Partially carboxylated prothrombins. I. Comparison of activation properties and purification of 1- and 0-carboxyglutamyl variants.

In addition to the 7-, 5- and 2-carboxyglutamyl varieties of dicoumarol-induced prothrombins (Malhotra, O.P. (1979) Thromb. Res. 15, 427-463), we have isolated two more atypical prothrombins, one containing 1.1 +/- 0.1 gamma-carboxyglutamic acid, '1-carboxyglutamyl prothrombin,' and the other less than 0.2, '0-carboxyglutamyl prothrombin.' Both variants showed a single component by analytical polyacrylamide-gel electrophoresis in the absence and in the presence of sodium dodecyl sulfate and contained antigenic activity indistinguishable from that of normal prothrombin. The pI of both proteins as assessed by electrofocusing was 4.835 +/- 0.015, compared with 4.58 for 10- and 7-, 4.75 for 5- and 4.81 for 2-carboxyglutamyl materials. By the two-stage prothrombin assay procedure, the 1- and 0-carboxyglutamyl variants generated thrombin, respectively 19 and 13% of normal prothrombin, and their activation times ranged from 4 to 7 h, compared with 7 min for normal. Kinetic studies, utilizing the one-stage coagulation assay, showed that both Km and tmin (minimal clotting time) increase proportionally with the decrease of gamma-carboxyglutamyl residues (from 10 to 7, 5, 2, 1 and 0 gamma-carboxyglutamic acids). Each of the five (partially) acarboxy prothrombins owe their clotting activity to gamma-carboxyglutamyl residues and not to the presence of some normal prothrombin molecules.