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

Objective: To address the role of CSPα in platelets.

Methods: We examined the platelet function and hemostatic phenotype of global CSPα^-/- mice using several in vitro and in vivo assays, including an IDEXX hematology analyzer to measure platelet count and size. Platelet activation and morphology were examined using cytometry and transmission electron microscopy. ATP secretion was measured using a sensitive luminescent plate assay. Hemostasis was assessed using tail bleeding and ex vivo a BioFlux microfluidics system. The levels of platelet SNARE machinery and cargo were quantified by western blotting. Protein localization was examined by 3-Dimensional Structured Illumination Microscopy (3D-SIM) and subcellular fractionation.

Results: Hematology data indicates that CSPα^-/- mice have normal platelet size and counts, but leukocytes counts were significantly lower in CSPα^-/- mice. CSPα^-/- platelets have defective α- and dense granule release with minimum effects on lysosomal granule release. CSPα^-/- platelets have reduced integrin activation in response to GPVI-specific agonists. 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. Loss of CSPα did not affect any of the proteins in the platelet secretory machinery, but it did interestingly cause a reduction in GPVI levels. Immunofluorescence experiments showed that CSPα colocalizes with markers for both α- and lysosomal granules. Subcellular fractionation studies showed that CSPα is present on membranes and not in lipid rafts. These results identify that CSPα is an essential element of the platelet secretory machinery and plays a significant role in thrombosis and hemostasis.

Conclusions: 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.