Summary
Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of
activated protein C (APC) in the inactivation of coagulation factors Va and Villa.
Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis.
This heterogeneity in molecular weight has been explained by limited proteolysis of
protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage
sites. Whether cleavage occurs at all three sites is not known. To study the role
of these arginine residues in human protein S, we have replaced them by leucine or
isoleucine. All seven possible variants were constructed: three variants with single
mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I,
R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS
polyacrylamide gels the single and double variants migrate as a doublet just like
the wild type protein S. The triple variant migrates as a single band at a molecular
weight corresponding to the upper band of the doublet. The upper band of the single
and double variants but not of the triple variant could be converted into the lower
band by thrombin treatment.
All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment,
this cofactor activity was abolished for the single (R49L, R60L, R70I) and double
variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I)
tested at several concentrations, retained its cofactor activity completely, suggesting
resistance to thrombin. This shows that thrombin can cleave at all three arginine
sites and that cleavage at each of these sites results in the loss of APC cofactor
activity. Finally, all variants bind to C4b-binding protein with an affinity similar
as the wild type recombinant molecule.
