RESUMEN
BACKGROUND: The hemostatic system is the major target of snake venom serine proteinases (SVSPs) that act on substrates of the coagulation, fibrinolytic and kallikrein-kinin systems. Bothrops protease A (BPA), the most glycosylated SVSP, is a non-coagulant, thermostable enzyme. A cDNA encoding BPA showed that the protein has a calculated molecular mass of 25 409 Da, implying that approximately 62% of its molecular mass as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (67 kDa) is due to carbohydrate moieties. RESULTS: Here we show that BPA is a potent fibrinogenolytic agent in vitro, as it readily degraded human and rat fibrinogen at a very low enzyme concentration. Partially N-deglycosylated BPA (p-N-d-BPA) generated similar fibrinogen products, but with enhanced fibrinogenolytic activity. In vivo, injection of 0.75 nmoles of BPA in rats completely avoided thrombus formation induced by stasis in the vena cava, or by endothelium injury in the jugular vein. Moreover, it decreased the fibrinogen plasma level and prolonged the recalcification time. Cleavage of fibrinogen in human and rat plasma was observed with native BPA and p-N-d-BPA by electrophoresis followed by western blot using an anti-fibrinogen antibody. BPA did not cause unspecific degradation of plasma proteins and did not cleave isolated albumin, vitronectin and fibronectin at the same concentration used with fibrinogen. Serine proteinase inhibitors failed to inhibit BPA, probably due to steric hindrance caused by its huge carbohydrate moieties. CONCLUSIONS: To the best of our knowledge, this investigation underscores a new, thermostable, specific defibrinogenating agent that may have an application in the prevention of thrombus formation.
Asunto(s)
Fibrinolíticos/farmacología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/farmacología , Venenos de Serpiente/enzimología , Venenos de Serpiente/farmacología , Animales , Fibrinógeno/metabolismo , Fibrinolíticos/química , Glicosilación , Humanos , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/farmacología , Venenos de Serpiente/química , Especificidad por Sustrato , Trombosis/prevención & controlRESUMEN
Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.
Asunto(s)
Metaloendopeptidasas/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Secuencia de Aminoácidos , Hidrólisis , Datos de Secuencia Molecular , Fosforilación , Especificidad por SustratoRESUMEN
Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the Ki's. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.