RESUMO
Activated Protein C (APC) is a serine-protease that displays antithrombotic and anti-inflammatory properties. In addition, cleavage of protease-activated receptor 1 (PAR1) by APC exerts endothelial cytoprotective actions. The effects of APC on endothelial cells may be reproduced by TR47, a PAR1-based peptide that mimics the novel N-terminus of PAR1 generated upon cleavage at Arg-46 by APC. In this study we demonstrate that wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), display similar inhibitory effects towards the transendothelial migration of A375 human melanoma cells. Consistent with this observation, APC and APC-2Cys significantly reduced the in vivo metastatic potential of the B16F10 murine melanoma cells. TR47 recapitulated the in vitro and in vivo protective profiles of APC and APC-2Cys. Treatment of EA.hy926 endothelial cells with TR47 (20 µM) significantly decreased the A375 cell migration. In addition, treatment of C57/BL6 mice with a single TR47 dose (125 µg/animal) strongly reduced the metastatic burden of B16F10 cells. Together, our results suggest that protection of the endothelial barrier by APC/TR47-mediated signaling pathways might be a valuable therapeutic approach to prevent metastasis.
Assuntos
Movimento Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Melanoma/metabolismo , Melanoma/secundário , Peptídeos/administração & dosagem , Receptor PAR-1/química , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/patologia , Peptídeos/químicaRESUMO
An algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor (F)Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and FXa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for FXa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes FXa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of FXa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with FXa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate FXa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the gamma-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Fator Xa/metabolismo , Galactanos/farmacologia , Anticoagulantes/química , Sítios de Ligação/genética , Cálcio/farmacologia , Cisteína Endopeptidases/metabolismo , Fator V/antagonistas & inibidores , Fator V/química , Fator V/metabolismo , Fator Xa/química , Fator Xa/genética , Inibidores do Fator Xa , Galactanos/química , Heparina/farmacologia , Humanos , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/farmacologia , Serpinas/sangueRESUMO
Ixolaris is a two-Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin-binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin-binding exosite (proexosite, HBPE) as the Ixolaris-binding site on the zymogen. Gel-filtration chromatography demonstrated 1:1 complex formation between fluorescein-labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (DeltaH) of -10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain-containing FX/FXa with comparable affinities ( approximately 1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction-Arg-93 >>> Arg-165 > or = Lys-169 > Lys-236 > Arg-125-which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs.
Assuntos
Fator X/química , Fator X/metabolismo , Fator Xa/metabolismo , Heparina/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/metabolismo , Animais , Sítios de Ligação , Calorimetria , Cromatografia em Gel , Fator VIIa/metabolismo , Heparina/química , Humanos , Cinética , Ligação Proteica , CarrapatosRESUMO
Ixolaris is a two-Kunitz TFPI (tissue factor pathway inhibitor) from the tick salivary gland. In contrast with human TFPI, Ixolaris binds tightly to the zymogen FX (Factor X) and to dansyl-Glu-Gly-Arg-chloromethyl ketone-treated FXa (DEGR-FXa; active-site-blocked FXa), indicating that exosites are involved in the FX(a)-Ixolaris interaction. Here we provide evidence that Ixolaris binds specifically to the FXa HBE (heparin-binding exosite), since (i) it markedly decreases the inhibition of FXa by the antithrombin-heparin but not the antithrombin-pentasaccharide complex, (ii) it impairs FXa binding to Sepharose-immobilized heparin, and (iii) it allosterically modulates the catalytic activity of FXa for small chromogenic substrates (S-2765). By using a series of recombinant FXa mutants in which the HBE is mutated, we have identified the importance of amino acids involved in the enzyme-inhibitor interaction as being in the following order: Arg-93>>Arg-165> or =Lys-169>Lys-236>Lys-96>Arg-240>Arg-125. Ixolaris at appropriate concentrations also inhibits thrombin formation in vitro by the assembled prothrombinase complex, a process that is critically dependent on the FXa HBE. Ixolaris is the first inhibitor characterized to date that binds specifically to the FXa HBE.