The epidermal growth factor-like domains of factor IX. Effect on blood clotting and endothelial cell binding of a fragment containing the epidermal growth factor-like domains linked to the gamma-carboxyglutamic acid region.
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The binding of factor IX to cultured bovine endothelial cells was characterized using isolated domains of bovine factor IX. An NH2-terminal fragment that consists of the gamma-carboxyglutamic acid (Gla) region linked to the two epidermal growth factor (EGF)-like domains bound to the endothelial cells with the same affinity as intact factor IX, indicating that the serine protease part of factor IX is not involved in binding. This fragment also inhibited the factor IXa beta'-induced clotting of plasma at a concentration that would suggest a competition for phospholipid binding sites. However, after proteolytic removal of the Gla region from the fragment, the two EGF-like domains inhibited clotting almost as effectively, suggesting a direct interaction between this part of the molecule and the cofactor, factor VIIIa. Using affinity-purified Fab fragments against the Gla region, the EGF-like domains, and the serine protease part, it was observed that the serine protease part of the molecule undergoes a large conformational change upon activation, whereas the Gla region and the EGF-like domains appear to be unaffected. All three classes of Fab fragments were equally efficient as inhibitors of the factor IXa beta'-induced clotting reaction. Part of factor Va and factor VIIIa have significant sequence homology to a lectin. We therefore investigated the effect on in vitro clotting of the recently identified unique disaccharide Xyl alpha 1-3Glc, that is O-linked to a serine residue in the NH2-terminal EGF-like domain of human factor IX (Hase, S., Nishimura, H., Kawabata, S.-I., Iwanaga, S., and Ikenaka, T. (1990) J. Biol. Chem. 265, 1858-1861). However, no effect on blood clotting was observed in the assay system used. Our results are compatible with a model in which the serine protease part provides the specificity of the binding of factor IXa to factor VIIIa-phospholipid, but that the EGF-like domain(s) also contributes to the interaction of the enzyme with its cofactor. (+info)
Protein structural requirements for Ca2+ binding to the light chain of factor X. Studies using isolated intact fragments containing the gamma-carboxyglutamic acid region and/or the epidermal growth factor-like domains.
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Coagulation factor X is a multidomain proenzyme of a serine protease. Calcium ions bind to the vitamin K-dependent gamma-carboxyglutamic acid (Gla) residues and to a site in the NH2-terminal of two epidermal growth factor (EGF)-like domains. To study structure-function relationships in the NH2-terminal part of factor X and to determine the structure of isolated domains, we have developed methods that allow the subsequent isolation of the first or both EGF-like domains with or without an attached Gla domain from controlled proteolytic digests of the protein. The Ca2(+)-induced changes of the intrinsic protein fluorescence were measured to elucidate whether the isolated fragments retain their native conformation. Changes in the fluorescence caused by Ca2+ binding were found to result from perturbations of the environment of the Trp residue in position 41. Calcium ion binding to the Gla-containing region linked to the NH2-terminal EGF-like domain was identical with that to intact factor X, indicating a native orientation of the ligand binding groups in the fragment. In contrast, the isolated Gla peptide had a lower affinity for Ca2+, suggesting that the NH2-terminal EGF-like domain serves as a scaffold for the folding of the Gla region. Similarly, the presence of the Gla region was found to increase the affinity of the Gla-independent site in the first EGF-like domain for Ca2+. The metal ion-induced resistance against chymotryptic cleavage COOH-terminal of Tyr-44 in intact factor X is similar in the isolated fragment that contains the Gla region linked to one EGF-like domain, indicating a native conformation of the fragment in the presence of Ca2+. Furthermore, the Gla-independent metal ion binding site binds Ca2+ but does not appear to bind Mg2+. (+info)
The gamma-carboxyglutamic acid and epidermal growth factor-like domains of factor X. Effect of isolated domains on prothrombin activation and endothelial cell binding of factor X.
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Factor Xa is the enzymatically active constituent of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. We have isolated fragments, from tryptic digests of factor X, that consists of the gamma-carboxyglutamic acid (Gla) region linked to one or two epidermal growth factor (EGF)-like domains. Calcium ion binding measurements indicated that these fragments have a native conformation. The factor X-GlaEGF fragments inhibit factor Xa-induced blood clotting in a manner suggesting that they compete with factor Xa for phospholipid binding sites. The same conclusion was reached when thrombin generation was studied in a system of purified components (factor Xa, factor Va, prothrombin, phospholipid, and Ca2+). There was no evidence for a strong interaction between the EGF-like domains of factor Xa and factor Va in either system. However, experiments in the purified system without phospholipid indicated a direct, albeit weak, interaction between the Gla region of factor Xa and factor Va and between the COOH-terminal EGF-like domain of factor Xa and factor Va. Using domain-specific Fab fragments, we have confirmed that the conformation of the serine protease region alters dramatically upon activation of factor X. Furthermore, we have demonstrated that the conformation of the Gla region is affected by the activation, whereas the EGF-like domains appear to be unaltered. The association constant for factor X binding to endothelial cells was two orders of magnitude lower than that for binding of factor IX to these cells. Binding of the Gla and GlaEGF fragments suggested Gla-mediated binding to phospholipid rather than binding to a specific receptor. (+info)
Recent estimates of the structure of the factor VIIa (FVIIa)/tissue factor (TF) and factor Xa (FXa) ternary complex.
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Characterization of a Gla-containing protein from calcified human atherosclerotic plaques.
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In this article we describe the isolation of a 4-carboxyglutamic acid (Gla)-containing protein from calcified human atherosclerotic plaques. The protein was extracted from pulverized calcified plaques by demineralization with ethylenediaminetetraacetate and was subsequently purified by anion-exchange fast protein and high-performance liquid chromatography by using ion-exchange and gel-filtration columns. The protein was designated as plaque Gla protein (PGP) and has an apparent mass of 23 kD as estimated from sodium dodecyl sulfate-polyacrylamide gel analysis. By determining the sequence of its first six amino acid residues, the protein was unequivocally demonstrated to be not related to any other known protein. Moreover, no immunological relationship (as tested by Western blot analysis) was found between PGP and other known Gla-containing proteins. (+info)