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

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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 Ca2+‐dependent. Ca2+‐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 Ca2+. Experiments evaluating platelet α‐granule secretion demonstrated that incubation of permeabilized platelets with 10 μM Ca2+ prior to exposure to ATP inhibited ATP‐dependent α‐granule secretion from permeabilized platelets. SNAP‐23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca2+‐mediated degradation of SNAP‐23 and reversed Ca2+‐mediated inhibition of ATP‐dependent α‐granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP‐23 and inhibits α‐granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.

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