Platelet Spreading Requires Exocytosis of a Subpopulation of α-Granules Expressing VAMP-7,

Abstract 3248

There has been recent controversy as to whether platelet α-granules represent a single, homogenous granule population or are composed of different subpopulations that contain different cargos and serve discrete functions. To evaluate this question, we have studied granule movement in spreading platelets. During platelet spreading, the majority of dense and α-granules coalesce in the central granulomere. However, some granules are observed in the platelet periphery. We evaluated whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Granules in spread platelets were stained with antibodies directed at vesicle-associated membrane proteins (VAMPs) −3, −7, and −8. We and others have shown that VAMP-3 and −8 function in granule exocytosis. VAMP-7 contains a profilin-like N-terminal extension capable of interacting with Arp2/3. Samples were imaged using confocal microscopy. Quantitative evaluation of antibody staining in the granulomere vs. the periphery demonstrated that 93+/−7% of VAMP-8, 86+/−14% of von Willebrand factor (an α-granule cargo), and 93+/−7% of serotonin (a dense granule cargo) localized to the central granulomere. In contrast, 77+/−8% of VAMP-7 localized to the periphery. To determine whether a VAMP-7⁺ α-granule subpopulation actively moves from the granulomere to the platelet periphery during spreading, we loaded platelet α-granules with Qdot 565 nanocrystals and performed time-lapse video microscopy of single platelets. While the majority of endocytosed nanocrystals remained in the central granulomere during spreading, a subpopulation of labeled granules actively moved from the granulomere towards the growing edge at the platelet periphery. Colocalization studies demonstrated that 73% of peripheral Qdot 565 nanocrystals colocalized with VAMP-7. Furthermore, 82% of granules expressing VAMP-7 colocalized with P-selectin (specific for α-granules). These results indicate that a subpopulation of VAMP-7⁺ α-granules actively moves to the platelet periphery during spreading. To determine whether α-granules are required for spreading, we evaluated spreading in platelets from a patient with gray platelet syndrome (GPS, a congenital absence of α-granules). Platelet surface area increased 4-fold with spreading in normal controls. In contrast, the increase in surface area of GPS platelets following plating for 15 min was minimal – suggesting that α-granules are required for platelet spreading. We also evaluated spreading in murine Jinx platelets, which lack functional Munc13-4. These platelets demonstrate normal morphology and intact proximal signaling, but have a severe granule exocytosis defect. Total surface area of Munc13-4 deficient platelets spread for 15 min was 47+/−3% that of wild-type controls. Platelet spreading was also evaluated using a grated optical biosensor capable of detecting membrane contact with its surface in real time. This technique showed that spreading was decreased in Munc13-4 deficient platelets to 48+/−17% of controls. These results identify a new α-granule subtype that expresses VAMP-7 and is required for platelet spreading. This observation supports the premise that α-granules are heterogeneous and demonstrates that different VAMP isoforms localize to functionally discrete α-granule subpopulations.