Decarboxylation of bovine prothrombin fragment 1 and prothrombin

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.     

Evolution of prothrombin: Isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.The nucleotide sequences reported in this paper have been submitted to the EMBL/Genbank database under the following secession numbers: M 81391 for Gallus gallus, M 81393 for Eptatretus stouti.Correspondence to: R.T.A. MacGillivray     

The submicrosomal site for the conversion of prothrombin precursor to biologically active prothrombin in rat liver

The submicrosomal site for the conversion of prothrombin precursor to prothrombin in rat lever has been investigated by subcellular fractionation techniques.Prothrombin precursor activity could be detected in the luminal as well as the membrane fraction of the rough microsomes. The corresponding fractions from smooth microsomes did not exhibit any activity. After warfarin treatment of the rats, the concentration of prothrombin precursor in rough microsomes increased rapidly from approx. 2 to 6–8 h, when a plateau was reached. In smooth microsomes, prothrombin precursor activity appeared 1 h after injection of warfarin, and increased to a plateau reached after about 4 h. The total activity of prothrombin precursor at the plateau obtained after warfarin treatment was 4–5 times higher in the rough luminal fraction than in the corresponding smooth fraction.The vitamin K-dependent carboxylase activity was localized to the rough microsomes. The enzyme system was associated with the membrane, mainly at the luminal side, whereas the substrate appeared to be localized in both the luminal and membrane fraction.The results indicate that the conversion of prothrombin precursor to biologically active prothrombin occurs at a late stage in the rough endoplasmic reticulum or at a transition between rough and smooth endoplasmic reticulum.     

Monoclonal antibodies against human abnormal (des-gamma-carboxy)prothrombin specific for the calcium-free conformer of prothrombin

A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis of the ability of this antibody to bind to abnormal prothrombin, but not to prothrombin, in the presence of calcium ions. The antibodies were purified by affinity chromatography in EDTA on columns of prothrombin-Sepharose. Bound antibodies were eluted with 15 mM CaCl2. The kinetics of dissociation of antibody from the antibody-prothrombin complex with the addition of calcium ions fit a first-order kinetic model. Increasing CaCl2 concentration increased the rate of antibody-prothrombin dissociation. Ca(II) and Mn(II) inhibited antibody-prothrombin interaction; half-maximal binding was observed at 0.9 and 4 mM, respectively. Mg(II) had little effect on antibody-antigen interaction. The JO1 X 1 antibody bound fragment 1, fragment (1-39), abnormal prothrombin, and prothrombin equivalently in the presence of EDTA, but did not bind to des(1-44)prothrombin in the presence of EDTA or prothrombin in the presence of CaCl2. These results indicate that the monoclonal antibody JO1 X 1 is conformation specific for the calcium-free conformer of prothrombin and directed against an antigenic determinant near the NH2 terminus of prothrombin expressed in the 1-39 region of the protein. This analysis provides confirmation of the presence of a metal-free conformer of prothrombin.     

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.     

Isolation and partial characterization of a human prothrombin variant: prothrombin Barcelona

An abnormal prothrombin variant, Prothrombin Barcelona, has been isolated by chromatography on DEAE Sephadex, from several members of the same family. In the absence of any normal component, it was eluted in two unequal peaks. The second peak was homogeneous. This component had the same molecular weight as normal prothrombin but migrated slightly faster on disc gel electrophoresis. The first peak, the smaller one, was heterogeneous: in addition to a minor band similar to that of the second peak, a major one with less anodic mobility and with a molecular weight of 32,000 was found. A possible chromatographic artefact has been eliminated. The family study gave good arguments for an heterozygote state of both parents, the siblings being homozygote.     

The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.