Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation.
OBJECTIVE
Several platelet proteins are palmitoylated, but whether protein palmitoylation functions in platelet activation is unknown. We sought to determine the role of platelet protein palmitoylation in platelet activation and thrombus formation.


RESULTS
Platelet proteins were depalmitoylated by infusing acyl-protein thioesterase 1 into permeabilized platelets. In intact platelets, platelet protein palmitoylation was blocked using the protein palmitoylation inhibitor cerulein. The effects of inhibiting platelet protein palmitoylation on platelet function and on thrombus formation in vivo were evaluated. When infused into permeabilized platelets, acyl-protein thioesterase 1 reduced total platelet protein palmitoylation and inhibited protease-activated receptor-1-mediated alpha-granule secretion with an IC50 of 175 nmol/L and maximal inhibition of > or = 90%. G(alpha q) and SNAP-23, membrane-associated proteins that are constitutively palmitoylated, translocated to the cytosol when permeabilized platelets were exposed to recombinant acyl-protein thioesterase 1. The protein palmitoylation inhibitor cerulein also inhibited platelet granule secretion and aggregation. Studies using intravital microscopy showed that incubation with cerulein decreased the rate of platelet accumulation into thrombi formed after laser-induced injury of mouse arterioles and inhibited maximal platelet accumulation by >60%.


CONCLUSIONS
These studies show that platelets possess a protein palmitoylation machinery that is required for both platelet activation and platelet accumulation into thrombi. These studies show that inhibition of platelet protein palmitoylation blocks platelet aggregation and granule secretion. In a murine model of thrombus formation, inhibition of protein palmitoylation markedly inhibits platelet accumulation into thrombi at sites of vascular injury.
Sim
Derek S
DS
Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02115, USA.
Dilks
James R
JR
Flaumenhaft
Robert
R
D000217:Acyltransferases / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001160:Arterioles / Q000601:surgery; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002108:Ceruletide / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D050766:Qb-SNARE Proteins / Q000378:metabolism; D050767:Qc-SNARE Proteins / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013927:Thrombosis / Q000097:blood / Q000378:metabolism* / Q000473:pathology
D048868:Adaptor Proteins, Signal Transducing; D009419:Nerve Tissue Proteins; D019007:P-Selectin; D010446:Peptide Fragments; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D044463:Receptor, PAR-1; D011994:Recombinant Proteins; C100493:SNAP23 protein, human; C082835:thrombin receptor peptide (42-47); D019308:Palmitic Acid; D002108:Ceruletide; D000217:Acyltransferases; C473038:ZDHHC17 protein, human; C402226:LYPLA1 protein, human; D013869:Thiolester Hydrolases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11