RESUMO
Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.
Assuntos
Proteínas Sanguíneas/fisiologia , Glicoproteínas/fisiologia , Glicosaminoglicanos/fisiologia , Inativadores de Plasminogênio/fisiologia , Inibidores de Serina Proteinase/fisiologia , Amidas/metabolismo , Humanos , Técnicas In Vitro , Radioisótopos do Iodo , Calicreínas/urina , Inativadores de Plasminogênio/urina , Inibidor da Proteína C , Sensibilidade e Especificidade , Inibidores de Serina Proteinase/urina , Calicreínas Teciduais , Ativador de Plasminogênio Tipo Uroquinase/urina , VitronectinaAssuntos
Glicosaminoglicanos/farmacologia , Inativadores de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Heparina/farmacologia , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/isolamento & purificação , Calicreínas/metabolismo , Cinética , Inativadores de Plasminogênio/isolamento & purificação , Inativadores de Plasminogênio/urina , Ligação Proteica , Inibidor da Proteína C , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Protein C inhibitor (PCI) inhibits tissue kallikrein by forming stable 1:1 complexes (k1 = 2.3 x 10(4)M-1s-1). Heparin inhibits the tissue kallikrein/PCI-interaction and complex formation of 125I-tissue kallikrein in serum. 125I-tissue kallikrein complexes formed in plasma can be immunoprecipitated with monoclonal anti-PCI IgG suggesting that PCI might be identical to the kallikrein binding protein described previously (J. Chao et al. 1986, Biochem. J. 239, 325-331).
Assuntos
Proteínas de Transporte/isolamento & purificação , Calicreínas/metabolismo , Inativadores de Plasminogênio/isolamento & purificação , Precipitação Química , Humanos , Técnicas In Vitro , Calicreínas/antagonistas & inibidores , Inativadores de Plasminogênio/farmacologia , Inativadores de Plasminogênio/urina , Proteína C/antagonistas & inibidores , Inibidor da Proteína C , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/farmacologiaRESUMO
Recent investigations have shown that in the murine kidney urokinase (uPA) and tissue-type plasminogen activator (tPA) are synthesized and released in urine by tubular epithelial cells, raising the possibility that plasminogen activators (PAs) may be involved in the maintenance of patency and fluidity in renal tubules. To further investigate the contribution of the PA system in renal pathology, we have determined the effects of LPS on the renal production of PAs: we localized PA-catalyzed proteolysis by zymographic analysis of tissue sections and studied the accumulation of mRNAs for PAs and their inhibitors (PAI-1 and PAI-2) by in situ hybridization. Both a single and two injections of LPS induced a dramatic reduction in urinary and renal uPA enzymatic activity; this decrease in catalytic activity was attributable to a reduction in uPA mRNA levels in both proximal and distal tubules. By contrast, we noticed a marked increase of tPA mRNA content in glomerular cells which was not accompanied by a concomitant increase in tPA-mediated proteolytic activity. In addition, a major up-regulation in PAI-1 mRNA levels was observed throughout the kidney, while PAI-2 mRNA was not detectable in the kidneys of control or LPS-injected animals. Our investigations document the profound alterations of the PA/PAI balance in renal tissue following in vivo LPS administration. They suggest that imbalanced extracellular proteolysis might participate in the alterations of kidney function observed in septic shock.