Immunological and functional analyses of the extracellular domain of human tissue factor.
Tissue factor (TF) initiates the extrinsic pathway of blood coagulation via formation of an enzymatic complex with coagulation factor VII/VIIa (FVII/VIIa). Although FVII is the only known ligand for TF, several reports in recent years have shown that the function of TF may not be limited to serving as a trigger of coagulation but that TF could also play a role in cellular signaling, metastasis, adhesion and embryogenesis. To explore the loci of the extracellular domain of TF important for its function, we analyzed the functional and immunological epitopes of TF1-219 by the use of both E. coli expressed TF variants encompassing various portions of the extracellular domain of TF and different anti-TF monoclonal antibodies (mAbs). N- and C-terminally truncated TF variants were analyzed for their VIIa-dependent procoagulant activity (PCA). The results obtained are in agreement with previously performed mutant and structural analyses of the interaction of FVII/FVIIa with the extracellular domain of TF. In addition, we observed that combination of two TF variants, Ec-TF1-122 and Ec-TF120-219, yields a soluble and active two-chain TF molecule with remarkable PCA. The reaction patterns of anti-TF mAbs with truncated TF variants and synthetic TF-derived peptides demonstrated that at least three distinct conformation-dependent epitope areas of TF (residues 1-25, 175-202, and 181 -214, respectively) are detected by these mAbs raised against native TF. In fact, mAbs, which are directed to the same epitope area of TF, behave very similar in various applications including immunohistochemistry and clotting tests. Since mAbs directed to the C-terminal epitope area of TF (residues 181-214) influence TF activity independent of FVIIa-binding, this region may be involved in functions of TF distinct from haemostasis.
Magdolen
V
V
Frauenklinik der Technischen Universität München, Germany.
Albrecht
S
S
Kotzsch
M
M
Haller
C
C
Bürgle
M
M
Jacob
U
U
Grosser
M
M
Kessler
H
H
Graeff
H
H
Müller
M
M
Schmitt
M
M
Luther
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D003029:Coagulants / Q000276:immunology / Q000378:metabolism; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003029:Coagulants; D018985:Epitopes, B-Lymphocyte; D011993:Recombinant Fusion Proteins; D013925:Thromboplastin