Slp1 inhibits platelet dense granule secretion in a dose-dependent manner. (A) Ca2+-induced dense granule secretion after incubation of permeabilized platelets with Slp1. Permeabilized platelets were incubated with the indicated concentrations of purified recombinant His6-tagged Slp1 or 1 μM boiled Slp1 and then stimulated with Ca2+ for 1 minute. For baseline serotonin secretion, platelets were left unstimulated in the absence or presence of Slp1. Baseline and Ca2+-induced secretion of dense granules was analyzed by measuring released serotonin (5-HT) as described in “Assay for secretion of platelet dense granules.” The results shown are expressed as mean ± SEM of 3 independent experiments performed in triplicate. *P < .05 (statistically significant); ***P < .001 (statistically significant). (B) GTP-γS–induced dense granule secretion after incubation of permeabilized platelets with Slp1. Permeabilized platelets were incubated with the indicated concentrations of purified recombinant His6-tagged Slp1 or 1 μM boiled Slp1 and then stimulated with GTP-γS for 5 minutes. Baseline and GTP-γS–induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are mean ± SEM of 3 independent experiments performed in triplicate. *P < .05 (statistically significant); ***P < .001 (statistically significant). (C) Ca2+-induced dense granule secretion after incubation of permeabilized platelets with the C2A domain of Slp1. Permeabilized platelets were incubated with 1 μM GST as control, GST-Slp1, GST-C2A, or with a combination of 1 μM GST-Slp1 and 5 μM GST-C2A and then incubated without or with Ca2+ for 1 minute. Baseline and Ca2+-induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are expressed as mean ± SEM of 6 independent experiments performed in triplicate. *P < .05 (statistically significant); **P < .01 (statistically significant).
