α–Granule contents must be released from their intracellular repository in order to achieve their physiologic function. α–Granule contents are release when the α–granule membrane fuses with surface-connected membranes of the OCS or the plasma membrane.69
SNAREs represent the core of the fusion machinery. They are membrane-associated proteins that are oriented to the cytosol (). SNAREs associated with granules are termed vesicular SNAREs (vSNAREs), while those associated with target membranes (e.g., OCS and plasma membrane) are termed tSNAREs. The association of vSNAREs with tSNAREs generates the energy required for membrane fusion.70
Role of SNAREs α–granule membrane fusion
Known platelet vSNAREs include VAMP-2, -3, -7, and -8; while known platelet tSNAREs include syntaxins 2, 4, 7, and 11 and SNAP-23, -25, and -29.71–77
Studies performed in mice deficient in specific VAMP isoforms indicate that VAMP-8 is the dominant vSNARE involved in α–granule release, while VAMP-3 and perhaps VAMP-2 play subordinate roles.76,78
Syntaxins 2 and 4 both appear to function in α–granule release, which is unusual in that two syntaxin isoforms do not typically mediate release of the same granule.72,79
There is no indication that SNAP-25 or SNAP-29 function in α–granule release, while the function of SNAP-23 is well-established.72,79,80
The distribution of SNAREs in platelets provides a basis for several characteristics of α–granule secretion, including homotypic α–granule fusion and the fusion of α–granules with the open canalicular system and plasma membrane.81
In addition to their roles in α–granule release, SNAREs likely participate in platelet granule formation. However, this SNARE function yet to be evaluated in detail.
The function of SNAREs in platelet granule secretion must be tightly regulated so as to prevent the indiscriminant release of α–granule cargo. The (Sec1/Munc) SM proteins function as clamps to regulate the function of SNAREs. SM protein isoforms found in platelets include Munc13-4 and Munc18a, b, and c. Of these, Munc18c has been found to function in α–granule release.82
Munc-18c is complexed primarily to syntaxin-4 in platelets.82
An antibody that prevents association of Munc-18c with syntaxin-4 augments α–granule release, raising the possibility that Munc18c serves as a negative regulatory of SNARE function.82
Munc13-4 functions in dense granule release as a downstream effector of Rab27;83
however, its role in α–granule release is not known. CDCrel-1 is another syntaxin binding protein implicated in the regulation of α–granule release. Platelets from mice lacking CDCrel-1 demonstrate enhanced secretion in response to collagen.84
Many other chaperone proteins that bind to and direct the function of SNARE proteins have been described. A subset of these proteins has been found in platelets, and some of these function in α–granule secretion. NSF is a hexameric ATPase that is essential for most forms of membrane-trafficking, including regulated granule secretion.85
The primary role of NSF is to disassemble SNARE complexes present on the same membrane (cis conformation) so that they are available to interact with cognate SNAREs on opposing membranes (trans conformation). Both inhibitory peptides and antibodies to NSF have been demonstrated to interfere with α–granule release from platelets.79,86
Further evidence suggests that nitric oxide inhibits NSF regulation of α–granule release.87
rotein (SNAP) α–SNAP binds and activates NSF.88
In platelets, wild-type α–SNAP augments granule secretion, whereas a dominant-negative α–SNAP mutant (α–SNAPL294A) and antibodies directed at α–SNAP inhibit granule secretion.
Rab proteins and their effectors are capable of docking opposing membranes and seem to modify SNARE protein function. Rab proteins are the largest branch of the ras superfamily of GTPases. Rabs 3b, 6c, and 8 are phosphorylated upon platelet activation.66,89
Rab GDP dissociation inhibitor (RabGDI), a general inhibitor of RabGTPases, inhibits α–granule but not dense granule release.90
In addition, a dominant-negative mutant of His-tagged Rab4S22N (but not mutant His-Rab3BT36N) inhibits α–granule secretion but fails to affect dense granule release.90
These data raise the possibility that Rab 4 is required for α–granule but not dense granule secretion. In nucleated cells, Rab proteins have been shown to function by binding to large effector proteins that have been proposed to interact with SNARE proteins directly or with proteins, such as NSF and Munc-18c, which mediate SNARE protein function91
The Rab effector proteins in platelets that mediate α–granule release have not yet been identified.