title: Synaptotagmin-like protein 4 and Rab8 interact and increase dense granule release in platelets.
abstract: BACKGROUND
  Platelets are highly specialized cells that regulate hemostasis and thrombosis in the vasculature. Upon activation, platelets release various granules that impact on platelets, the coagulation system, other blood cells and the vessel wall; however, the mechanisms controlling granule release are only partially known. We have shown previously that synaptotagmin-like protein (Slp)1 decreases dense granule release in platelets.
  
  
  OBJECTIVE
  To determine the role of other Slps and their binding partners on platelet dense granule release.
  
  
  METHODS
  RT-PCR and immunoblotting were used to identify Slps in human platelets. Interaction between Slp4 and Rab8 was investigated with pull-down assays, coimmunoprecipitation, and confocal microscopy. Secretion assays on permeabilized platelets were performed to investigate the effects of Slp4 and Rab8 on dense granule release.
  
  
  RESULTS
  Slp4 mRNA and protein are expressed in human platelets. Slp4 interacts with Rab8 in transfected cells and at endogenous protein levels in platelets. We mapped the Rab interaction site to the Slp-homology domain of Slp4, and showed preferential binding of Slp4 to the GTP-bound form of Rab8. Live microscopy showed colocalization of green fluorescent protein-Slp4 and mCherry-Rab8 at the plasma membrane of transfected cells. Endogenous platelet Slp4 and Rab8 colocalized in the center of activated platelets, where granule secretion takes place. Secretion assays revealed that Slp4 and Rab8 enhance dense granule release and that the Slp4 effect is dependent on Rab8 binding.
  
  
  CONCLUSIONS
  Slp4 and Rab8 are expressed and interact in human platelets, and might be involved in dense granule release.
authors.affiliation: UCD Conway Institute, UCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
authors.forename: A
authors.initials: A
authors.lastname: Hampson
authors.forename: A
authors.initials: A
authors.lastname: O'Connor
authors.forename: A
authors.initials: A
authors.lastname: Smolenski
mesh_terms: D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D015539:Platelet Activation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000378:metabolism*; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C512779:SYTL4 protein, human; D033921:Vesicular Transport Proteins; C082377:RAB8A protein, human; D020691:rab GTP-Binding Proteins