ABSTRACT
Activated factor X is a key component of the coagulation cascade, but whether it directly regulates pathological cardiac remodeling is unclear. In mice subjected to pressure overload stress, cardiac factor X mRNA expression and activity increased concurrently with cardiac hypertrophy, fibrosis, inflammation and diastolic dysfunction, and responses blocked with a low coagulation-independent dose of rivaroxaban. In vitro, neurohormone stressors increased activated factor X expression in both cardiac myocytes and fibroblasts, resulting in activated factor X-mediated activation of protease-activated receptors and pro-hypertrophic and -fibrotic responses, respectively. Thus, inhibition of cardiac-expressed activated factor X could provide an effective therapy for the prevention of adverse cardiac remodeling in hypertensive patients.
ABSTRACT
JNJ-64179375 (JNJ-9375) is a recombinant human IgG4 monoclonal antibody engineered to mimic an IgA antibody that was identified in a patient who exhibited markedly prolonged clotting times but without spontaneous bleeding episodes over several years of follow-up. The crystal structure of the JNJ-9375 antigen-binding fragment/thrombin complex showed an almost identical binding mode to that of the patient IgA. In the current study, we characterized the in vitro and in vivo properties of JNJ-9375. Surface plasmon resonance studies demonstrated that JNJ-9375 binds to α-thrombin with high affinity and specificity (K D: 0.8 nM for human thrombin). JNJ-9375 produced concentration-dependent prolongation of in vitro clotting assays in human plasma, including thrombin time (TT), ecarin clotting time, prothrombin time, and activated partial thromboplastin time, with EC2X values of 4.4, 12.4, 172.6, and 202.7 µg/ml, respectively. JNJ-9375 inhibited thrombin-induced platelet aggregation in human plasma with an IC50 value of 52.6 nM (7.8 µg/ml) and produced concentration-dependent prolongation of reaction time tested by thromboelastography. JNJ-9375 pretreatment resulted in dose-dependent reduction in thrombus formation in the rat arteriovenous (AV) shunt model of thrombosis. Robust efficacy was observed at 0.3 mg/kg accompanied by 1.5× of TT. Bleeding was increased at 3 mg/kg in a rat tail transection bleeding model demonstrating a therapeutic index of 10× compared with 1× for apixaban in the same models. Our data suggest that thrombin exosite I inhibition is efficacious against thrombosis in a pretreatment prevention animal model. SIGNIFICANCE STATEMENT: JNJ-9375 is a novel, fully human monoclonal antibody that binds to the exosite I region of thrombin with high affinity and specificity. JNJ-9375 concentration dependently prolonged clotting times and inhibited thrombin-induced platelet aggregation in in vitro assays based on its mechanism of action. In an in vivo rat AV shunt model, JNJ-9375 prevented thrombus formation in a dose-dependent fashion while demonstrating reduced bleeding risk. The present study demonstrated the antithrombotic effects of inhibiting the exosite I region of thrombin when given in a prevention mode in preclinical animal models.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antithrombins/pharmacology , Immunoglobulin G/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Recombinant Proteins/pharmacology , Animals , Antibodies, Monoclonal/metabolism , Antithrombins/metabolism , Blood Platelets/drug effects , Blood Platelets/metabolism , Dose-Response Relationship, Drug , Humans , Immunoglobulin G/metabolism , Macaca fascicularis , Male , Mice , Platelet Aggregation Inhibitors/metabolism , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Factor V (FV) B-domain contains an acidic region (FV-AR2) and a basic region (FV-BR), which interact with each other and maintain FV in a procofactor form; removal of either region via deletion/proteolysis results in an active FVa molecule. Tissue factor pathway inhibitor type-1 (TFPI) and type-2 (TFPI2) each contain a C-terminus basic segment homologous to FV-BR; this region in TFPI (and predicted in TFPI2) binds to FV-AR2 in platelet FVa (that lacks FV-BR) with high affinity and inhibits FVa function. OBJECTIVES: To understand molecular interactions between FV-AR2 with FV-BR, TFPI-BR and TFPI2-BR. METHODS: Circular dichroism (CD) and molecular modeling approaches. RESULTS AND CONCLUSIONS: CD experiments reveal the presence of â¼20% helical content in both FV-AR2 and FV-BR but each lacks beta-sheet. Predicted structures of FV-AR2 and FV-BR, obtained using threading (I-TASSER), are consistent with the CD data and have compact folds with hydrophobic residues in the interior and charged residues on the surface. Scores from QMEAN and ModFOLD servers indicate a very high probability for each structure to be native. Predicted models of Kunitz domain-3 of TFPI and TFPI2 each with C-terminal basic tail are consistent with known homologous structures. Docking experiments using ClusPro indicate that the acidic groove of FV-AR2 has high shape complementarity to accommodate the conserved basic residues in FV-BR (1002-RKKKK-1006), TFPI-BR (256-RKRKK-260) or TFPI2-BR (191-KKKKK-195). Further, similar electrostatic interactions occur in each case. These models, in the absence of experimentally determined structures, provide a guiding point for proper mutagenesis studies in FV, TFPI and TFPI2.
