Evidence for a physiological role for membrane rafts in human platelets.
We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.
Gousset
Karine
K
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, 2315 Haring Hall, University of California-Davis, Davis, CA 95616, USA. kgousset@ucdavis.edu
Wolkers
Willem F
WF
Tsvetkova
Nelly M
NM
Oliver
Ann E
AE
Field
Cara L
CL
Walker
Naomi J
NJ
Crowe
John H
JH
Tablin
Fern
F
D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015153:Blotting, Western; D018955:CD36 Antigens / Q000737:chemistry / Q000378:metabolism; D018958:CD55 Antigens / Q000737:chemistry / Q000378:metabolism; D002232:Carbocyanines; D002469:Cell Separation; D002784:Cholesterol / Q000737:chemistry; D003505:Cyclodextrins / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D015539:Platelet Activation / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013109:Sphingomyelins / Q000737:chemistry; D013696:Temperature; D047392:beta-Cyclodextrins
D018955:CD36 Antigens; D018958:CD55 Antigens; D002232:Carbocyanines; D003505:Cyclodextrins; D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013109:Sphingomyelins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C024286:3,3'-dioctadecylindocarbocyanine; D002784:Cholesterol