Date Available

6-7-2017

Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Sidney W. Whiteheart

Abstract

Endocytosis is key to fibrinogen (Fg) uptake, receptor trafficking of integrins (αIIbβ3, αvβ3) and purinergic receptors (P2Y1, P2Y12), and thereby for normal platelet function. However, platelet endocytosis could potentially be critical for actively sensing changes in vascular micro-environments and responding accordingly to what is being taken up. This is a more dynamic view of platelets as active surveyors of the vasculature; extending the importance of platelet endocytosis beyond granule biogenesis and perhaps even hemostasis. The mechanistic underpinnings of endocytosis, its importance in platelets, and the molecular machinery required and possible trafficking routes are however understudied, in part due to a lack of experimental tools. The work presented here, puts forth new players that regulate platelet endocytosis and mediate cargo loading and hemostasis as well as provides a novel mechanistic understanding of how endocytosis allows platelets to act as immune cells and become the first responders to pathogens in the vasculature.

Previously we showed the importance of ADP-ribosylation factor 6 (Arf6), which regulates αIIbβ3-mediated Fg uptake/storage and affects acute platelet functions e.g., clot retraction and spreading. To further identify elements of this endocytic machinery, we examined the role of a vesicle-residing Soluble N-ethylmaleimide Factor Attachment Protein Receptor (v-SNARE) called Cellubrevin/Vesicle Associated Membrane Protein-3 (VAMP-3) in platelet function. VAMP-3 KO mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Loss of VAMP-3 led to defective uptake of fluorescently-tagged Fg and low-molecular dextran in platelets though it had a greater negative effect on receptor-mediated Fg uptake than on the fluid-phase marker uptake. Additionally, we followed the time-dependent trafficking of Fg and dextran into platelets using 3D-Structured Illumination Microscopy. Wild-type platelets endocytosed both cargo but quickly sorted them into distinct compartments with partial overlap occurring only at early time points. Sorting was unaffected in VAMP-3KO platelets. VAMP-3 loss did affect some acute platelet functions leading to enhanced spreading on Fg and faster clot retraction compared to wild-type. Additionally, the rate of JAK2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3 KO platelets.

The idea that platelets can act as immune cells and contribute to innate immunity has been increasingly gaining ground. Groups have correlated thrombocytopenia with clinical outcomes of viremia and bacteremia. Chronic viral infections, e.g., HIV-1, severely increase the risk of acute myocardial infarction (MI), possibly via some level of platelet activation, contributing to increased thrombotic potential. Platelets do endocytose viruses and bacteria, but the molecular machinery is ill-defined. In nucleated cells, responses to HIV-1 are mediated by virus phagocytosis/endocytosis, degradation to release Toll-like Receptor ligands, and subsequent receptor activation. Is this process recapitulated in platelets? Here we show that platelets indeed use VAMP-3 and Arf6-dependent pathways to endocytose HIV-1 virions, degrade retroviral particles to release TLR ligands, which initiate platelet activation and secretion. HIV-1 uptake and subsequent activation is abolished in VAMP-3 and Arf6 KO mice.

Collectively, our studies shed light on how platelets act at the early stage of pathogen recognition and are able to process them to initiate an immune response.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.235

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