Construction and characterization of thrombin-resistant variants of recombinant human protein S.
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 VIIIa. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chang
G T
GT
Department of Haematology, University Hospital Utrecht, The Netherlands.
Aaldering
L
L
Hackeng
T M
TM
Reitsma
P H
PH
Bertina
R M
RM
Bouma
B N
BN
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D002352:Carrier Proteins; D002460:Cell Line; D015935:Complement C4b / Q000378:metabolism; D003169:Complement Inactivator Proteins; D014644:Genetic Variation; D006023:Glycoproteins; D006801:Humans; D007532:Isoleucine; D007930:Leucine; D008969:Molecular Sequence Data; D011486:Protein C; D017293:Protein S / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D011951:Receptors, Complement / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013917:Thrombin / Q000378:metabolism*
D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D011486:Protein C; D017293:Protein S; D011951:Receptors, Complement; D011994:Recombinant Proteins; D007532:Isoleucine; D015935:Complement C4b; D001120:Arginine; D013917:Thrombin; D007930:Leucine