SNAP-23 is a target for calpain cleavage in activated platelets.
The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation.
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Tara W
TW
Rutledge
S W
SW
Whiteheart
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000502:physiology*; D002154:Calpain / Q000097:blood*; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D003594:Cytoplasmic Granules / Q000502:physiology; D004151:Dipeptides / Q000737:chemistry; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry; D015539:Platelet Activation / Q000502:physiology*; D017027:Protein Tyrosine Phosphatases / Q000097:blood; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D004151:Dipeptides; D010446:Peptide Fragments; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D017027:Protein Tyrosine Phosphatases; D002154:Calpain