Om P. Malhotra

Purification and characterization of dicoumarol-induced prothrombins. Barium citrate atypical (7-GLA) prothrombin

Abstract Barium citrate atypical prothrombin purified by our modified procedure, showed a single component by several criteria of purity. The variant was similar to normal prothrombin in: molecular mass (70,000 daltons), antigenic activity, carboxyl terminal glutamic acid, carbohydrate composition, pI (pH 4.55 or 4.6), and thrombin generation by Echis carinatus venom. Despite these similarities, in the presence of thromboplastin, calcium ions, and factors V, VII, and X, the variant activated slowly taking 90 min rather than 7 min for normal protein. Specific bioactivity approximated 52% of normal. This anomaly is due to the decreased ability of the atypical to bind calcium ions, which resulted from the reduction of 10 γ -carboxyglutamyl residues (Gla) for normal prothrombin to 7 residues for atypical prothrombin. Therefore, the electrophoretic mobility of the variant was influenced by calcium ions to a lesser extent than that of the normal prothrombin.

Purification and characterization of dicoumarol-induced prothrombins. III. Alumina pH 4.6 atypical (2-Gla)+ variant

Abstract Dicoumarolized bovine plasma containing 15% normal biological and 100% normal immunological activity, when treated consecutively with barium citrate and barium oxalate, removed 40% of the plasma prothrombin. The remaining antigen was removed by alumina Cγ gel with approximately three-fifths of the antigenic material precipitating at 50 to 67% ammonium sulfate saturation. The “alumina” variant which was highly purified from this fraction showed a single component by several physicochemical criteria of purity and contained 2 γ-carboxyglutamyl residues (Glas). Its antigenic activity was comparable to that of normal prothrombin. In the presence of Echis carinatus (EC) venom, its thrombin-generating capacity equalled that of normal; however, by bioassay it approximated only 18% of that of normal prothrombin. Under physiological conditions, the alumina (2-Gla) variant activated in 9 hr ( vs 3 hr for 5-Gla, 1.5 hr for 7-Gla, and 7 min for 10-Gla (normal) prothrombin). Mobility by disc-gel electrophoresis was somewhat slower and pH (pH 4.8 or 4.82) by electrofocusing was somewhat greater in comparison with our other prothrombins. By agar gel electrophoresis, alumina prothrombin moved between the α 1 and α 2 macroglobulins, with its mobility seemingly unaffected by calcium ions. Further, we observed that this variant bound no calcium ions by the ultrafiltration technique.

Purification and characterization of dicoumarol-induced prothrombins. II. Barium oxalate atypical (5-Gla) variant

Abstract Purified dicoumarol-induced prothrombin, which adsorbed onto barium oxalate but not onto barium citrate, showed a single component by several criteria of purity and contained 100% antigenic activity of normal prothrombin. In the presence of Echis carinatus venom, the barium (Ba) oxalate variant generated thrombin comparable to that from normal; but when activated physiologically, the thrombin yield was 30% of normal and about 58% of barium (Ba) citrate atypical protein. The time required for bio-activation was approximately 3 hr, compared to 7 min for normal and approximately 90 min for Ba citrate atypical prothrombin. The differences in physiological activation were due to a reduced content of γ -carboxyglutamic acids; five were present in the Ba oxalate variant compared to ten for normal and seven for the Ba citrate variant. This Gla reduction decreased the Ba oxalate variants calcium-binding capacity and electrophoretic mobility.

Partially carboxylated prothrombins II. Effect of γ-carboxyglutamyl residues on the properties of prothrombin fragment 1

Purified prothrombin fragments 1 derived from normal (10-carboxyglutamyl) and dicoumarol-induced 7-, 5-, 2-, 1-, and 0-carboxyglutamyl prothrombins contained the same number of gamma-carboxyglutamyl residues as their respective parent molecules. The effect of gamma-carboxyglutamyl residues was more pronounced on the fragments 1 than on the prothrombins. Consequently, the pI values of the fragments 1 were very well differentiated, with normal fragment 1 focusing at pH 3.58, 7-carboxyglutamyl fragment 1 at 3.79, 5- at 3.97, and 2- at pH 4.29. Similarly, by agar gel electrophoresis, normal fragment 1 was the most mobile, followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1. Because of Ca2+ being bound to the carboxyglutamyl residues, the electrophoretic mobility of normal fragment 1, in the presence of Ca2+, was reduced the most, followed by 7-, 5- and then 2-carboxyglutamyl fragment 1, while the mobilities of the 1- and 0-carboxyglutamyl fragments 1 were not affected. In contrast to their parent molecules, all of the fragments 1 in the presence of EDTA gave negative immunoprecipitation reactions against antibodies produced against normal prothrombin. In the presence of Ca2+, conversely, the fragments 1 containing comparable amounts of antigenic activity all gave positive reactions. However, the intensity of the immunoprecipitates varied, as normal fragment 1 gave the most prominent immunoprecipitation reaction, consecutively followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1 where the precipitation was so faint that it was hardly visible.

