RESUMO
Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.
Assuntos
Aptâmeros de Nucleotídeos , Fator IXa , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Fator IXa/metabolismo , Fator IXa/química , Fator IXa/antagonistas & inibidores , Humanos , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologiaRESUMO
Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 µm) and activated factor XI (Ki = 94 ± 11 µm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 µm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.
Assuntos
Fator XIa/antagonistas & inibidores , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Zea mays , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Tripsina/metabolismoRESUMO
Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbß3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbß3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.
Assuntos
Plaquetas/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Agregação Plaquetária , Trombospondinas/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Western Blotting , Feminino , Citometria de Fluxo , Humanos , Microscopia Confocal , Oligopeptídeos/farmacologia , Fosfatidilserinas/metabolismo , Polimerização/efeitos dos fármacos , Trombastenia/sangue , Trombastenia/metabolismo , Trombose/metabolismo , Transglutaminases/metabolismoRESUMO
Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.
Assuntos
Aptâmeros de Nucleotídeos , Domínio Catalítico , Hirudinas , Trombina , Humanos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Trombina/antagonistas & inibidores , Trombina/metabolismo , Trombina/química , Hirudinas/química , Hirudinas/farmacologia , Anticoagulantes/farmacologia , Anticoagulantes/química , Fator Xa/metabolismo , Fator Xa/química , Inibidores do Fator Xa/química , Inibidores do Fator Xa/farmacologia , Animais , Sítios de Ligação , Coagulação Sanguínea/efeitos dos fármacosAssuntos
Fator VIII , Hemofilia A , Humanos , Fator VIII/imunologia , Fator VIII/antagonistas & inibidores , Hemofilia A/tratamento farmacológico , Hemofilia A/sangue , Hemofilia A/imunologia , Coagulação Sanguínea/efeitos dos fármacos , Animais , Inibidores dos Fatores de Coagulação Sanguínea/sangue , Inibidores dos Fatores de Coagulação Sanguínea/imunologiaRESUMO
Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 µM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.