12871315

pubmed:Citation

6

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.

eng

IM

MEDLINE

1538-7933

Print

NLM

1161-73

pubmed-meshheading:12871315-Antigens, CD, pubmed-meshheading:12871315-Antigens, CD63, pubmed-meshheading:12871315-Blood Platelets, pubmed-meshheading:12871315-Cell Size, pubmed-meshheading:12871315-Cholesterol, pubmed-meshheading:12871315-Cyclodextrins, pubmed-meshheading:12871315-Fibrinogen, pubmed-meshheading:12871315-Humans, pubmed-meshheading:12871315-Immunohistochemistry, pubmed-meshheading:12871315-Membrane Microdomains, pubmed-meshheading:12871315-Phosphorylation, pubmed-meshheading:12871315-Phosphotransferases, pubmed-meshheading:12871315-Platelet Activation, pubmed-meshheading:12871315-Platelet Membrane Glycoproteins, pubmed-meshheading:12871315-Protein Transport, pubmed-meshheading:12871315-Protein-Tyrosine Kinases, pubmed-meshheading:12871315-Proto-Oncogene Proteins, pubmed-meshheading:12871315-Pseudopodia, pubmed-meshheading:12871315-Receptors, Thrombin, pubmed-meshheading:12871315-beta-Cyclodextrins

Concentration of rafts in platelet filopodia correlates with recruitment of c-Src and CD63 to these domains.

Journal Article, Research Support, Non-U.S. Gov't