Figure 6
Figure 6. Rap1GAP2 enhances platelet dense granule secretion by binding to Slp1. (A) Ca2+- and GTP-γS–induced dense granule secretion after incubation of permeabilized platelets with Rap1GAP2. Permeabilized platelets were incubated without or with the indicated concentrations of purified recombinant His6-tagged Rap1GAP2 and then stimulated with Ca2+ for 1 minute or with GTP-γS for 5 minutes. For baseline serotonin secretion, platelets were left unstimulated (−stim) in the absence or presence of Rap1GAP2. Baseline and 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). (B) Ca2+- and GTP-γS–induced dense granule secretion after incubation of permeabilized platelets with mutant Rap1GAP2 that is deficient in Slp1 binding. Permeabilized platelets were incubated without or with 1 μM purified recombinant His6-tagged Rap1GAP2ΔEVTKTT mutant, which does not bind to Slp1. Then, platelets were stimulated with Ca2+ for 1 minute or with GTP-γS for 5 minutes. For baseline serotonin secretion, platelets were left unstimulated (-stim) in the absence or presence of Rap1GAP2ΔEVTKTT. Baseline and induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are mean ± SEM of 7 independent experiments performed in triplicate. (C) Ca2+-induced dense granule secretion after incubation of permeabilized platelets with Rap1GAP2 peptides. Permeabilized platelets were incubated with Rap1GAP2 wild-type peptide (RG2wt peptide) or Rap1GAP2 peptide lacking the Slp1-binding TKXT motif (RG2ΔEVTKTT) at 100 μM each. The solvent dimethyl sulfoxide was used as control. Baseline and Ca2+-induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are mean ± SEM of 5 independent experiments performed in triplicate. *P < .05 (statistically significant).

Rap1GAP2 enhances platelet dense granule secretion by binding to Slp1. (A) Ca2+- and GTP-γS–induced dense granule secretion after incubation of permeabilized platelets with Rap1GAP2. Permeabilized platelets were incubated without or with the indicated concentrations of purified recombinant His6-tagged Rap1GAP2 and then stimulated with Ca2+ for 1 minute or with GTP-γS for 5 minutes. For baseline serotonin secretion, platelets were left unstimulated (−stim) in the absence or presence of Rap1GAP2. Baseline and 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). (B) Ca2+- and GTP-γS–induced dense granule secretion after incubation of permeabilized platelets with mutant Rap1GAP2 that is deficient in Slp1 binding. Permeabilized platelets were incubated without or with 1 μM purified recombinant His6-tagged Rap1GAP2ΔEVTKTT mutant, which does not bind to Slp1. Then, platelets were stimulated with Ca2+ for 1 minute or with GTP-γS for 5 minutes. For baseline serotonin secretion, platelets were left unstimulated (-stim) in the absence or presence of Rap1GAP2ΔEVTKTT. Baseline and induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are mean ± SEM of 7 independent experiments performed in triplicate. (C) Ca2+-induced dense granule secretion after incubation of permeabilized platelets with Rap1GAP2 peptides. Permeabilized platelets were incubated with Rap1GAP2 wild-type peptide (RG2wt peptide) or Rap1GAP2 peptide lacking the Slp1-binding TKXT motif (RG2ΔEVTKTT) at 100 μM each. The solvent dimethyl sulfoxide was used as control. Baseline and Ca2+-induced secretion of dense granules was analyzed by measuring released serotonin (5-HT). The results shown are mean ± SEM of 5 independent experiments performed in triplicate. *P < .05 (statistically significant).

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