title: Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains.
abstract: The molecular mechanism that causes non-adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains--also known as rafts--play a crucial role in this process. We have used a non-cytolytic derivative of perfringolysin-O, a cholesterol binding cytolysin, that binds selectively to cholesterol-rich membrane domains, combined with confocal- and immunoelectron microscopy to visualize cholesterol-raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of alpha-granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion-dependent raft aggregation was accompanied by concentration of the tyrosine kinase c-Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c-Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low-density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP-induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co-cluster with the tyrosine kinase c-Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.
authors.affiliation: Department of Hematology, Division of Thrombosis and Hemostasis, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. h.f.g.heijnen@azu.nl
authors.forename: H F G
authors.initials: HF
authors.lastname: Heijnen
authors.forename: M
authors.initials: M
authors.lastname: Van Lier
authors.forename: S
authors.initials: S
authors.lastname: Waaijenborg
authors.forename: Y
authors.initials: Y
authors.lastname: Ohno-Iwashita
authors.forename: A A
authors.initials: AA
authors.lastname: Waheed
authors.forename: M
authors.initials: M
authors.lastname: Inomata
authors.forename: G
authors.initials: G
authors.lastname: Gorter
authors.forename: W
authors.initials: W
authors.lastname: Möbius
authors.forename: J W N
authors.initials: JW
authors.lastname: Akkerman
authors.forename: J W
authors.initials: JW
authors.lastname: Slot
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D001792:Blood Platelets / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure*; D000081247:CSK Tyrosine-Protein Kinase; D048429:Cell Size; D002784:Cholesterol / Q000378:metabolism / Q000502:physiology; D003505:Cyclodextrins / Q000494:pharmacology; D005340:Fibrinogen; D006801:Humans; D007150:Immunohistochemistry; D021962:Membrane Microdomains / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011554:Pseudopodia / Q000737:chemistry*; D018179:Receptors, Thrombin; D060149:Tetraspanin 30; D047392:beta-Cyclodextrins; D019061:src-Family Kinases
chemical_list: D015703:Antigens, CD; C554586:CD63 protein, human; D003505:Cyclodextrins; D010980:Platelet Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D018179:Receptors, Thrombin; D060149:Tetraspanin 30; D047392:beta-Cyclodextrins; D005340:Fibrinogen; D002784:Cholesterol; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C031215:betadex