Identification of regions of bovine factor VII essential for binding to tissue factor.
Initiation of the extrinsic blood coagulation pathway is mediated by a complex formed between plasma-derived factor VII/VIIa and cell-derived tissue factor (TF). To identify the site(s) of interaction, zymogen VII and VIIa were enzymatically and chemically modified, and their affinities for TF were estimated by measuring their inhibitory effects on the amidolytic activity enhanced after formation of the VIIa-TF complex. We found that the VIIa-light chain (Ki = 3.5 x 10(-7) M) and its fragment consisting of the gamma-carboxyglutamic acid (Gla)-domain and the first epidermal growth factor (EGF)-like domain (Gla-EGF1 peptide; Ki = 1.0 x 10(-6) M) have an affinity for TF. Therefore, one of the binding sites of VII with TF is probably located in the Gla-EGF1 region. On the other hand, a dansyl-Glu-Gly-Arg chloromethyl ketone-treated Gla-domainless VIIa (Ki = 0.7 x 10(-7) M) showed a high affinity for TF, whereas the corresponding Gla-domainless VII similarly treated showed no binding potential, thereby indicating that binding site(s) other than in the Gla-EGF1 region are present in VIIa but not in VII. Acetylation or carbamylation of the alpha-amino group of the NH2-terminal Ile-153 of VIIa resulted in the loss of binding affinity for TF; such modifications convert VIIa into a zymogen-like inactive form by destroying the salt bridge between Ile-153 and Asp-343 in VIIa. The rate of carbamylation of VIIa was reduced in the presence of TF. Protection of the alpha-amino group of Ile-153 from carbamylation after complex formation was consistent with salt bridge formation between Ile-153 and Asp-343 in the VIIa-TF complex. Therefore, binding of TF with the heavy chain of VIIa may induce a conformational change that brings the alpha-amino group of Ile-153 close to the beta-carboxyl group of Asp-343 to make a stable salt bridge.
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
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Higashi
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Nishimura
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Aita
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Iwanaga
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D003485:Cyanates / Q000737:chemistry; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding; D013925:Thromboplastin / Q000378:metabolism*
D003485:Cyanates; D015055:1-Carboxyglutamic Acid; D013925:Thromboplastin; D002918:Chymotrypsin; D015942:Factor VIIa; C024835:potassium cyanate