Abstract
To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (1982) J. Clin. Invest. 69, 1253-1260). The three variant prothrombins, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from prothrombin in molecular weight, amino acid composition, and NH2-terminal amino acid sequence, with the exception of Gla residues. Variant prothrombin 1, with 8 Gla residues, had 66% of the coagulant activity of prothrombin, one high affinity metal-binding site (Kd = 15 nM), and three lower affinity sites (Kd = 2.7 microM); prothrombin contained two high affinity (36 nM) and four lower affinity sites (Kd = 1 microM). Ca(II) induced a 23% decrease in the intrinsic fluorescence of variant prothrombin 1 fragment 1, compared to a 35% decrease in that of prothrombin fragment 1. The phospholipid binding activity of variant prothrombin 1 was 44% that of prothrombin. Variant prothrombin 2 and variant prothrombin 3, with 4 and 6 Gla residues, respectively, had about 5% of prothrombin coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant prothrombin 3 fragment 1 and variant prothrombin 2 fragment 1 demonstrated 18 and 13% of Ca(II)-induced fluorescence quenching, respectively. Abnormal prothrombin, with 1 Gla residue, had 8% of prothrombin coagulant activity, a single lower affinity (1 microM) metal-binding site, and 13% Ca(II)-induced fluorescence quenching of the fragment 1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin. Most, if not all, of the Gla residues are required for complete prothrombin function, and the prothrombin coagulant activity correlates to the phospholipid binding activity of the prothrombin species.
Highlights
To study thespecific role of y-carboxyglutamic acid found in a class of calcium-binding proteins which require (Gla)residues in prothrombin, we have isolated a series vitamin K for their complete synthesis
We considered that thisabnormality single high affinity and twloower affinity metal-bind- might give rise to y-carboxyglutamic acid-deficient prothroming sites and exhibitendo phospholipid binding activ- bins more amenable to purification and characterization than ity
Affinity (1 MM) metal-bindingsite,and 13% Ca(I1)induced fluorescence quenching of the fragment1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin
Summary
F1 t to abnormal prothrombin (variant 2). From densitometer scans of these gels stained with Coomassie Blue R-. In the present of EDTA, the migration of variant prothrombins was indistinguishable from that of prothrombin and abnormal prothrombin. Variant prothrombins 2 and 3 were eluted by decreasing the calcium concentration in the buffer to 1mM. Variant prothrombin 1 remained bound to the column until eluted by buffer containing mM EDTA. These partially purified variants were purified to homogeneity by preparative gel electrophoresis in the presence of calcium (Fig. 1B). The final yields of the variant prothrombin species from 200 ml of plasma were as follows: variant prothrombin 1, 1.6mg; variant prothrombin 2, 1.4mg; and variant prothrombin 3, 0.9 mg. The molecular weights of thevariant species, as measured by their migration in dodecyl sulfate gels,were identical to that of prothrombin and abnormal prothrombin (Fig. 2).
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