ABSTRACT
Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.
Subject(s)
Antibodies, Monoclonal/pharmacology , Hemophilia A/prevention & control , Immunoglobulin Fab Fragments/immunology , Protein C Inhibitor/pharmacology , Protein C/antagonists & inhibitors , Animals , Antibodies, Monoclonal/classification , Antibodies, Monoclonal/immunology , Bleeding Time , Cell Membrane Permeability/drug effects , Cells, Cultured , Crystallography, X-Ray , Hemophilia A/blood , Hemorrhage/prevention & control , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Immunoglobulin Fab Fragments/metabolism , Macaca fascicularis , Male , Protein C/chemistry , Protein C/immunology , Protein C/metabolism , Protein C Inhibitor/blood , Protein C Inhibitor/pharmacokineticsABSTRACT
Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa-BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Neutralizing/chemistry , Factor XIa/chemistry , Allosteric Regulation , Amino Acid Sequence , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neutralizing/pharmacology , Blood Coagulation , Catalysis , Epitopes/chemistry , Factor XIa/antagonists & inhibitors , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Structure-Activity Relationship , Thrombelastography , Thrombin/metabolismABSTRACT
To elucidate the molecular mechanisms of cell death, we have cloned a new gene, designated death-upregulated gene (DUG), from rat insulinoma cells. DUG is constitutively expressed at very low levels in normal cells but is dramatically upregulated in apoptotic cells following serum/glucose starvation or death receptor ligation by Fas ligand. The DUG mRNA is present in two splicing forms: a long form that encodes a protein of 469 amino acids and a short form that gives rise to a polypeptide of 432 amino acids. The predicted DUG protein sequence contains two putative nuclear localization signals and multiple phosphorylation sites for protein kinases and two conserved MA3 domains. Importantly, DUG is homologous to eukaryotic translation initiation factor (eIF) 4G and binds to eIF4A presumably through MA3 domains. Upon transfection, DUG inhibits both intrinsic and extrinsic pathways of apoptosis. Thus, DUG is a novel homologue of eIF4G that regulates apoptosis.
