Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

Abstract

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation

“…These variants are incorrectly processed by the signal peptidase at their amino‐terminal residues. Furthermore, a proportion of the R‐1C variant is known to be present as heterodimers with α 1 ‐microglobulin [45]. Neither variant was able to bind to sEPCR or be efficiently activated by thrombin over the endothelial cell surface.…”

“…The purification of variant protein C from the plasma of patients who were heterozygous for the mutations R‐1L and R‐1C has been described previously [45,54]. Briefly, the method utilized two immunoaffinity columns directed against protein C. The first column resulted in efficient purification of protein C from other plasma components, whereas the second separated normal from variant protein C. The characterization of these APC variants has also been reported previously [45,54] and consequently details of the purification and characterization will not be replicated here. The activities of these variants were compared to human plasma protein C from Enzyme Research Laboratories (ERL, South Bend, IN, USA).…”

Laboratory tests for protein C deficiency

“…Heterozygous FV Leiden did not appear to affect the results of an RVVbased assay [14]. With chromogenic assays, it is important to note that a rare type II protein C variant has been reported that is not detected in the chromogenic activity assay but is detected in the clot-based assay [18,19]. A study by the ECAT Foundation shows significantly lower inter-laboratory and intra-laboratory variation using chromogenic, as opposed to clotting based, assays of protein C activity [20].…”

Targeted Gene Sequencing Identifies Variants in the Protein C and Endothelial Protein C Receptor Genes in Patients With Unprovoked Venous Thromboembolism

“…The PCArg-1Cys and PCArg9Cys variants, both of which carry a free cysteine residue, have been shown to form a complex with alpha 1-microglobulin 25 . Although, we did not observe complexes between these PC variants and alpha 1-microglobulin based on our Western Blot analysis (presumably because HEK293 cells do not synthesize alpha 1-microglobulin), it is possible that these complexes exist in the patient plasma.…”