ABSTRACT
Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.
Subject(s)
Antibodies, Bispecific/biosynthesis , Immunoglobulin G/immunology , Neoplasms/therapy , Animals , Antibodies, Bispecific/immunology , Humans , Immunoglobulin G/genetics , Immunoglobulin G/therapeutic use , Mice , Models, Animal , Neoplasms/genetics , Neoplasms/immunology , Point Mutation/genetics , Point Mutation/immunology , Rats , Xenograft Model Antitumor AssaysABSTRACT
Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pM) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with K(d) ~9 nM and binds FVa with K(d) ~100 nM. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with K(d) ~36-48 nM and bind FVa with K(d) ~252-456 nM. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (~7 nM) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.
Subject(s)
Blood Platelets/cytology , Fibrinolysis/physiology , Glycoproteins/metabolism , Lipoproteins/metabolism , Megakaryocytes/cytology , Platelet Membrane Glycoprotein IIb/metabolism , Blood Coagulation , Blood Platelets/enzymology , Bone Marrow Cells/cytology , Female , Fetal Blood/enzymology , Gene Expression Regulation , Glycoproteins/genetics , Hemostasis , Humans , Ligands , Lipoproteins/genetics , Pregnancy , Protease Inhibitors/chemistry , Protein Binding , Protein Structure, Tertiary , Surface Plasmon ResonanceABSTRACT
Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIß. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIß and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIß in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIß acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.
Subject(s)
Blood Coagulation/physiology , Lipoproteins/metabolism , Thromboplastin/antagonists & inhibitors , Amino Acid Sequence , Base Sequence , Blotting, Western , Computational Biology , Conserved Sequence/genetics , Dose-Response Relationship, Drug , Factor Xa/metabolism , Fluorescence Polarization , Humans , Lipoproteins/pharmacology , Molecular Sequence Data , Sequence AlignmentABSTRACT
Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg(1545), ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule.
Subject(s)
Factor Va/chemistry , Factor Xa/chemistry , Peptides/chemistry , Proteolysis , Factor Va/antagonists & inhibitors , Factor Va/genetics , Factor Va/metabolism , Factor Xa/genetics , Factor Xa/metabolism , Humans , Peptides/genetics , Peptides/metabolism , Protein Binding , Protein Structure, TertiaryABSTRACT
BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.
