Effect of antihuman cellubrevin antibody on Ca++-induced P-selectin surface expression in SL-O–permeabilized platelets.
(A) Gel-filtered platelets (20 μL/sample) in PIPES/EGTA buffer supplemented with 5 mM MgATP were permeabilized with 3 U/mL SL-O in the presence or absence of the indicated concentration of antihuman cellubrevin antibody at pH 6.9 for 20 minutes. Platelets were then exposed to 10 μM Ca++ for 10 minutes. P-selectin surface expression was assayed by incubating the sample (10 μL) with a phycoerythrin-conjugated AC1.2 anti–P-selectin antibody (5 μL) for 20 minutes. PBS (500 μL) was added to the sample, and the platelets were analyzed immediately by flow cytometry. Data are expressed as the percentage inhibition of P-selectin expression in samples exposed to anticellubrevin antibody compared with samples exposed to buffer alone. Error bars represent the SE of 3 to 6 independent experiments. (B) Gel-filtered platelets in PIPES/EGTA buffer supplemented with 5 mM MgCl2 were preincubated with either no addition or 1 μg/mL human cellubrevin peptide in the presence or absence of 25 μg/mL antihuman cellubrevin antibody, as indicated. Platelets were then permeabilized with 3 U/mL SL-O at pH 6.9 for 20 minutes and were subsequently exposed to buffer or 10 μM Ca++ for 10 minutes. Platelets were assayed for P-selectin expression by flow cytometry. Error bars represent the SE of 3 to 6 independent experiments.
