Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

J Biol Chem. 2018 Mar 9;293(10):3593-3606. doi: 10.1074/jbc.RA117.000140. Epub 2018 Jan 19.


Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [3H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [3H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.

Keywords: SNAP-23; acyltransferase; cardiovascular; cerulenin; lipid raft; membrane fusion; palmostatin B; posttranslational modification (PTM); syntaxin; thrombosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acylation / drug effects
  • Acyltransferases / antagonists & inhibitors
  • Acyltransferases / metabolism
  • Blood Platelets / drug effects
  • Blood Platelets / enzymology
  • Blood Platelets / metabolism*
  • Cysteine / chemistry
  • Cysteine / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Exocytosis* / drug effects
  • Humans
  • Hydroxylamine / pharmacology
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism
  • Oxidation-Reduction
  • P-Selectin / metabolism
  • Palmitic Acid / metabolism
  • Platelet Activation / drug effects
  • Platelet Aggregation / drug effects
  • Protein Processing, Post-Translational* / drug effects
  • Protein Transport / drug effects
  • Qa-SNARE Proteins / chemistry
  • Qa-SNARE Proteins / metabolism*
  • Qb-SNARE Proteins / chemistry
  • Qb-SNARE Proteins / metabolism*
  • Qc-SNARE Proteins / chemistry
  • Qc-SNARE Proteins / metabolism*
  • Reducing Agents / pharmacology
  • Surface Properties / drug effects
  • Thiolester Hydrolases / antagonists & inhibitors
  • Thiolester Hydrolases / metabolism
  • Tritium


  • Enzyme Inhibitors
  • P-Selectin
  • Qa-SNARE Proteins
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • Reducing Agents
  • SELP protein, human
  • SNAP23 protein, human
  • STX11 protein, human
  • Tritium
  • Hydroxylamine
  • Palmitic Acid
  • Acyltransferases
  • Thiolester Hydrolases
  • Cysteine