RESUMEN
Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.
Asunto(s)
Proteínas de Insectos/genética , Sistema Calicreína-Quinina/efectos de los fármacos , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales/genética , Triatoma/genética , Secuencia de Aminoácidos , Animales , Coagulación Sanguínea/efectos de los fármacos , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Factor XII/antagonistas & inhibidores , Factor XII/química , Factor XII/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/farmacología , Cinética , Cininas/antagonistas & inhibidores , Cininas/sangre , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Calicreína Plasmática/antagonistas & inhibidores , Precalicreína/antagonistas & inhibidores , Precalicreína/química , Precalicreína/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas y Péptidos Salivales/metabolismo , Proteínas y Péptidos Salivales/farmacología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Triatoma/metabolismo , Tiempo de Coagulación de la Sangre Total , Zinc/farmacologíaRESUMEN
A new kallikrein-kinin system inhibitor, designated anophensin, was identified in the salivary glands of the malaria vector mosquito, Anopheles stephensi. In vitro reconstitution experiments showed that anophensin inhibits activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII (FXII) and prekallikrein (PK), and subsequent release of bradykinin. Additionally, anophensin inhibits activation of the kallikrein-kinin system on cultured human umbilical vein endothelial cells (HUVECs). Direct binding assays show that this inhibitory effect is due to Zn(2+)-dependent specific binding of anophensin to both FXII and high molecular weight kininogen (HK). Furthermore, anophensin interacts with both the N-terminus of FXII and domain D5 of HK, which are the binding domains for biological activating surfaces. These results suggest that anophensin inhibits activation of the kallikrein-kinin system by interfering with the association of FXII and HK with biological activating surfaces, resulting in the inhibition of bradykinin release in a host animal during insect blood-feeding.