PB0870 - The Role of Cysteine String Protein-α in Platelet Exocytosis

Background: Platelets use SNARE-mediated exocytosis to maintain vascular integrity via the release of three types of granules: dense, α, and lysosomal. To understand how the process of exocytosis is regulated, we probed the role of Cysteine String Protein-α (CSPα), a potential t/Q-SNARE chaperone and assessed how it affects thrombosis and hemostasis. This abundant protein is the only detectible isoform from its respective family in platelets.

Aims: To address the role of CSPα in platelets.

Methods: We have examined the phenotype of CSPα-/- mice using an IDEXX hematology analyzer to measure platelet count and size. Platelet activation and morphology were examined using cytometry. Hemostasis was assessed using tail bleeding and a BioFlux microfluidics system. The levels of platelet SNARE machinery were quantified by western blotting, and protein localization was examined by Super-Resolution Immunofluorescence microscopy.

Results: Hematology data indicates that CSPα-/- mice have normal platelet size and counts, but erythrocytes and leukocytes counts were lower in CSPα-/- mice. CSPα-/- platelets have defective α and lysosomal granule release, but integrin activation was unaffected. Tail bleeding in CSPα-/- and CSPα+/- mice was significantly increased compared to wild-type littermates. Thrombus formation was also defective in CSPα-/- mice at low-shear rates in microfluidics assays. Microscopy experiments suggest that CSPα is localized to a granule population, but it also is cytoplasmic as well. The dominant t-SNAREs, SNAP-23 and Syntaxin-11 were reduced in CSPα-/- platelets while the remaining SNAREs were unchanged. Further experimentation is underway to better determine how CSPα interacts with elements of the platelet secretory machinery.

Conclusion(s): These experiments indicate that CSPα has a role in platelet secretion and thus in thrombosis and hemostasis. These data fill gaps in our knowledge of CSPα’s physiological role and our understanding of how platelet exocytosis is controlled.

Funding: HL56652, HL138179, HL150818, VA, and an NSF HRD 2004710.