Table 3. Agonist-induced [ 3 H]-DAG... | American Society of Hematology

Table 3.

Agonist-induced [³H]-DAG generation, [Ca²⁺]_i mobilization, and pleckstrin phosphorylation in washed platelets

	[³H]DAG, cpm increase over resting			[Ca²⁺]_i nM, maximum above resting values
Agonist	N	P	N	P	N	P
10 μM ADP	—	—	132 ± 5	133 ± 4	—	—
0.2 U/mL thrombin	2430 ± 308	2237 ± 260	598 ± 6	552 ± 8	7889	8379
10 μg/mL collagen	—	—	—	—	4032	3828
3 μM U46619	—	—	—	—	3940	3700
100 nM PMA	—	—	—	—	5227	5439

	[³H]DAG, cpm increase over resting			[Ca²⁺]_i nM, maximum above resting values
Agonist	N	P	N	P	N	P
10 μM ADP	—	—	132 ± 5	133 ± 4	—	—
0.2 U/mL thrombin	2430 ± 308	2237 ± 260	598 ± 6	552 ± 8	7889	8379
10 μg/mL collagen	—	—	—	—	4032	3828
3 μM U46619	—	—	—	—	3940	3700
100 nM PMA	—	—	—	—	5227	5439

Washed platelets prepared from normal/healthy (N) subjects or from the patients (P) were preloaded with either [³H]arachidonic acid (1 μCi/mL [0.037 MBq] for DAG measurements), fura-2 (2 μM for calcium measurements) or [³²P]PO₄ (1 mCi/mL [37 MBq] for pleckstrin phosphorylation studies) were prepared as described in “Patient, materials, and methods.” For DAG studies, thrombin incubation was for 1 minute and lipids were extracted, followed by separation of neutral lipids using TLC. The separated DAG was identified using appropriate markers and was visualized with iodine before being cut out and analyzed for radioactivity. In phosphorylation studies, agonist stimulation was for 3 minutes. Following electrophoretic separation of [³²P]-labeled proteins, areas on the gel corresponding to the 47-kDa protein (pleckstrin) were cut out and analyzed for associated radioactivity. For 47-kDa phosphorylation measurements the values shown are the averages from 2 separate experiments. The values shown for DAG and calcium measurements are mean values ± SEM (n = 3). — indicates not done.

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