TABLE 4. Kinetic and dissociation equilibrium constants for the interactions of the normal pentasaccharide with native, latent and cleaved antithrombin at 10°C, ionic strength 0.15 and pH 7.4.

Kinetic constants were determined by stopped-flow fluorimetry, monitoring the change in tryptophan or TNS fluorescence induced by pentasaccharide binding, as described in Experimental Procedures. k_on and k_off were determined form the slopes and ordinate intercepts of linear plots of observed pseudo-first order rate constants, k_obs, versus pentasaccharide concentration (Fig. 3A). k_on and k_off values ± SE were calculated by linear regression. K₁, k₊₂ and k_-2 were determined by fitting the data from the plots of Fig. 3B to the rectangular hyperbolic equation (37). These values ± SE were calculated by nonlinear regression. K_D values are the means ± SE of three fluorescence titrations conducted at 10°C as described in Experimental Procedures

Antithrombin form	kon (106 M-1s-1)	koff (s-1)	k-2 (s-1)	K1 (μM)	k+2 (s-1)	Kd (nM)
Native	33 ± 1	0.1 ± 0.2	0.3 ± 0.9	6 ± 0.4	200 ± 5	4 ± 2
Latent	30 ± 4	1.5 ± 0.9	3 ± 1	9 ± 1	200 ± 15	150 ± 50
Cleaved	14 ± 6a	2 ± 2 b	9 ± 2	14 ± 4	200 ± 30	160 ± 90

^aCalculated from K₁ and k₊₂

^bCalculated from K₁, k₊₂ and K_D