Biospecific interactions of vitamin K-dependent factors with phospholipid-like polystyrene derivatives. Part II: factor IX.

We previously demonstrated that phosphorylated polystyrene derivatives exhibit phospholipid-like behaviour and therefore are able to interact with factor II, one of the vitamin K-dependent coagulation factors. Under the same conditions as for factor II, we examined the interactions of factor IX with phosphorylated resins of various compositions in phosphate groups: these studies were carried out with or without albumin precoating of the polymers and either in the presence or absence of calcium ions. Adsorption experiments show that, in the absence of calcium ions, only one class of adsorption sites of factor IX can be evidenced with the interactions taking place through the formation of binary complexes, whereas in the presence of calcium ions, the affinity of factor IX for phosphorylated resins becomes very high and two types of adsorption sites have been evidenced with biospecific ternary complexes being formed. The domains of predominance of these complexes were determined. Moreover, the only functional groups borne by the phosphorylated polystyrene resins involved in factor IX-polymer interactions are phosphodiester groups. Comparison between factor II and factor IX adsorption onto the same polymers leads to the conclusion that the observed differences probably reflect the differences in the Gla domains of the vitamin K-dependent factors. Finally, this study demonstrates that phosphorylated polystyrene derivatives can be used as stationary phases for purification of factor IX by highly specific liquid biochromatography.

Biospecific polymers: recognition of phosphorylated polystyrene derivatives by anti-DNA antibodies.

The recognition of DNA-like phosphorylated polymers by anti-DNA antibodies from the plasma of systemic lupus erythematosus patients was evidenced a few years ago by our research group. However, the radioimmunological Farr assay used for the assessment of anti-DNA antibodies adsorption was not sensitive enough to give accurate results, particularly in the case of weak levels of antibodies. An alternative method based on the use of radiolabelled species was set up in order to check the validity of previous results. Polystyrene resins with different levels in phosphate groups substitution were assessed with regard to their interactions with anti-DNA antibodies. Results show that the anti-DNA antibodies affinity is dependent on the composition of the polymers and reaches a maximum for a composition of 17.5-22.5 mol of phosphorus per 100 mol of monomeric units. This composition corresponds to the DNA-like polymer previously described. A computer-assisted method was used in order to have an insight into the structure of the active sites responsible for the DNA-like behaviour of this polymer. Numerical simulations of the phosphorylation reaction were performed using a Monte Carlo method, taking the structure predictions and the environment of the phosphorylated units into account. A number of thus generated virtual polymers correlated with the experimental results of the adsorption of anti-DNA antibodies. The chemical structure of the active site was determined by computations introducing selected hypotheses on the structure of the phosphorylated units. Moreover, since the number of active sites is directly related to the number of adsorbed anti-DNA antibodies in the experimental results, the most probable structure of the active sites is proposed and compared to a fragment of DNA. Conclusions are that the distances between the phosphate groups in the active sites of the DNA-like polymer and in the DNA fragment are similar. Optimal conditions for the purification of SLE sera by highly specific liquid chromatography using phosphorylated polystyrene resins of precise compositions as stationary phases can thus be envisaged, as well as a new method for the detection of anti-DNA antibodies.