RESUMO
Previously we investigated the tissue factor (TF)-dependent coagulation pathway and key haemostatic cofactors in white women with preeclampsia (P-EC) and suggested that plasma factor VII (FVII) levels can differentiate women with P-EC from healthy nonpregnant women or normal pregnant women, at the same trimester, with high sensitivity, specificity, positive and negative predictive values. Here we re-examine the TF-dependent pathway in a large cohort of Brazilian women. A total of 240 women were studied. These included healthy nonpregnant women (nâ=â79), normotensive pregnant women (nâ=â80) and women with severe P-EC (nâ=â81). Commercially available enzyme-linked immunosorbent assays were used to measure plasma FVII, activated factor VII (FVIIa), TF and tissue factor pathway inhibitor (TFPI). All study participants were matched for age. Pregnant women (with/without P-EC) were matched for gestational age and parity. Plasma levels of FVII, FVIIa and TFPI were significantly increased in women with severe P-EC compared with healthy nonpregnant women (Pâ<â0.01) or normotensive pregnant women (Pâ<â0.01). FVIIa was also higher in normotensive pregnant women compared with nonpregnant women (Pâ<â0.01). However, no such significant trends were observed for plasma TF levels (Pâ=â0.074). In conclusion, circulating FVII, FVIIa and TFPI were significantly elevated in women with severe P-EC in the absence of comparable changes in plasma TF levels. The present work is in agreement with our previous report on FVII levels in white women with P-EC. Thus, this lends further support to the notion that plasma FVII levels are potentially valuable diagnostic marker for P-EC, irrespective of ethnicity.
Assuntos
Fator VII/genética , Fator VIIa/genética , Lipoproteínas/genética , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Tromboplastina/genética , Adulto , Coagulação Sanguínea , Pressão Sanguínea , Brasil , Estudos de Casos e Controles , Estudos de Coortes , Fator VII/metabolismo , Fator VIIa/metabolismo , Feminino , Expressão Gênica , Humanos , Lipoproteínas/sangue , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Índice de Gravidade de Doença , Tromboplastina/metabolismoRESUMO
Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.
Assuntos
Coagulação Sanguínea , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Progesterona/metabolismo , Tromboplastina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator VIIa/metabolismo , Fator X/metabolismo , Feminino , Humanos , Microdomínios da Membrana/metabolismo , Invasividade Neoplásica , Transporte Proteico , Receptores de Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
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
NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K(i)=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.
Assuntos
Ancylostoma/química , Fator Xa/química , Proteínas de Helminto/química , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Bovinos , Fator VIIa/química , Fator VIIa/metabolismo , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tromboplastina/química , Tromboplastina/metabolismoRESUMO
Thrombovascular diseases result from imbalanced haemostasis and comprise important health problems in the aging population worldwide. The activity of enzymes pertaining to the coagulation cascade of mammalians exhibit several control mechanisms in order to maintain a proper balance between bleeding and thrombosis. For instance, human coagulation serine proteases carrying a F225 or Y225 are allosteric modulated by the binding of Na+ in a water-filled channel connected to the primary specificity pocket (S1 subsite) of these enzymes. We have characterized the structure, topography and lipophilicity of this channel in the ligand-free fast (sodium-bound) and slow (sodium-free) forms of thrombin, in the sole available structure of activated protein C and in several structures of the coagulation factors VIIa, IXa and Xa, differing in the nature of the bound inhibitor and in the occupancy of exosite-I as well as the Ca2+ and Na+ binding sites. Opposite to thrombin, the aqueous channels in all other coagulation enzymes sheltering a Na+ binding site do not have an aperture on the enzyme surface opposite to the S1 subsite entrance. In these enzymes, the lack of the three-residue insertion in loop 1 (183-189) as found in thrombin allied to compensatory mutations in the positions 187-185 and 222 effects a constriction in the water-filled channel that ends up by segregating the ion binding site from the S1 subsite. We also disclosed major topographical changes on the thrombin's surface upon sodium release and transition to the slow form that culminate in the narrowing of the S1 subsite entrance and, strikingly, in the loss of communication between the primary specificity pocket and the exosite-I. Such observation is in accordance with existing experimental data demonstrating thermodynamic linkage between these distant regions on the thrombin surface. Conformational changes in F34, L40, R73 and T74 were the main responsible for this effect. A path by which these changes in the vicinity of exosite-I could be transmitted to the S1 subsite and, consequently, to the sodium binding site is proposed.