Abstract
Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF(2-219) in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N- and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg(74) mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.
Highlights
Tissue factor (TF)1 is an integral membrane cofactor that upon exposure to circulating blood binds with high affinity to factor VIIa to catalyze the rapid activation of procoagulant zymogens factors IX and X, thereby initiating the blood clotting cascade [1,2,3]
All three domains of TF are required for the physiological function of the cofactor, previous studies have indicated that the two extracellular domains expressed by recombinant DNA methods as a soluble protein can bind to factor VIIa (fVIIa) with a high affinity to enhance both the amidolytic and proteolytic activities of the protease (6 –9)
The structural data have indicated that the extracellular domains of TF make extensive interactions with both the light and heavy chains of fVIIa [10]
Summary
Construction, Mutagenesis, and Expression of Soluble Tissue Factor in Bacteria—Construction and expression of soluble TF lacking both the trans-membrane and cytoplasmic domains (sTF2–219) in the pINIIIpelB bacterial periplasmic expression/purification vector system has been described previously [6]. FVIIa (0.3 nM), PC/PS vesicles (0.3 mM), and saturating concentrations of sTF2–219 mutants (at least 10ϫ Kd(app) values) were incubated for 10 min in 96-well assay plates and the reactions were initiated by adding increasing concentrations of factor X (20 –2630 nM). The absorbance at 405 nm was monitored over 5 min using a Vmax kinetic microplate reader, and the initial rates of chromogenic substrate hydrolysis (⌬A405/min) were converted to the nanomolar of product by reference to a standard curve prepared with purified human factor Xa. The apparent Km and kcat values for substrate hydrolysis were calculated from the Michaelis-Menten equation, and the catalytic efficiencies were expressed as the ratio of kcat/Km. Factor IX Activation—Steady-state kinetics of factor IX activation by fVIIa in complex with wild-type and mutant sTF2–219 derivatives was performed by the same methods described above for factor X. Following incubation for 5 min at room temperature, the reactions were terminated by adding 50 mM EDTA and the rate of factor Xa generation was determined as described above
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