Isolation and some properties of 11- and 9-Gla prothrombins from normal plasma.
The relationship of prothrombin structure to function with respect to gamma-carboxyglutamic acid (Gla) residues can be effectively evaluated by characterizing the behavior of prothrombin isomers differing in Gla content. In addition to the isolation of a whole spectrum of Gla-deficient, 0- to 9-Gla isomers from dicoumarol-treated plasma, prothrombin isomers containing 11 (10.90) and 9 (8.85) Gla residues have now been isolated from normal bovine plasma. The isomers were isolated by barium citrate adsorption, elution, and finally by heparin-agarose, DEAE-cellulose, and immuno-affinity chromatographies. Each of the purified isomers showed a single component by agar gel and sodium dodecyl sulfate - polyacrylamide gel electrophoresis. By agar gel electrophoresis, the 11-Gla prothrombin isomer moved the fastest, followed by the 10-, and lastly the 9-Gla isomer, independent of Ca2+. The corresponding 9-, 10-, and 11-Gla prothrombin fragments 1 exhibited similar migration tendencies. By gel electrofocusing, 11- and 9-Gla fragments 1, respectively, focused anodal and cathodal to 10-Gla fragment 1. The Ca2+-induced decrease in the intrinsic fluorescence in 11-, 10-, and 9-Gla fragments 1 was 48, 40, and 45%, respectively. This metal-induced structural change did not correlate with the functional, thrombin-generating property of the isomers, as the 9-Gla variant exhibited 75%, and the 11-Gla 110-115%, of normal coagulant activity.
Malhotra
O P
OP
Medical Research Service, Department of Veterans Affairs Medical Center, Cleveland, OH 44106.
D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002847:Chromatography, Agarose; D001728:Dicumarol / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007535:Isomerases; D011516:Prothrombin / Q000737:chemistry* / Q000302:isolation & purification
D015055:1-Carboxyglutamic Acid; D001728:Dicumarol; D011516:Prothrombin; D007535:Isomerases; D002118:Calcium