Navegando por Palavras-chave "plasminogen"
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- ItemSomente MetadadadosAssembly of high molecular weight kininogen and activation of prekallikrein on cell matrix(F K Schattauer Verlag Gmbh, 2001-09-01) Motta, G.; Shariat-Madar, Z.; Mahdi, F.; Sampaio, Claudio AM [UNIFESP]; Schmaier, A. H.; Universidade Federal de São Paulo (UNIFESP); Univ MichiganInvestigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-RK binding to ECV304 cells or their matrix requires greater than or equal to 50 muM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding, Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a K-d of 15.8 or 9.0 nM and a B-max of 2.6 X 10(7) or 21.4 X 10(7) sites/cell, respectively. PK activation un ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum. indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.
- ItemSomente MetadadadosCathepsin V, but not cathepsins L, B and K, may release angiostatin-like fragments from plasminogen(Walter de Gruyter & Co, 2008-02-01) Puzer, Luciano; Barros, Nilana M. T. [UNIFESP]; Paschoalin, Thaysa [UNIFESP]; Hirata, Izaura Y. [UNIFESP]; Tanaka, Aparecida S. [UNIFESP]; Oliveira, Marcelo C.; Broemme, Dieter; Carmona, Adriana K. [UNIFESP]; Univ British Columbia; Universidade Federal de São Carlos (UFSCar); Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)Cathepsin V is a lysosomal cysteine peptidase highly expressed in corneal epithelium; however, its function in the eye is still unknown. Here, we describe the capability of cathepsin V to hydrolyze plasminogen, which is also expressed in human cornea at levels high enough to produce physiologically relevant amounts of angiostatin-related molecules. the co-localization of these two proteins suggests an important role for the enzyme in the maintenance of corneal avascularity, essential for optimal visual performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of plasminogen digestion by cathepsin V revealed the generation of three major products of 60, 50 and 40 kDa, which were electrotransferred to polyvinylidene difluoride membranes and excised for characterization. NH2-terminal amino acid sequencing of these fragments revealed the sequences EKKVYL, TEQLAP and LLPNVE, respectively. These data are compatible with cleavage sites at plasminogen F94-E95, S358-T359 and V468-L469 peptide bonds generating fragments of the five-kringle domains. in contrast, we did not detect any plasminogen degradation by cathepsins B, K and L. Using a Matrigel assay, we confirmed the angiogenesis inhibition activity on endothelial cells caused by plasminogen processing by cathepsin V. Our results suggest a novel physiological role for cathepsin V related to the control of neovascularization in cornea.
- ItemSomente MetadadadosHeparin modulation of human plasma kallikrein on different substrates and inhibitors(Walter de Gruyter & Co, 2006-08-01) Gozzo, Andrezza Justino [UNIFESP]; Nunes, Viviane A. [UNIFESP]; Cruz-Silva, Ilana [UNIFESP]; Carmona, Adriana K. [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Faljoni-Alario, Adelaide; Sampaio, Misako U. [UNIFESP]; Araujo, Mariana S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein ( huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. the catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12 x 10(4) M-1 S-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40 x 10(5) M-1 S-1 for H-D-ProPhe-Arg-p-nitroanilide, 2.25 x 10(4) M-1 S-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24 x 10(2) M-1 S-1 for factor XII and 5.58 x 10(2) M-1 S-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates ( by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis ( 7.7- and 1.4-fold, respectively). the second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40 x 10(2) M-1 S-1 and 1.70 x 10(4) M-1 S-1, respectively. Heparin improved the inhibition of huPK by these inhibitors ( 3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.
- ItemSomente MetadadadosPlasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor(Walter de Gruyter Gmbh, 2010-05-01) Coppini, Larissa Pereira [UNIFESP]; Barros, Nilana M. T. [UNIFESP]; Oliveira, Marcela [UNIFESP]; Hirata, Izaura Y. [UNIFESP]; Alves, Marcio F. M. [UNIFESP]; Paschoalin, Thaysa [UNIFESP]; Assis, Diego M. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Puzer, Luciano; Broemme, Dieter; Carmona, Adriana K. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Triangulo Mineiro; Univ British ColumbiaPlasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively We have recently reported that cathepsin V. but not cathepsins L. B, and K. can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. in contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. the peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cathepsin V at the Phe-Glu bond. is a selective substrate for the enzyme when compared with cathepsins B. L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. the demonstrated importance of the S-3'-P-3' interaction indicates the significance of the extended subsites for enzyme specificity and affinity.