title: SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23.
abstract: In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.
authors.affiliation: Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Katharine Crane
authors.initials: KC
authors.lastname: Lai
authors.forename: Robert
authors.initials: R
authors.lastname: Flaumenhaft
mesh_terms: D001792:Blood Platelets / Q000378:metabolism; D002154:Calpain / Q000008:administration & dosage / Q000494:pharmacology*; D002352:Carrier Proteins / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D015539:Platelet Activation / Q000502:physiology*; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050600:SNARE Proteins; D013997:Time Factors; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins; D010447:Peptide Hydrolases; D002154:Calpain