Subject(s)
Anticoagulants/pharmacology , Arthropod Proteins/pharmacology , Blood Coagulation/drug effects , Factor V/metabolism , Factor Xa/metabolism , Salivary Proteins and Peptides/pharmacology , Animals , Anticoagulants/blood , Anticoagulants/chemistry , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Blood Coagulation/physiology , Blood Coagulation Tests , Dose-Response Relationship, Drug , Factor V/antagonists & inhibitors , Factor Xa Inhibitors , Feeding Behavior , Fibrinogen/metabolism , Humans , Ixodes/chemistry , Ixodes/genetics , Ixodes/physiology , Mutagenesis , Protein Structure, Tertiary , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Salivary Proteins and Peptides/chemistry , Salivary Proteins and Peptides/genetics , Surface Plasmon Resonance , Thrombin/metabolismABSTRACT
Inhibitory antibodies to factors VIII or IX represent a serious complication for hemophilia patients. Treatment involves products that bypass the intrinsic pathway and promote thrombin generation. Direct infusion of factor Xa should also restore hemostasis; however, it has a short half-life in plasma and could activate systemic coagulation in an uncontrolled fashion. Here we show that factor Xa mutants with zymogen-like properties (FXa(I16L) and FXa(V17A)) circumvent these limitations. In the absence of factor Va, the FXa variants are poor enzymes for a range of physiological ligands and are resistant to inactivation by antithrombin III and tissue factor pathway inhibitor. Notably, assembly of FXa(I16L) and FXa(V17A) on activated platelets with factor Va to form prothrombinase completely restores biologic activity. In hemophilic plasma, FXa(I16L) and FXa(V17A) have prolonged half-lives compared with wild-type factor Xa (approximately 60 minutes vs approximately 1 minute) and promote robust thrombin generation that bypasses the intrinsic pathway. The variants require factor Va generated in situ for procoagulant function, and cofactor inactivation by the protein C pathway regulates their activity. The efficacy, extended half-life, and mechanism of action suggest that novel zymogen-like forms of factor Xa might prove useful as new therapeutic procoagulants to treat deficiencies upstream of the common pathway.
Subject(s)
Factor Va/metabolism , Factor Xa/genetics , Factor Xa/metabolism , Genetic Variation , Mutation/genetics , Thrombin/metabolism , Antithrombin III/pharmacology , Blood Coagulation/drug effects , Hemophilia A/genetics , Hemophilia A/metabolism , Hemophilia A/pathology , Humans , Lipoproteins/pharmacology , Thromboplastin/metabolismABSTRACT
Phosphoinositide signaling pathways regulate numerous processes in eukaryotic cells, including migration, proliferation, and survival. The regulatory lipid phosphatidylinositol 4,5-bisphosphate is synthesized by two distinct classes of phosphatidylinositol phosphate kinases (PIPKs), the type I and II PIPKs. Although numerous physiological functions have been identified for type I PIPKs, little is known about the functions and regulation of type II PIPK. Using a yeast two-hybrid screen, we identified an interaction between the type IIbeta PIPK isoform (PIPKIIbeta) and SPOP (speckle-type POZ domain protein), a nuclear speckle-associated protein that recruits substrates to Cul3-based ubiquitin ligases. PIPKIIbeta and SPOP interact and co-localize at nuclear speckles in mammalian cells, and SPOP mediates the ubiquitylation of PIPKIIbeta by Cul3-based ubiquitin ligases. Additionally, stimulation of the p38 MAPK pathway enhances the ubiquitin ligase activity of Cul3-SPOP toward multiple substrate proteins. Finally, a kinase-dead PIPKIIbeta mutant enhanced ubiquitylation of Cul3-SPOP substrates. The kinase-dead PIPKIIbeta mutant increases the cellular content of its substrate lipid phosphatidylinositol 5-phosphate (PI5P), suggesting that PI5P may stimulate Cul3-SPOP activity through a p38-dependent signaling pathway. Expression of phosphatidylinositol-4,5-bisphosphate 4-phosphatases that generate PI5P dramatically stimulated Cul3-SPOP activity and was blocked by the p38 inhibitor SB203580. Taken together, these data define a novel mechanism whereby the phosphoinositide PI5P leads to stimulation of Cul3-SPOP ubiquitin ligase activity and also implicate PIPKIIbeta as a key regulator of this signaling pathway through its association with the Cul3-SPOP complex.
