The cytoplasmic portions of VAMP 3 and VAMP 8, but not VAMP 2, inhibit granule secretion in permeabilized human platelets.
Streptolysin O–permeabilized platelets were preincubated with various concentrations of soluble rVAMP proteins lacking the C-terminal transmembrane domains. Granule secretion was induced by increasing the amount of Ca++ to 10 μM. (A) Secretion of α granules was monitored by measuring P-selectin expression with flow cytometry. P-selectin expression induced with 1 U/mL thrombin in nonpermeabilized platelets was considered total (100%). (B) Secretion of14C-serotonin from dense granules of platelets. Release of14C-serotonin was measured by scintillation counting. The total 14C-serotonin content (100%) was measured in platelets lysed in 0.4% Triton X-100. All sets of experiments contained samples with no addition of Ca++ for measurement of nonspecific leakage of 14C-serotonin. The amount of nonspecific leakage, typically less than 10% to 15% of the total14C-serotonin, was subtracted from all data points before calculation of the percent secretion. (A,B) The values shown (mean ± SD) are representative results from 3 independent experiments performed in duplicate. In samples with no rVAMP, the α- and dense-granule secretion in permeabilized platelets from different donors represented 30% to 60% of the total inducible by thrombin in nonpermeabilized platelets.
