The cofactor function of the N-terminal domain of tissue factor.
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.
Kittur
Farooqahmed S
FS
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Manithody
Chandrashekhara
C
Morrissey
James H
JH
Rezaie
Alireza R
AR
D000494:Allosteric Regulation; D001120:Arginine / Q000235:genetics; D002384:Catalysis; D003461:Crystallography; D015942:Factor VIIa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D013925:Thromboplastin; D001120:Arginine; D015942:Factor VIIa