Date Published: July 17, 2015
Publisher: Public Library of Science
Author(s): Keiko Maruyama, Masashi Akiyama, Koichi Kokame, Akiko Sekiya, Eriko Morishita, Toshiyuki Miyata, Pablo Garcia de Frutos.
Protein S (PS) acts as a cofactor for activated protein C in the plasma anticoagulant system. PS Lys196-to-Glu (K196E) mutation is a genetic risk factor for venous thromboembolism in Japanese individuals. Because of the substantial overlap in PS anticoagulant activity between KK (wild-type) and KE (heterozygous) genotypes, it is difficult to identify PS K196E carriers by measuring PS activity. Here, we generated monoclonal antibodies specific to the PS K196E mutant and developed a simple and reliable method for the identification of PS K196E carriers. We immunized mice with a keyhole limpet hemocyanin-conjugated synthetic peptide with Glu196. The hybridoma cells were screened for the binding ability of the produced antibodies to recombinant mutant EGF-like domains of PS (Ile117–Glu283). We obtained three hybridoma cell lines producing PS K196E mutation-specific antibodies. We established a sandwich enzyme-linked immunosorbent assay (ELISA) system in which the PS K196E mutation-specific monoclonal antibody was used as a detection antibody. We measured human plasma samples by using this system and successfully discriminated 11 individuals with the KE genotype from 122 individuals with the KK genotype. The ELISA system using the PS K196E mutation-specific antibody is a useful tool for the rapid identification of PS K196E carriers, who are at a higher risk for venous thromboembolism.
Protein S (PS) is an anticoagulant protein that acts as a cofactor for activated protein C in the proteolytic inactivation of activated coagulation factors Va and VIIIa and as a cofactor for tissue factor pathway inhibitor to efficiently inhibit factor Xa [1–4]. Thus, the reduced PS anticoagulant activity observed in congenital PS deficiency is a genetic risk for venous thromboembolism (VTE). PS circulates in human plasma at a concentration of approx. 25 μg mL−1 (~350 nM). Approximately 60% of PS forms a non-covalent 1:1 stoichiometric complex with C4b-binding protein, which results in loss of cofactor function for activated protein C [1,2].
We screened supernatants of hybridoma cells from 1,672 wells by preferential binding to a recombinant mutant PS EGF-like domain, EGF-PS-E, compared to its wild-type counterpart, EGF-PS-K. We obtained three monoclonal antibodies, 4B1, 15C8 and 16E3, all of which bound to mutant EGF-PS-E but not to wild-type EGF-PS-K.