TABLE 1.

Parameters for binding of FV and FVa to [5F]FFR-NotD and [OG]EGR-FXa

Dissociation constants (K_D ligand), stoichiometric factors (n), initial anisotropy (r_o), maximum anisotropy (r_max), and maximum fluorescence intensity (F_max/F_o) are listed from direct titrations of [5F]FFR-NotD or [OG]EGR-FXa with FV or FVa. Experiments were performed in either the absence or presence of 50 μm PC/PS, 350 μm PC/PS/PE, or 50 μm PC/PS/PE SUVs. Results of the competitive binding studies for the indicated ligand and competitor were analyzed by simultaneous fitting of the titration in the absence and presence of two fixed concentrations of competitor to obtain the dissociation constant for the ligand (K_D ligand) and competitor (K_D competitor), with the stoichiometric factor for the ligand interaction (n) and competitor interaction (m) experimentally determined. For analysis of [OG]EGR-FXa binding to FV and [OG]EGR-FXa competition with FFR-NotD for FVa, n was fixed at 1. Experimental error in the parameters represents ± 2 S.D. Binding studies were performed and the data were analyzed as described under “Experimental Procedures.”

Probe	Ligand	Competitor	SUVs	KD ligand	n	KD competitor	m	ro	rmax	Fmax/Fo
				nm	mol/mol	nm	mol/mol
[5F]FFR-NotD	FVa			0.002 ± 0.01	0.79 ± 0.02			0.22 ± 0.002	0.27 ± 0.008	1.38
[5F]FFR-NotD	FVa	Native NotD		0.002 ± 0.04	0.76 ± 0.06	0.002 ± 0.04	1.6 ± 0.01	0.22 ± 0.002	0.27 ± 0.0008	1.34
[5F]FFR-NotD	FVa		+a	0.01 ± 0.02	0.85 ± 0.06			0.22 ± 0.002	0.27 ± 0.001	1.43
[5F]FFR-NotD	FV			12 ± 1	0.79 ± 0.08			0.21 ± 0.002	0.27 ± 0.001	1.24
[5F]FFR-NotD	FV		+a	0.22 ± 0.06	0.69 ± 0.06			0.22 ± 0.002	0.28 ± 0.001	1.31
[OG]EGR-FXa	FV		+b	1110 ± 250	1			0.23 ± 0.005	0.38 ± 0.001	1.06
[OG]EGR-FXa	FVa	FFR-NotD	+a	0.3 ± 0.2	1	0.011 ± 0.008	1.4 ± 0.08	0.23 ± 0.004	0.29 ± 0.004	1.50
[OG]EGR-FXa	FVa	FFR-NotD	+c	0.4 ± 0.3	1	0.007 ± 0.007	1.37 ± 0.07	0.23 ± 0.004	0.29 ± 0.004	1.54
[OG]EGR-FXa	FVa		+c	0.8 ± 0.5	1.26 ± 0.07			0.22 ± 0.004	0.28 ± 0.002	1.44

^a 50 μm PC/PS.

^b 350 μm PC/PS/PE.

^c 50 μm PC/PS/PE.