![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=1724255777&Signature=qsy-t-ltqf~FNj2jlKa~KZ8JiwW8DeiG5Jcpd9zcS3ynLtTQdiW10Wiw50AM0zkKluDN4LAJgel2xBFuXytRZ74fkVrohabViW4ZyYPl6632N-Z3~bchZP3ek-BpZrhs3wnH4o5Ljm47ufW1EbBFLx3cka0RS5vljzQWL5KE1SSbBXd55eGUKq2~GowdPnPvTICJ1V35dwSvsvVsdRzaigmgEmREW7LjNNOXDS6zTnL16mDDpvee-EKMkilZpfG-3ZteNloYRlyrubQdhoeKxqLk-UecQPljwve20cjrxVV48qaHJnG1yzRuW8-tw-JOk7dskwYAgR2meIiqoGu1Lw__&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).
