Author ORCID Identifier

https://orcid.org/0000-0001-8160-9210

Date Available

9-15-2024

Year of Publication

2024

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

Advisor

Dr. Sidney W. Whiteheart

Abstract

Platelets play many critical roles in the vasculature ensuring proper hemostasis and maintaining vascular integrity. They can mediate these different processes via the release of different cargo molecules from granules. Cargo release from the granules is mediated by a family of proteins called Soluble NSF Attachment Protein Receptors (SNAREs). This process is complex and is regulated by several protein-protein interactions that are not completely characterized. SNARE chaperones have been identified for the t-SNAREs such as Munc18b/STXBP2 for Syntaxin-11, but none have been identified for the VAMPs and SNAP proteins. Proteins of interest as potential chaperones for VAMPs and SNAP proteins are respectively, α-synuclein and its interacting partner Cysteine String Protein-α (CSPα).

To address the roles of both α-synuclein and CSPα in secretion and hemostasis, we examined the platelet and hemostatic phenotype of α-synuclein-/- mice and CSPα-/- mice using several in vitro assays and in vivo models. Measurements of granule secretion show that α-synuclein-/- mice only have a mild activation-dependent dense granule secretion defect with minimal effects on α and lysosomal granule release. Consistent with the mild secretion phenotype α-synuclein-/- mice did not have defective thrombus formation in the tail bleeding, FeCl3 carotid injury or jugular vein puncture models. CSPα-/- mice, on the other hand, have defective granule secretion from both α and dense granules with minimum effects on lysosomal granule release. Consistent with the significant secretion defect, CSPα-/- mice had a significant tail bleeding defect and attenuated thrombus formation under flow. Loss of CSPα reduced GPVI levels and reduced αIIbβ3 activation especially in response to GPVI-specific agonists. Neither loss of α-synuclein nor CSPα affected proteins in the platelet secretory machinery. Immunofluorescence co-localization studies showed that both α-synuclein and CSPα co-stained with markers for both α and lysosomal granules. Subcellular fractionation studies showed that α-synuclein is mostly cytosolic while CSPα is membrane associated but is not found in lipid rafts.

The data presented in this dissertation identified two new regulatory elements in the platelet secretory machinery, α-synuclein and CSPα. α-Synuclein has a limited role while data for CSPα shows that is essential. Thus, my work gives insight into how platelet secretion is regulated and will help us better understand what the proper threshold of platelet function is for proper thrombosis and hemostasis.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.52

Funding Information

  • National Science Foundation Division of Human Resource Development Grant (no.: 2004710) from 2020-2022.
  • National Institute of Health (no.: 5R01HL138179) from 07/01/2017 - 03/31/2020.
  • National Institute of Health (no.:5R01HL056652) from 04/01/2018 - 03/31/2020.
  • Veteran Affairs Merit Award (no.: I01BX003877) from 10/01/17 – 12/31/22.
  • National Institute of Health (no.: 1R35HL150818-01) from 4/1/2020 – 3/31/2027.

Download

Included in

Cell Biology Commons

Share

COinS