![Fig. 5. The effects of Ca++, -SNAP, and NSF on [3H]5-HT release. / (A) Increasing Ca++(10−8-10−3 mol/L final) was used to induce exocytosis in the presence of 50 μg/mL of wild-type α-SNAP or mutant α-SNAP–L294A. The released [3H]5-HT was measured as in Figure 4. In an additional titration, 30 μg/mL of apyrase was added to deplete ATP from the reaction (n = 6). (B) In a separate, summary experiment, the effects of 50 μg/mL bovine wild-type α-SNAP, 60 μg/mL anti–α-SNAP antibody, and 60 μg/mL rabbit IgG on the 10 μmol/L Ca++-triggered [3H]5-HT secretion were compared with the control (100%) (n = 6). (C) [3H]5-HT–labeled and SLO-permeabilized platelets were incubated with increasing amounts of the 2E5 monoclonal antibody (anti-NSF; 0-0.32 mg/mL). The release of [3H]5-HT was measured as before and normalized to the control (no addition) (n = 5). (D) The 2E5 inhibitory effect can be reversed by the addition of recombinant NSF. Radiolabeled and SLO-permeabilized platelets were incubated on ice for 30 minutes with buffer (control), 80 μg/mL 2E5, 80 μg/mL plus 0.75 mg/mL recombinant NSF, or 0.75 mg/mL recombinant NSF alone. Platelets were activated by 10 μmol/L Ca++, and the release of [3H]5-HT was measured and normalized to the control group (n = 5).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/95/3/10.1182_blood.v95.3.921.003k17_921_929/6/bloo00317005x.jpeg?Expires=1735514422&Signature=x8mtCcyrC299QnKTBIsJ0dRnNYLZMPA1QMvL42FpISSL3j~oTq4njl-pUgCLvWazDmjslLGN2HwtxAj-QK~uiT5NGx4zAwYloaEVsGmzVPMMRXSn39ILOM1Lc1F2W65sySVVKgft9Dvp6e6~T8tt6aDJILGshg68L6z-njOQEW5tTaSwVUB~TGHbeMveaHkYyEPsX~FLwUO5F2o9mWJdxA8augHMgk71o7OdSFNokR2q5muV5HMtfVkwVWiA4FxSCWGuUOr34XOJ3XZKRNtoRymQvLxTBu~qYfhRNmccltzPQAKYgj1xP7lO31I8crZFLfUFvHDc-bEvlMlj9cz0Dw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The effects of Ca++, -SNAP, and NSF on [3H]5-HT release.
(A) Increasing Ca++(10−8-10−3 mol/L final) was used to induce exocytosis in the presence of 50 μg/mL of wild-type α-SNAP or mutant α-SNAP–L294A. The released [3H]5-HT was measured as in Figure 4. In an additional titration, 30 μg/mL of apyrase was added to deplete ATP from the reaction (n = 6). (B) In a separate, summary experiment, the effects of 50 μg/mL bovine wild-type α-SNAP, 60 μg/mL anti–α-SNAP antibody, and 60 μg/mL rabbit IgG on the 10 μmol/L Ca++-triggered [3H]5-HT secretion were compared with the control (100%) (n = 6). (C) [3H]5-HT–labeled and SLO-permeabilized platelets were incubated with increasing amounts of the 2E5 monoclonal antibody (anti-NSF; 0-0.32 mg/mL). The release of [3H]5-HT was measured as before and normalized to the control (no addition) (n = 5). (D) The 2E5 inhibitory effect can be reversed by the addition of recombinant NSF. Radiolabeled and SLO-permeabilized platelets were incubated on ice for 30 minutes with buffer (control), 80 μg/mL 2E5, 80 μg/mL plus 0.75 mg/mL recombinant NSF, or 0.75 mg/mL recombinant NSF alone. Platelets were activated by 10 μmol/L Ca++, and the release of [3H]5-HT was measured and normalized to the control group (n = 5).
