Biochemical Journal

Research article

A role for Sec1/Munc18 proteins in platelet exocytosis

Todd D. SCHRAW, Paula P. LEMONS, William L. DEAN, Sidney W. WHITEHEART

Abstract

A critical aspect of haemostasis is the release of clot-forming components from the three intra-platelet stores: dense-core granules, α granules and lysosomes. Exocytosis from these granules is mediated by soluble proteins [N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs)] and integral membrane proteins [vesicle and target SNAP receptors (v- and t-SNAREs)]. Three Sec1/Munc18 proteins (SM proteins) are present in platelets (Munc18a, Munc18b and Munc18c) and they bind to and potentially regulate specific syntaxin t-SNAREs. In resting platelets, these SM proteins associate with granules and open canalicular system membranes predominantly but not with the plasma membrane. Munc18a binds to syntaxin 2 alone and does not associate with other members of the core SNARE complex. Munc18b associates with a larger complex that contains synaptosome-associated protein of 23 kDa (SNAP-23) and cellubrevin/vesicle-associated membrane protein 3. Munc18c associates with both syntaxins 2 and 4, with synaptosome-associated protein of 23 kDa (SNAP-23) and with a v-SNARE. On stimulation, most of the platelet SM proteins are still found in membrane fractions. Phosphorylation of each Munc18 increases in thrombin-treated cells and phosphorylated Munc18c remains associated with syntaxins 2 and 4, but its affinity for the SNAREs appears to be reduced. To determine the functional role of the platelet SM proteins, we examined the effects of Munc18-based peptides (Munc18a peptide 3 and Munc18c peptide 3). Addition of the peptides to permeabilized platelets inhibits secretion from all three platelet granules. These peptides also inhibit agonist-induced aggregation in saponin-permeabilized platelets. These studies demonstrate a clear role for SM proteins in platelet exocytosis and aggregation and suggest a dominant role for Munc18c in all three granule-release events.

  • secretion
  • soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)
  • syntaxin

Footnotes

  • Abbreviations used: AMP-PNP, adenosine 5′-[β,γ-imido]triphosphate; 18a/pep3, Munc18a peptide 3; GST, glutathione S-transferase; 5-HT, hydroxytryptamine; mAb, monoclonal antibody; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry; NSF, N-ethylmaleimide-sensitive fusion protein; OCS, open canalicular system; pAb, polyclonal antibody; PF4, platelet factor 4; phospho-Munc18c, phosphorylated Munc18c; PKC, protein kinase C; SLO, streptolysin O; SM proteins, Sec1/Munc18 proteins; SNAP-23, synaptosome-associated protein of 23 kDa; SNARE, soluble NSF attachment protein receptor; s-Sec1, squid homologue of Sec1 protein; s-syntaxin, squid homologue of syntaxin 1; t- and v-SNARE, target and vesicle SNARE; VAMP, vesicle-associated membrane protein.