Abstract
SNARE proteins are a family of membrane-associated proteins that mediate granule secretion in many cell types including platelets. These proteins are oriented such that all but the membrane spanning region of the protein resides in the cytosol. Complex formation between SNARE proteins brings granule membranes in close apposition to surface-connected membranes, facilitating membrane fusion and granule release. Initial studies evaluated the subcellular localization of SNARE proteins in resting platelets by electron microscopy using a pre-embedding immunonanogold technique. These studies demonstrated that VAMP 3 is associated primarily with granules, the majority of SNAP-23 is associated with plasma membrane, and syntaxin 2 is distributed relatively equally between granules, plasma membranes, and membranes of the open canalicular system. Following activation of platelets with either the thrombin receptor activating peptide, SFLLRN, or the phorbol ester, PMA, we observed staining of SNAP-23 and syntaxin 2 on the extracellular surface of platelets. VAMP 3 remained intracellular. Extracellular localization of SNARE proteins has not previously been observed in any cell type. We therefore sought to confirm extracellular localization. Flow cytometry of intact platelets using antibodies directed at SNAP-23, syntaxin 2, and VAMP 3 demonstrated extracellular localization of SNAP-23 and syntaxin 2, but not VAMP 3, following activation of platelets with either SFLLRN or PMA. Incubation of intact pacified platelets with trypsin resulted in degradation of SNAP-23 and syntaxin 2 as detected by immunoblotting. In contrast, trypsin failed to cleave the intracellular proteins VAMP 3 or Rab4. SNAP-23 associates with membranes via palmitoyl moieties. Incubation of intact platelets with acyl-protein transferase 1 (APT1), an enzyme that removes palmitate from proteins, resulted in release of SNAP-23 from the platelet membrane. Similarly, incubation of intact platelets with botulinum toxin C, which specifically cleaves certain syntaxin isoforms, released syntaxin 2 from the platelet surface. Of note, APT1 and botulinum toxin released SNAP-23 and syntaxin 2, respectively, from the surface of pacified, resting platelets. These data suggested that SNAP-23 and syntaxin 2 reside on the surface of the resting platelet. We therefore sought to evaluate whether increased surface expression of SNAP-23 and syntaxin 2 following platelet activation results from epitope unmasking of SNARE proteins already present on the platelet surface. Incubation with trypsin prevented PMA-induced stimulation of SNAP-23 and syntaxin 2 expression, but not PMA-induced P-selectin expression. Furthermore, incubation of resting intact platelets with botulinum toxin C resulted in release of SNAP-23, suggesting that SNAP-23 and syntaxin 2 form a complex on the resting platelet surface. These results indicate that enhanced detection of SNARE proteins on the platelet surface following activation results not from translocation of SNARE proteins from internal stores, but rather from activation-induced epitope exposure of SNARE proteins localized to the extracellular surface in resting platelets. These data represent the first demonstration of extracellular SNARE proteins and suggest that SNAP-23 and syntaxin 2 localize to the platelet surface during granulopoiesis.
SNAP-23 and syntaxin-2 localize to the extracellular surface of the platelet plasma membrane
