Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymic activity of the complex

Abstract

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.

Comparison of natural and recombinant tissue factor proteins: new insights

“…Owing to the threshold nature of the TF-triggered thrombin generation, this < 5-fold difference in the catalytic efficiency leads to dramatic changes in thrombin generation in complex systems representing the blood coagulation process (Figure 4 ). These data indicate that, in contrast to previously published statements suggesting that post-translational modifications have no effect on TF activity (Paborsky and Harris , 1990 ;Waxman et al , 1992 ), TF glycosylation has a pronounced effect on factor X proteolysis by the factor VIIa-TF complex and, as a consequence, on thrombin generation.…”

“…A possible lack of interest to TF glycosylation could be explained by the influence of several studies published approximately two decades ago. In those studies, the carbohydrates were reported to be insignificant for the activity of TF (Waxman et al , 1992 ;Stone et al , 1995 ;Rickles et al , 1996 ) owing to a similar activity of the non-glycosylated recombinant protein from Escherichia coli and the glycosylated one from kidney 293 cells (Paborsky and Harris , 1990 ). In contrast to those publications, data from other laboratories (Pitlick , 1975 ;Shands , 1985 ;Bona et al , 1987 ) suggested that carbohydrates could play a role in the activity of TF.…”

Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

“…Our results (Table 1) on intact FVII, intreaction with detergent solubilized TF (KTF = 0.6 + 0.1 nM) and with TFl_21 s (Kaw = 6.0 -+ 0.4 nM) agrees reasonably well with previous values [22,27]. Much lower values (KvF = 7--27 pM) have, however, been obtained in recent studies with TF in (PC/PS) phospholipid vesicles [27,30] thus indicating that membrane constituents may contribute to stabilization of the complex.…”

“…It has been shown that binding of TF to factor VII~ in the presence of Ca 2+ induces a pronounced enhancement of the activity with peptidyl substrates [25], and this [5,16,22,[26][27][28] as well as other methods [29,30] have been used for the assessment of the dissociation constant, KTF. Our results (Table 1) on intact FVII, intreaction with detergent solubilized TF (KTF = 0.6 + 0.1 nM) and with TFl_21 s (Kaw = 6.0 -+ 0.4 nM) agrees reasonably well with previous values [22,27].…”

Fluorogenic assay of activated factor VII. Plasma factor VIIa levels in relation to arterial cardiovascular diseases in Japanese.

“…Recombinant soluble human tissue factor (sTF, residues 1 to 217) was expressed in yeast and purified according to a published method. 33 The sTF concentration was determined by using a 1% extinction coefficient at 280 nm of 14.9 34 and a molecular weight of 37 000. 33 The clot-based assay for FVIIa was originally developed by Morrissey et al 31 We modified it as follows.…”