Since the original description of SNAREs in neurons [18
] much has been learned. Recent models of SNARE function posit that vesicle/granule-target membrane fusion is governed, in part, by the matching of a vesicle SNARE (v-SNARE), with a heterodimeric or trimeric, protein complex in the target membrane (t-SNAREs). This trans
-membrane complex is the minimal element required for membrane fusion [19
]. v-SNAREs are type II integral membrane proteins of generally low molecular weight (15
kDa) which contain a characteristic 60–70 amino acid SNARE motif [20
]. Human and mouse platelets contain VAMP-2/synaptobrevin, VAMP-3/cellubrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin, with the later being the most abundant [21
]. t-SNAREs are of two types: the SNAP-23/25 type (SNAP-23, SNAP-25, SNAP-29, Sec9p), and the syntaxin type (syntaxin 1–19, etc.
). The SNAP-23-like proteins have two SNARE motifs, lack transmembrane domains, but are generally anchored to the membrane through thioester-linked acyl groups. The type II membrane protein, syntaxins, contain the characteristic SNARE motif and an N-terminal regulatory domain [25
]. Human platelets contain syntaxin 2, 4, 7, and 11 [26
] as well as SNAP-23 [30
] and SNAP-29 [31
]. The presence of SNAP-25 is perhaps controversial [31
]. SNAREs employ the SNARE motifs to form a four-helix bundle arranged as a parallel coiled-coil [32
]. This structure is stabilized by core hydrophobic interactions running the length of the bundle. Based on this inherent stability, the temporal and spatial control of SNARE complex formation and disassembly represent important tasks for the secretory machinery.
SNAREs in Platelets
Three groups have contributed to our present knowledge of the function of platelet SNAREs (for reviews see [33
]). Our group [28
] first showed that SNARE proteins are present in platelets and Flaumenhaft et al.
] demonstrated a role for syntaxin 4 and SNAP-23 in α-granule release. Syntaxin 4 is also involved in lysosome release [27
]. Subsequent work showed that SNAP-23 is involved in all three release events as is syntaxin 2 [26
]. No role has been detected for syntaxin 7 and syntaxin 11’s role remains to be addressed. Based on these data, syntaxin 2 solely mediates dense core granule release but functionally overlaps with syntaxin 4 in mediating α-granule and lysosome release. This dual usage of syntaxin 2 and 4 might explain how differential release of subclasses of α-granules could occur. Perhaps syntaxin 2 promotes release of pro-angiogenic α-granules while syntaxin 4 mediates release of anti-angiogenic granules. This would imply that t-SNARE regulation may be the essential discriminator of which classes of cargo are released. Given the general usage of the v-SNARE, VAMP-8 (discussed below), this is an attractive model to explain the observations of Italiano et al.
] and Ma et al.
]; but, more direct analysis with knockout mice will be required to fully understand how the t-SNAREs could facilitate differential α-granule release.
Assigning roles for the v-SNAREs had been more ambiguous. Initial studies [30
] showed that P-selectin exposure (α-granule release) was sensitive to Tetanus neurotoxin (a heterodimeric, zinc-dependent, endopeptidase specific for VAMP-1, -2, and -3 [36
]). Subsequently, Feng et al.
] showed that an anti-peptide antibody to the divergent N-terminus of VAMP-3 could inhibit α-granule release, suggesting a role for VAMP-3. However, these data were not consistent with the lack of a secretion defect in platelets from VAMP-3−/−
]. Polgar et al.
] showed that the cytoplasmic domains (containing the SNARE motif) of VAMP-3 and VAMP-8 could inhibit release from dense core and α-granules. Since the isolated cytoplasmic domains are promiscuous in their associations in vitro
], it was uncertain whether the inhibitory effects were illustrative of a v-SNARE’s role or of the t-SNARE heterodimer’s binding preferences.
