VARP associates with VAMP-7 and Arp2/3 in an activation-dependent manner. (A) VAMP-7 and VARP were immunoprecipitated (IP) from human platelets before or after exposure to 5 μM SFLLRN. Immunoprecipitated proteins were separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis and evaluated for VARP, Arp2/3, and VAMP-7 by immunoblot analysis. (B) Double immunofluorescence microscopy of actin and either VAMP-7, VARP, Arp2/3, or VAMP-8 was performed, and images were analyzed as previously described⁴⁰  to demarcate the granulomere and periphery of spread platelets. The percentage of VAMP-7, VARP, Arp2/3, and VAMP-8 fluorescence in the granulomere and periphery was quantified. Measurements represent the standard deviation of 25 individual platelet measurements per condition. (C) Representative images of double immunofluorescence microscopy of actin and VAMP-7, VARP, Arp2/3, or VAMP-8. Scale bars represent 5 microns. (D) Model of putative role for VARP in linking platelet granule exocytosis and actin polymerization. In the resting state (top left), VARP binds VAMP-7 and Arp2/3, localizing the granule exocytosis machinery and the actin polymerization machinery to the same location and maintaining them in an inactive state. Following platelet activation (bottom left), VAMP-7 and Arp2/3 are released from VARP. VAMP-7 interacts with target (t)-SNAREs on the plasma membrane and Arp2/3 functions in actin reorganization. Fusion of granules with the plasma membrane provides extra membrane to cover growing actin structures during spreading (right).