisation, as these MAPs showed protein level changes after platelet activation. Next, I investigated whether these selected candidate MAPs can affect microtubule dynamics. Using biochemistry and confocal imaging techniques, I found that RMDN-3 does not interact with microtubules. I observed that KIF13A can form tubular structures and move along microtubules within cells. I discovered that the full-length MAP1B and some truncated counterparts can co-localise with microtubules. I examined protein interactions of these MAPs using co-immunoprecipitation and mass spectrometry. Furthermore, I interrogated the motor proteins and cytoskeletal components in platelets using small molecule inhibitors.

Finally, I examined whether human platelets could synthesise and degrade proteins using pulsed stable isotope labelling by amino acids (pulse-SILAC). Although platelets do not have a nucleus, low levels of protein synthesis have been detected using radiolabelled amino acids. I first improved a protocol to isolate high-purity platelets from human blood by depleting leukocytes and adding extra washing steps. I then used pulse-SILAC to monitor de novo protein synthesis in resting and activated platelets. I found that platelets do not synthesise new proteins. Overall, this thesis highlights MAPs as regulators for platelet microtubule dynamics and provides insights into platelet proteome.