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.

Mechanism of the calcium-dependent self-association of bovine prothrombin. Use of a covalent cross-linking reagent to study the reaction.

The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin and has demonstrated that the covalent dimerization reaction involves the gamma-carboxyglutamic acid region of prothrombin (1-42 of 582). An essential role for the gamma-carboxyglutamic acid residues of prothrombin in the association reaction was demonstrated by experiments that converted gamma-carboxyglutamic acid residues to gamma-methylene glutamic acid or glutamic acid and resulted in a prothrombin species that was inactive in our cross-linking assay. Other experiments showed that very high concentrations of calcium ion inhibit the cross-linkage of prothrombin. This result is most consistent with an essential gamma-carboxyglutamic acid-calcium ion-gamma-carboxyglutamic acid bridge(s) in the calcium-dependent self-associated form of prothrombin.

Use of high-performance size-exclusion chromatography to measure protein molecular weight and hydrodynamic radius. An investigation of the properties of the TSK 3000 SW column.

We have conducted a study of the TSK 3000 SW high-performance size-exclusion column to define under what conditions proteins would migrate most consistently with their known hydrodynamic properties. Our findings include the following: 1) the residual negative charge of the column does cause charge-exclusion or charge-retention effects at low ionic strengths; with elution in deionized water several anionic proteins elute approximately in the void volume; 2) at mu greater than or equal to 0.5, protein migration is not only independent of ionic strength, but consistent with protein molecular weight and hydrodynamic volume; 3) small hydrophobic peptides are retarded by the column; and 4) very asymmetric proteins and other hydrodynamic particles are likely to be retarded by an "end-on insertion" mechanisms.

Purification of human prothrombin fragment 1 using hydrophobic interaction chromatography on phenyl-sepharose.

Hydrophobic interaction chromatography on Phenyl-Sepharose for the purification of prothrombin fragment 1 is described. The results suggest that this method is both easier and more effective than the use of anion-exchange chromatography for the purification of fragment 1. In addition, the results presented here suggest that prothrombin has rather extensive hydrophobic properties.

Calcium-dependent changes in properties of human prothrombin: a study using high-performance size-exclusion chromatography and gel-permeation chromatography.

High-performance size-exclusion chromatography using a TSK 3000 SW column and aqueous gel filtration with Sephacryl S-200 SF have been used to characterize the effects of calcium ions on the hydrodynamic properties of human prothrombin and prethrombin 1. The results suggest that the effective hydrodynamic radius of prothrombin is less in the presence than in the absence of calcium ions. In addition, when using the TSK-3000 SW column, Ca2+-dependent formation of a hydrophobic site in the fragment 1 region of prothrombin results in an apparent further decrease in hydrodynamic radius.

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin.

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombin's catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.

Self-association of bovine prothrombin fragment 1 in the presence of metal ions. Use of a covalent cross-linking reagent to study the reaction.

The interaction of metal ions with prothrombin fragment 1 has been shown by other investigators to result in conformational change(s). Previous studies based on hydrodynamic measurements from other laboratories have suggested that prothrombin fragment 1 will, in addition to undergoing conformational change, self-associate in the presence of metal ions but there is a question regarding the specificity and role of metal ions in this process. The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin fragment 1 and has focused on factors influencing the covalent dimerization of bovine prothrombin fragment 1 during reaction with this reagent. Optimal dimerization of bovine prothrombin fragment 1 required the involvement of cations in two separable processes. The "first role" is probably a reflection of a slow conformational change in the bovine prothrombin fragment 1 representing isomerization of a proline-containing peptide bond (Marsh, H. C., Scott, M. E., Hiskey, R. G., and Koehler, K. A. (1979) Biochem. J. 183, 513-517). The "second role" most likely reflects ion bridging with another prothrombin fragment 1 molecule. The ion specificities of these two roles are clearly different. Ions stabilizing the proper conformation (first role) are Ca2+, Sr2+, Mg2+, Mn2+, with Ba2+ having less capability in filling this role. Ions which have activity in the second role (ion bridge formation) are Ca2+, Sr2+, and Ba2+. Mn2+ and Mg2+ cannot fulfill the requirements of this second role and actually inhibit the function of Ca2+.

Influence of metal ions on prothrombin self-association. Demonstration of dimer formation by intermolecular cross-linking with dithiobis(succinimidylpropionate).

Interaction of certain metal ions with prothrombin and prothrombin fragment 1 has been shown to result in conformational change(s). Self-association of prothrombin and prothrombin fragment 1 in the presence of divalent cations has been reported. The present study has made use of a covalent cross-linking reagent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin in the presence of divalent cations. In the presence of certain divalent cations, prothrombin dimer is the product of such cross-linking. Optimal dimerization of bovine prothrombin requires preincubation with calcium ions prior to the addition of cross-linking reagent. Calcium ions are also required for dimerization after this preincubation period. A similar time dependence is not seen with human prothrombin. The dimerization of bovine prothrombin is also supported by strontium and gadolinium to an extent comparable to that of calcium and to a lesser extent by barium or manganese. Magnesium ions do not support dimerization. The results suggest that certain divalent cations either induce or stabilize a conformation of prothrombin which can self-associate to form dimers. The results further suggest that divalent cations are also necessary for the actual cross-linking process subsequent to this conformational change.