Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions.
Protein-phospholipid as well as protein-protein interactions may be critical for tight binding of the serine protease factor VIIa (VIIa) to its receptor cofactor tissue factor (TF). To elucidate the role of protein-protein interactions, we analyzed the interaction of VII/VIIa with TF in the absence of phospholipid. Binding of VII occurred with similar affinity to solubilized and phospholipid-reconstituted TF. Lack of the gamma-carboxyglutamic acid (Gla)-domain (des-(1-38)-VIIa) resulted in a 10- to 30-fold increase of the Kd for the interaction, as did blocking the Gla-domain by Fab fragments of a specific monoclonal antibody. These results suggest that the VII Gla-domain can participate in protein-protein interaction with the TF molecule per se rather than only in interactions with the charged phospholipid surface. Gla-domain-independent, low affinity binding of VII to TF required micromolar Ca2+, indicating involvement of high affinity calcium ion binding sites suggested to be localized in VII rather than TF. Interference with Gla-domain-dependent interactions with TF did not alter the TF. VIIa-dependent cleavage of a small peptidyl substrate, whereas the proteolytic activation of the protein substrate factor X was markedly decreased, suggesting that the VIIa Gla-domain not only participates in the formation of a more stable TF. VIIa complex but contributes to extended substrate recognition.
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Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Kalnik
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MW
Lund-Hansen
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Edgington
T S
TS
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D015942:Factor VIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007457:Iodine Radioisotopes; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000378:metabolism; D012996:Solutions; D013925:Thromboplastin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007457:Iodine Radioisotopes; D010455:Peptides; D012996:Solutions; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium