The Platelet Actin Cytoskeleton Associates Directly with Syntaxin-4 and Participates in α-Granule Secretion.
Authors: Flaumenhaft Robert , Dilks James R. , Rozenvayn Nataliya , Woronowicz Kamil 
Published: April 2020

Abstract
Following platelet activation, platelets undergo a dramatic shape change directed by the actin cytoskeleton and accompanied by secretion of granule contents. Secretion of platelet granules requires soluble NSF Attachment Protein Receptors (SNAREs) that mediate fusion events required for granule release. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism of this influence is not known. We sought to determine whether actin controls α-granule release by interacting with platelet SNAREs. We found that disruption of the actin cytoskeleton by latrunculin A prevented pseudopodia formation and inhibited thrombin-induced α-granule secretion by 90±4% without significantly affecting activation-induced platelet aggregation. Latrunculin A inhibited α-granule secretion induced by either calcium ionophore A23187 or the phorbol ester PMA, indicating that latrunculin A blocked a relatively distal component of the secretory signaling pathway. To study distal mechanisms of α-granule release, we developed a cell-free secretory system using an α-granule-enriched membrane preparation isolated from a metrizamide gradient. When incubated with either platelet cytosol alone or ATP alone, P-selectin expression was not observed in this system. However, upon incubation of the membrane preparation with both platelet cytosol and ATP, a 7.1±2.5-fold increase in P-selectin expression was observed, indicating α-granule secretion. Release of β-thromboglobulin, a soluble α-granule component, was also observed following incubation of the membrane preparation with ATP and cytosol. α-Granule secretion in the cell-free secretory system required the distal secretory machinery as evidenced by the fact that anti-syntaxin-4 antibody inhibited P-selectin expression by 61±19%. To determine whether the α-granule preparation supported actin polymerization, FITC-phalloidin was used to monitor F-actin formation by flow cytometry. Incubation of the α-granule-enriched preparation with platelet cytosol plus ATP resulted in a 3.4±0.3-fold increase in the binding of FITC-phalloidin. Inhibition of actin polymerization using cytochalasins prevented both FITC-phalloidin binding and P-selectin expression in the cell-free platelet system. Incubation of the α-granule-enriched preparation with purified platelet actin and ATP resulted in actin polymerization and α-granule release. These results showed that actin polymerization was required for α-granule release in the cell-free secretory system and that purified platelet actin could substitute for platelet cytosol to facilitate granule release. To determine whether platelet SNAREs associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with the actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with actin cytoskeletons isolated from either resting or activated platelets. Association of all SNAREs was inhibited by cytochalasin B, indicating that SNAREs precipitated secondary to association with the actin cytoskeleton rather than with lipid microdomains or protein aggregates. Since syntaxin-4 and SNAP-23 bound the actin cytoskeleton in both the resting and activated platelets, we evaluated whether they bound purified polymerized platelet actin directly. Recombinant syntaxin-4 bound directly to polymerized platelet actin to approximately the same extent as purified α-actinin, a known actin-binding protein. In contrast, recombinant SNAP-23 failed to bind significantly to polymerized platelet actin. Since SNAP-23 binds syntaxin-4 in resting platelets, its association with the resting actin cytoskeleton is likely mediated via its interaction with syntaxin-4, which binds actin directly. Based on these data, we propose a model whereby activation-induced actin polymerization facilitates SNARE complex formation and membrane fusion through the direct association of actin with syntaxin-4.