RESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a multidomain protein that negatively regulates the coagulation cascade. TFPI inhibits the tissue factor (TF)-activated factor VII-activated FX (FXa) complex during TF-mediated coagulation initiation. The aptamer BAX 499 binds specifically to TFPI and inhibits its function, mediating a procoagulant effect in both in vitro and in vivo models of hemophilia. OBJECTIVES: This study sought to identify the regions of TFPI that are critical for BAX 499 binding, and to determine how binding mediates aptamer inhibition of TFPI. METHODS AND RESULTS: In vitro biochemical methods were used to evaluate the BAX 499 interaction with and inhibition of TFPI. Binding experiments indicated that the full-length TFPI protein is required for tight aptamer binding. Binding-competition experiments implicated the Kunitz 1, Kunitz 3 and C-terminal domains of TFPI in aptamer binding, a finding that is supported by hydrogen-deuterium exchange experiments, and indicated that aptamer and FXa can bind simultaneously to TFPI. In enzymatic assays, BAX 499 inhibited TFPI in a manner that is distinct from domain-specific antibodies, and aptamer inhibitory activity is reduced in the presence of the TFPI cofactor protein S. CONCLUSIONS: These studies demonstrate that BAX 499 binds to TFPI via multiple domains of the protein in a manner that is distinct from other TFPI inhibitors, mediating a mechanism of inhibition that does not involve direct competition with FXa. With this unique inhibitory mechanism, BAX 499 provides a useful tool for studying TFPI biology in health and disease.
Assuntos
Aptâmeros de Nucleotídeos/química , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/química , Tromboplastina/química , Anticorpos/química , Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/química , Medição da Troca de Deutério , Ensaio de Imunoadsorção Enzimática , Fator Xa/química , Hemofilia A/tratamento farmacológico , Humanos , Hidrogênio/química , Concentração Inibidora 50 , Peptídeos/química , Ligação Proteica , Proteína S/química , Estrutura Terciária de Proteína , Tromboplastina/antagonistas & inibidoresRESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the major inhibitor of tissue factor-initiated coagulation, making it an interesting and novel therapeutic target in hemophilia treatment. The aptamer BAX499 (formerly ARC19499) is designed to improve hemostasis by specifically inhibiting TFPI. OBJECTIVES: The aim of the study was to examine the concentration-dependent augmentation of clotting by BAX499. METHODS: Whole blood clot formation was quantified by rotational thromboelastometry and thromboelastography, and thrombin generation in platelet-poor plasma was assessed with the calibrated automated thrombogram, in samples from patients with congenital hemophilia A (N=55) and B (N=11), patients with acquired hemophilia A (N=1), and healthy controls (N=37). RESULTS: BAX499 significantly improved clotting of samples from hemophilic patients in a concentration-dependent manner, resulting in clotting profiles in samples from patients with severe hemophilia that were similar to those of healthy controls. CONCLUSION: BAX499 improved ex vivo clotting parameters in blood and plasma from patients with hemophilia A and B with different severity of disease, and also in a patient with acquired hemophilia. These results further support the contention that anti TFPI strategies may be an effective treatment for hemophilic patients.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Hemofilia A/sangue , Hemofilia B/sangue , Hemostáticos/farmacologia , Lipoproteínas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Adolescente , Adulto , Idoso , Áustria , Coagulação Sanguínea/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Hemofilia A/diagnóstico , Hemofilia A/genética , Hemofilia B/diagnóstico , Hemofilia B/genética , Humanos , Índia , Lipoproteínas/sangue , Lipoproteínas/genética , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Tromboelastografia , Trombina/metabolismo , Tempo de Coagulação do Sangue Total , Adulto JovemRESUMO
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro- and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high-affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. OBJECTIVE: To examine the BAX499 effect on clotting in a spatial, reaction-diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). METHODS: Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: BAX499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m(-2) by shortening lag time and increasing clot size by up to ~2-fold. The effect was TFPI specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m(-2) , both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. CONCLUSIONS: BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.
