Prothrombin plays a pivotal role in blood coagulation (Fig. 1, 2). Its functional properties relate to the presence of gamma- carboxyglutamyl (Gla) residues, which result from Vitamin K- dependent post-translational carboxylation of ten specific glutamyl residues (Fig. 3,4). My bovine studies (e.g. examining the linkage between metal-ion binding and protein function within the """"""""prothrombinase complex"""""""") will differentiate between liganding and the subsequent conformational involvements of the Gla residues in the normal physiological process. Within this framework, I previously isolated and characterized six different Gla-deficient (0-,1-,2-,3-,5- and 7-Gla) prothrombin variants. Each generated thrombin quantitatively but at a markedly slower rate (high K/m) than 10-Gla prothrombin because of the sharp decrease in Ca/2+- and phospholipid-binding as compared to normal 10-Gla prothrombin. Seven- and less-Gla variants revealed only 3% of normal prothrombin activity, when measured by their abilities to accelerate the clotting of prothrombin and Factor VII-deficient plasma. Newly isolated 8- and 9-Gla prothrombins possess, respectively, and 18 and 75% of normal activity, providing a transition between the 7- and 10-Gla prothrombins. The isolation of 8- and 9-Gla variants became possible through utilization on monoclonal antibodies (McAb) against the Ca2+-stabilized structure (Ca II Ab) of prothrombin for immunoaffinity chromatography. My immunochemical studies with Ca II Ab, polyclonal and monoclonal, have shown that they (polyclonal) are not specific for normal prothrombin as they do cross-react with 7-, 8- and (particularly) 9-Gla variants. Furthermore, my preliminary data with 5- and 7-Gla prothrombins suggest that Glu positions 15 and 17 are not modified, and selective positions (residues 7,8,20,30,33) may be preferentially carboxylated. The proposed research will reveal whether the Gla- deficient prothrombins are indeed selectively deficient, indicating the order of """"""""in vivo"""""""" carboxylation of Glu residues. My expertise in describing how the position and/or number of Gla residues affect the functional, physicochemical (conformational) and immunochemical properties of prothrombin and Gla-containing Fragment 1 forms the basis for the isolation of human materials both for basic and clinical studies. I shall isolate normal and the most crucial Gla-deficient prothrombins. The latter will then be divided into two (or more) groups according to their physiological activity. The goal will be to monitor the plasma of patients on warfarin therapy for normal, 9-Gla (if this is the only one which is functionally-active and total (normal plus Gla-deficient) prothrombins by immunoassays with specific monoclonal antibodies to assess the hemostatic state of these patients.
The aim i s to determine why some patients exhibit thrombotic episodes while others remain more prone to hemorrhage, even though their plasm prothrombin bioactivity is maintained at clinically acceptable levels. Furthermore, Gla-deficient prothrombins may provide another marker for liver dysfunction.
|Malhotra, O P; Valencic, F; Fossel, E T et al. (1991) Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules. J Protein Chem 10:31-41|
|Malhotra, O P (1991) Isolation and some properties of 11- and 9-Gla prothrombins from normal plasma. Biochem Cell Biol 69:404-8|
|Malhotra, O P (1990) Dicoumarol-induced 9-gamma-carboxyglutamic acid prothrombin: isolation and comparison with the 6-, 7-, 8-, and 10-gamma-carboxyglutamic acid isomers. Biochem Cell Biol 68:705-15|
|Malhotra, O P (1989) Dicoumarol-induced prothrombins containing 6, 7, and 8 gamma-carboxyglutamic acid residues: isolation and characterization. Biochem Cell Biol 67:411-21|