Recent work [24
] has shown that VAMP-8/endobrevin is the primary v-SNARE in mouse platelets. VAMP-8−/−
platelets have a clear secretion defect which is not seen in VAMP-3−/−
platelets or from either permeabilized human or VAMP-3−/−
platelets that have been treated with the catalytic Tetanus toxin light chain. The secretion defect in VAMP-8−/−
platelets however was not complete. α-Granule and lysosome release were significantly affected even at high agonist doses and upon extended incubation; however, dense core granule release eventually approached wild-type levels. The VAMP-8-independent release could be eliminated by treatment with Tetanus toxin, suggesting that the secondary mechanism employed either VAMP-2 or -3. Preliminary experiments, using permeabilized human platelets and VAMP-8-specific Fab fragments, show that VAMP-8’s roles in mouse and human platelets are similar (Ren Q and Whiteheart SW, unpublished data).
The redundancy in v-SNARE usage is analogous to chromaffin cells [40
], where there is a primary v-SNARE (VAMP-2) required for rapid and efficient, agonist-induced release, and a secondary v-SNARE (VAMP-3) which functions, albeit less efficiently, in the absence of the primary v-SNARE. Full ablation of release from chromaffin cells required deletion of both VAMP-2 and VAMP-3. The redundancy in VAMP usage observed in ex vivo
platelet secretion assays is consistent with the in vivo
thrombosis phenotype of the VAMP-8−/−
mice. Upon laser injury, the VAMP-8−/−
mice show a delay in thrombus formation and an increase in embolization, though the overall extent of platelet accumulation is not statistically different from wild-type (Graham et al.
In light of VAMP-8’s role in platelets, it is striking to note recent reports indicating that a Single Nucleotide Polymorphism (SNP) in the human VAMP-8 gene strongly correlates with early onset myocardial infarction (MI) [41
]. The authors suggest that this SNP might affect VAMP-8 expression levels given its position in the gene’s 3’-untranslated region. One could hypothesize that heightened VAMP-8 levels would increase a platelet’s secretion ability, making it more likely to release its cargo and thus hyperthrombotic. However, further biochemical and functional analysis will be required to fully understand the ramifications of this genetic connection between VAMP-8 and early onset MI.
SNARE regulators control how and when SNARE proteins interact. Some are required to stabilize and sort SNAREs to appropriate membranes. Other regulators play key roles in promoting and selecting SNARE-SNARE interactions. Many of these regulators are sensitive to second messengers such as diacylglycerol and Ca2+
and others are substrates for kinases. While much is known about some of these SNARE regulators in other systems, relatively little is known about their precise roles in platelets. However, it has been possible to determine if the families are represented and in some cases whether they are important for platelet secretion. Syntaxin-binding proteins of the Munc18 family (a, b, and c [43
]) are present in platelets. Antibodies that affect Munc18c binding to syntaxin 4 have a positive effect on release from permeabilized platelets [44
], while peptides based on regulatory sequences of Munc18a and c are inhibitory [43
]. Munc18 family members are phosphorylated in platelets and, though the significance of this is not yet clear, phosphorylation does affect Munc18 binding to syntaxins [43
]. At least two members of the Munc13 family are present in platelets (Munc13-1 and Munc13-4; Schraw TD, Ren Q, and Whiteheart SW, unpublished data). In neurons, chromaffin cells, and cytotoxic T-cells, these proteins are important for granule docking/priming and SNARE engagement through interactions with the syntaxin t-SNAREs [45
]. Munc13-4 has been shown to be involved in dense core granule release via
its interaction with the small GTP-binding proteins, Rab27a and b [48
]. Other potential SNARE regulators such as DOC2α nd tomosyn are present in platelets (Ye S, Ren Q, Schraw, TD, and Whiteheheart SW, unpublished data) but their roles in secretion have not yet been probed.
Secretory Machinery as a Potential Therapeutic Target
The goal of anti-thrombotic therapies is a regimen that dampens thrombosis without inducing excessive bleeding. The secretory machinery might prove an effective target to accomplish this goal. In vivo analysis of the VAMP-8−/− mice shows a clear defect in laser-induced arteriole thrombosis but one not as complete as in an HPS mouse (Graham et al. submitted). Thrombi in VAMP-8−/− mice show a significant delay in initiation and well as a lack of stability. However, thrombi do form, and there is no significant difference in tail bleeding times in these animals. These data suggest that targeting the platelet’s primary, secretory machinery might be an effect way to manage thrombosis in vivo and thus limit clot formation without inducing excessive bleeding. Future analysis of other mouse strains with secretion deficient platelets is ongoing to validate this concept and to probe the additional roles that platelet exocytosis might play in the vasculature.