Biological and nonbiological activation of normal and dicoumarol-treated prothrombin☆

Abstract Similar to normal, prothrombin obtained from dicoumarol-treated steers required factor X for activation in addition to factor V, thromboplastin and Ca 2+ . The shortest clotting time, however, persisted for approximately an hour as compared to 10 minutes for normal prothrombin. Factor X from dicoumarol-treated plasma also corrected the clotting (activation) defect. Poly-L-lysin, protamine sulfate and 25% sodium citrate solution activated both normal and dicoumarol-treated preparations containing factor X within a few hours. Thrombin yield for normal was half that of its potential but 50% greater than the potential for dicoumarol-treated preparations. This greater yield from dicoumarol-treated prothrombin represented the (nonbiological) activation of “abnormal” molecules. In the absence of factor X, poly-L-lysin and protamine sulfate, on the other hand, did not activate the two prothrombins. In 25% sodium citrate, such preparations activated very slowly taking days or weeks rather than hours. These results indicate the requirement of factor X even for nonbiological activation of prothrombin.

Terminal amino acids of normal and dicoumarol-treated prothrombin

Abstract Selective tritium labelling method for the determination of C-terminal amino acids was applied successfully to proteins whose carboxyl residue is not available to carboxypeptidases A & B or to hydrazinolysis methods. The carboxyl and amino residues of prothrombin were found to be glutamic acid (or glutamine) and alanine, respectively. The C-terminal amino acid of prothrombin did not change by DEAE-cellulose chromatography. Prothrombin obtained from dicoumarol-treated steers also had NH 2 -terminal alanine and C-terminal glutamic acid (or glutamine). The similar characteristics of the two preparations (normal and dicoumarol-treated) suggest that the polypeptide chain of the abnormal molecules was not significantly different from that of normal.

Partially carboxylated prothrombins: III. Carboxylation studies

Abstract Vitamin K-dependent carboxylation of partially carboxylated prothrombins, containing 7-, 5-, 2-, 1- and 0γ-carboxyglutamic acid residues, was studied with Sepharose-bound carboxylase isolated from the microsomal fraction of warfarin-treated bovine liver. The 14CO2 incorporation was slowest for the 7-carboxyglutamyl variant, intermediate for the 5- and fastest for the 2-, 1- and 0-carboxyglutamyl proteins, these latter three being similar to each other. The carboxylation kinetics for the protein substrate(s) were complex because of the presence of multiple sites. The apparent Km of the 7-carboxyglutamyl variant was, however, the lowest. The CO2 incorporation requiring vitamin K occurred in the specific glutamic acid residues, as the 10-carboxyglutamyl (normal) prothrombin did not undergo carboxylation.

Protein m and the activation of dicoumarol induced atypical prothrombin

Abstract Under carefully controlled conditions and with standard comparable amounts of purified Factor Xa + Factor V + Phospholipid and calcium ions, the yield of thrombin was incomplete and approximately the same from purified prothrombin, purified prethrombin 1, purified 2-Gla prothrombin, purified 5-Gla prothrombin, or purified 7-Gla prothrombin. By adding purified Protein M to the activation mixture, the yield of thrombin and the rate of formation was increased. The procoagulant effect of Protein M compensates for reduced γ-carboxyglutamic acid content of modified prothrombin. It enhances the activation of normal prothrombin.

Monoclonal antibodies to prothrombin

Hybridoma technology was used for the production of murine monoclonal antibodies to bovine normal prothrombin. Hybrid cell cultures were assayed for the production of antibodies, both in the absence and presence of calcium ions, by Enzyme-Linked Immunosorbent Assay (ELISA). Antibody-producing cell lines were cloned two times and grown as ascites tumors. Monoclonal antibodies (McAb), isolated by affinity chromatography (Protein A-Sepharose), were tested for their affinity for normal (10-Gla) and dicoumarol-induced abnormal prothrombins containing 2, 5, 7, 8 and 9 gamma-carboxyglutamyl (Gla) residues. A total of 24 McAb were obtained and the immunoglobulins were of the IgG1 subclass. Nine of the twenty-four McAb did not require Ca2+ for the formation of Ag-Ab complexes, and reacted equally with normal and Gla-deficient prothrombins. These antibodies had affinity for prethrombin1 (P1) but not for the Gla-containing prothrombin fragment1 (F1) portion of the molecule. In contrast, the 15 Ca2+-dependent McAb reacted with F1 but not with P1. They discriminated the abnormal prothrombins based upon their Gla content. For example, though all the Ca2+-dependent McAb formed Ag-Ab complexes with 9-, essentially none formed with 5- or less-Gla prothrombins. [Some reacted equally with 9- and 10-Gla (normal) prothrombin while others had only 25% of normal affinity for 9-Gla isomer]. Only four and twelve of the 15 McAb had some affinity for 7- and 8-Gla variants, respectively. These results show that antibodies which react with the Ca2+-stabilized conformation of prothrombin are not specific for normal prothrombin, as has been reported in the literature.

Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules.

Ca2+ titrations of the intrinsic fluorescence of a series of γ-carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibitTm(the (Ca2+)total concentration at which ln (B/F)=0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase inTmto 5.4 mM is observed for 8-GLA fragment 1.Tmdecreases to 3.8 mM for the 7- and 6-GLA proteins. The value ofh, about 2.8±0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2–1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca2+, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes inTmandh response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.