Characterization of the Monoclonal Antibodies Against Human Protein C Specific for Calcium Ion-induced Conformers

Abstract

Five Ca2+-dependent monoclonal antibodies against human protein C: HPC 1 to 5, have been produced. Calcium binding to the light chain (HPC 1, 2) or to the heavy chain (HPC 3, 4) elicited the epitopes for these antibodies. HPC 5 lost its binding activity upon disulfide bridge reduction. HPC 1 and 5 did not react with γ-carboxyglutamic acid (Gla)-domainless protein C nor with isolated Gla-domain while others retained the reactivity even after the Gla-domain had been removed. Half maximal binding of these antibodies were observed 0.35-1.5mM Ca2+. Other divalent cations including Mg2+ and Mn2+ could substitute for Ca2+. Thus the low affinity or secondary metal binding sites rather than the high affinity Ca2+-binding site may have participated in the epitope induction. Furthermore, HPC 1, 4 and 5 reacted with prothrombin and factor X. The cross-reactivities to prothrombin were lost in accordance with the extent of modification of the Gla residues, indicating that certain Gla-residues had been involved in the expression of conserved epitopes. It is notable that the conformational change in the Gla-domain also influenced the induction of a Ca2+-dependent epitope in the serine protease portion of the molecule (HPC 4). The antibodies did not affect the reactions in the fluid phase, but they, except HPC 2, interfered with the cell surface-mediated activation of protein C by the thrombin-thrombomodulin complex and inactivation of factor Va by activated protein C (APC). We therefore conclude that certain concerted conformational changes induced by Ca2+ in both the light and heavy chain of protein C are important for the functions of protein C on the surface of endothelial cells.