Studies of secretion using permeabilized platelets.
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Tara W Rutledge
Publications and source records attributed to Tara W Rutledge.
Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.
It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [3H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice.
Animals↗
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.
Amino Acid Sequence↗