SNARE protein degradation upon platelet activation: Calpain cleaves SNAP‐23

Abstract

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.

Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release

“…There are several reasons for this series of negative results, but the most likely explanation is that synaptobrevin/VAMP-2 is expressed at low levels and is degraded during sample preparation. Consistently, Lai and Flaumenhaft 29 have shown that cellubrevin/VAMP-3 can be degraded in activated platelets by an unknown protease, therefore degradation appears to be a key problem when examining platelet v-SNAREs. Taking this into consideration, we now demonstrate that synaptobrevin/ VAMP-2 is present in human platelets.…”

IκB kinase phosphorylation of SNAP-23 controls platelet secretion

“…The degradation of SNAP-23 and phospho-SNAP-23 in A23187-stimulated platelets is consistent with reports that calpain cleaves SNAP-23 in highly activated platelets. 36,37 m-Calpain cleaves between Gly164 and Asn165 (supplemental Figure 1) but Ser95 phosphorylation does not affect cleavage. These data confirm that SNAP-23 is phosphorylated on Ser95 and show that SNAP-23 phosphorylation occurs in response to a range of agonists.…”

The actin cytoskeleton differentially regulates platelet α-granule and dense-granule secretion

“…40 If platelets are permeabilized for 15 minutes or more in the absence of ATP, they will no longer secrete granules in response to Ca 2ϩ ( Figure 7). 41 Loss of responsiveness is secondary to diffusion of cytosolic ATP from the permeabilized platelet and can be reversed by addition of ATP to the incubation mixture ( Figure 7). If the resting cytoskeleton serves as a barrier to granule secretion, we reasoned that disruption of this barrier may substitute for ATP in priming platelets for granule secretion triggered by Ca 2ϩ or GTP-␥-S.…”