Isolation and characterization of single-chain protein S.
Protein S is a vitamin K-dependent cofactor of activated protein C in the proteolytic cleavage and concomitant inactivation of the coagulation factors Va and VIIIa. Protein S is sensitive to proteolysis by thrombin which reduces its functional activity. Uncontrolled proteolytic breakdown, leading to the generation of a two-chain molecule, is commonly encountered during the purification of both human and bovine protein S. In this study we demonstrate that human, single-chain, intact protein S can be isolated from plasma in a single step by affinity chromatography using a monoclonal antibody, CLB PS 52, directed to an epitope located within the thrombin-sensitive region of protein S. The product of purification was readily cleaved by thrombin after Arg49, resulting in a two-chain molecule which demonstrated a lower reactivity towards CLB-PS 52 than the parent single-chain protein. This study for the first time shows that intact protein S can be isolated directly from plasma using a monoclonal antibody selected for its ability to recognize uncleaved protein S.
Meijer-Huizinga
F
F
Department of Blood Coagulation, Central Laboratory of The Netherlands, Amsterdam.
Mertens
K
K
van Mourik
J A
JA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D003169:Complement Inactivator Proteins; D000939:Epitopes / Q000276:immunology; D006023:Glycoproteins; D006801:Humans; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D017293:Protein S / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D000939:Epitopes; D006023:Glycoproteins; D017293:Protein S; D013917:Thrombin