Table 1.

Binding of C domain proteins by flow cytometry

Labeling method	C2 or C1C2 final concentration, μM	% platelets labeled with C2		% platelets labeled with C1C2
		Before activation	Activated	Before activation	Activated
ESH8	1.7	<4	26	18	47
	3.4	4.5	31	24	58
	6.8	5.6	42	31	66
Biotin	0.3	ND	ND	19	39
	0.6	ND	ND	37	63
	1.2	ND	ND	52	81
Direct	0.025	0.1	7	9	32
	0.05	0.2	22	15	73
	0.125	0.5	33	25	83
	0.25	1.1	55	35	91

Results are representative of determinations carried out for at least 4 different platelet preparations. Labeling methods were (a) ESH8 followed by PE-anti–murine IgG, (b) biotinylated C1C2-2296C labeled with PE-streptavidin, and (c) C2-2296C or C1C2-2296C labeled directly with a sulfhydryl-fluorescein probe. C2 binding did not increase when higher concentrations were added (not shown). The percentage of cells labeled for ESH8- and biotin-labeling methods was calculated from flow cytometer output after subtracting background binding and gating as described in “Flow cytometry to quantitate platelet binding.” C2 and C1C2 binding were compared at equimolar concentrations using the same platelet preparation for each set of measurements for ESH8- and fluorescein-labeled experiments.

Activated indicates platelets following SFLLRN activation; ND, not determined.