PB0868 - Dynamics of alpha-granule secretion and cytoskeletal arrangements in spreading platelets

Background: α-granule secretion is a key element of platelet function in haemostasis, thrombosis and inflammation. In suspended platelets, agonist-induced α-granule secretion requires dynamic changes of the actin cytoskeleton. Since the actin cytoskeleton also changes dramatically during platelet adhesion and spreading, the question arises if and how α-granule secretion and platelet spreading influence each other through the shared actin cytoskeleton.

Aims: To understand the spatiotemporal coordination of actin cytoskeletal arrangements and α-granule secretion in adherent platelets.

Methods: Washed platelets from healthy human donors were seeded onto fibrinogen or collagen coated glass coverslips in the presence of different agonists. Platelets were fixed after 2…60 minutes, fluorescently labelled for f-actin and α-granule components, imaged using STED microscopy or 3D STORM, and analysed using custom ImageJ and MATLAB scripts.

Results: The super-resolution microscopy techniques used here unambiguously distinguished single α-granules from adjacent granules or dispersed/secreted staining, membrane components (P-selectin as rings) from internal components (Thrombospondin-1 as solid spheres, von Willebrand factor within granule sub-sections), and upwards from downwards secretion. Unstimulated platelets on fibrinogen spread and rearranged their cytoskeleton over 20 minutes before secreting α-granules. In contrast, secretion on collagen started within 5 minutes even before completion of spreading and simultaneously with cytoskeletal rearrangements. Soluble agonists accelerated platelet spreading and granule secretion on fibrinogen depending on agonist strength (Thrombin>CRP-XL>ADP). Especially PAR-agonists (TRAP-6 and PAR-4 AP) induced excessive filopodia formation. In all cases, α-granules remained located centrally and were secreted upwards and/or downwards but were never observed in lamellipodia nor in filopodia. Downwards secreted thrombospondin was contained below platelets, sealed off by the lamellipodium.

Conclusion(s): Based on our preliminary data, the cross-talk between major actin cytoskeletal structures and α-granule secretion in adherent platelets is limited to a mutual excluded volume effect. Further experiments will probe the nanoscale rearrangement of f-actin surrounding secreting granules.