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The Platelet Actin Cytoskeleton Associates Directly with Syntaxin-4 and Participates in α-Granule Secretion
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 rele...
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Published in: | Blood 2008-11, Vol.112 (11), p.1839-1839 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Online Access: | Get full text |
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Summary: | 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-f |
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ISSN: | 0006-4971 1528-0020 |
DOI: | 10.1182/blood.V112.11.1839.1839 |