Subject(s)
Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , Nuclear Proteins/metabolism , Phosphatidylinositol Phosphates/metabolism , Repressor Proteins/metabolism , Active Transport, Cell Nucleus , Cell Cycle Proteins/genetics , Cell Line , Cullin Proteins/genetics , Enzyme Activation , Humans , Imidazoles/pharmacology , MAP Kinase Kinase 6/metabolism , Nuclear Proteins/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Binding , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Repressor Proteins/genetics , Sensitivity and Specificity , Signal Transduction/drug effects , Substrate Specificity , Ubiquitination , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Phosphoinositides regulate numerous cellular processes required for growth, proliferation, and motility. Whereas phosphoinositide signal transduction pathways within the cytosol have been well characterized, nuclear signaling pathways remain poorly understood. Accumulating experimental data have now started to uncover critical functions for nuclear phosphoinositides. In particular, phosphoinositides modulate the activity of the tumor suppressor protein ING2 in response to extracellular stress. These findings highlight a previously uncharacterized function for phosphoinositides and implicate their metabolism in signaling pathways critical for cell survival.
Subject(s)
Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Signal Transduction , Animals , Cell Survival , Humans , Models, Biological , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Over the last decades, evidence has accumulated suggesting that there is a distinct nuclear phosphatidylinositol pathway. One of the best examined nuclear lipid pathways is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) by PLC resulting in activation of nuclear PKC and production of inositol polyphosphates. However, there is a growing number of data that phosphoinositides are not only precursor for soluble inositol phosphates and diacylglycerol, instead they can act as second messengers themselves. They have been implicated to play a role in different important nuclear signaling events such as cell cycle progression, apoptosis, chromatin remodeling, transcriptional regulation and mRNA processing. This review focuses on the role of specifically PI4,5P(2) in the nucleus as a second messenger as well as a precursor for PI3,4,5P3, inositol polyphosphates and diacylglycerol.
Subject(s)
Cell Nucleus/metabolism , Cell Physiological Phenomena , Nuclear Envelope/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Signal Transduction/physiology , Type C Phospholipases/metabolism , Animals , HumansABSTRACT
Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKIalpha isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKIalpha, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKIalpha and PIP2 participate in ruffling. To explore this, PIPKIalpha was overexpressed in serum-starved cells and stimulated with PDGF. In serum-starved cells, PIPKIalpha expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKIalpha-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKIalpha in PDGF-stimulated cells resulted in membrane ruffling. The PDGF- and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKIalpha. Combined, these data support a model where the localized production of PIP2 by PIPKIalpha is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.
Subject(s)
Cell Membrane/ultrastructure , Phosphotransferases (Alcohol Group Acceptor)/metabolism , rac GTP-Binding Proteins/metabolism , Amino Acid Substitution , Base Sequence , Cell Line , Cell Membrane/drug effects , Chromones/pharmacology , Cloning, Molecular , DNA Primers , Enzyme Inhibitors/pharmacology , Fibroblasts/cytology , Fibroblasts/enzymology , Humans , Microscopy, Confocal , Minor Histocompatibility Antigens , Morpholines/pharmacology , Mutagenesis, Site-Directed , Platelet-Derived Growth Factor/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal Transduction , rac GTP-Binding Proteins/chemistryABSTRACT
The ability of cells to form cell contacts, adhere to the extracellular matrix, change morphology, and migrate is essential for development, wound healing, metastasis, cell survival and the immune response. These events depend on the binding of integrin to the extracellular matrix, and assembly of focal adhesions, which are complexes comprising scaffolding and signalling proteins organized by adhesion to the extracellular matrix. Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulates interactions between these proteins, including the interaction of vinculin with actin and talin. The binding of talin to beta-integrin is strengthened by PtdIns(4,5)P(2), suggesting that the basis of focal adhesion assembly is regulated by this lipid mediator. Here we show that the type I phosphatidylinositol phosphate kinase isoform-gamma 661 (PIPKI gamma 661), an enzyme that makes PtdIns(4,5)P(2), is targeted to focal adhesions by an association with talin. PIPKI gamma 661 is tyrosine phosphorylated by focal adhesion associated kinase signalling, increasing both the activity of phosphatidylinositol phosphate kinase and its association with talin. This defines a mechanism for spatial generation of PtdIns(4,5)P(2) at focal adhesions.