Abstract
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.
Highlights
From the $Departmentof Immunology and the 7Departmentof Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037 and the **NovoNordisk Biopharmaceutical Division, DK-2820 Gentofte, Denmark
This monoclonal antibody (mAb) does not interfere with VI1 binding to cell surface tissue factor (TF) (15).binding of detergent-solubilized lZ5I-TFor soluble 1251-TF1.21t9o the mAb TF9-10H10 was not inhibitedin the presence of 300 nM VI1(Fig. 1).If VI1 binding to TF is analyzed, the TF .VI1 complexes bound to themAb should reflect the concentration of complexes formed in solution
The preferential cleavage of factor X that is associated with the phospholipid surface may contribute largely to the increase in catalytic activity of the TF.VIIa complex in a phospholipid environment(6)
Summary
VI1 (VII) to its receptor tissue factor (TF)’ (1, 2) and the subsequent rapidconversion of bound VI1 to the active se protease factor VIIa (VIIa) (3) is the trigger for cell surface activation of the coagulationcascades. Analysis of IZ5I-TFand of '251-TF1.21b9inding to TF9-lOH10 was performed in the presence of 5 mM CaC12,0.5% BSA in TBS with or without 300 nM VII. Residual bound radioactivity was determined after washes as described above This analysis demonstrated a slow dissociation of the bound T F . Characterizationof the Anti- VI1mAb F4-2.1B-Binding of solution phase VI1 to mAb F4-2.1Bwas analyzed by coating Falcon MicroTest 111 plates with purified IgG at 10 pg/ml overnight, followedby blocking with 5% nonfat dry milk in TBS. Des-(l-38)-VIIa (5 nM) were incubated for 60 min followed by rapid washes with ice-cold buffer. VI1 binding to immobilized phospholipid was performed as described (6).Inhibition of VI1 binding by F4-2.1B was tested by including the mAb (1p ~ in) the incubation mixture of VII, 5 mM CaC12,and theimmobilizedphospholipid. 3.5 mM CaCL was added which yielded approximately 1 mM free CaZ+,since 2.5 mM phosphate precipitated upon the addition of CaCI2.The second addition of 9 mMCa" yielded a net Ca2+concentration of 10 mM
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