Navegando por Palavras-chave "Plasma kallikrein"
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- ItemAcesso aberto (Open Access)Ação de proteínas isoladas de plantas na hemostasia e trombose(Universidade Federal de São Paulo (UNIFESP), 2017-06-29) Salu, Bruno Ramos [UNIFESP]; Oliva, Maria Luiza Vilela [UNIFESP]; Maffei, Francisco Humberto de Abreu; Brito, Marlon Vilela de [UNIFESP]; Francisco Humberto de Abreu Maffei : http://lattes.cnpq.br/4372618586567842; Marlon Vilela de Brito : http://lattes.cnpq.br/1760304906917885; http://lattes.cnpq.br/5832778223847349; http://lattes.cnpq.br/4217215760789690; Universidade Federal de São Paulo (UNIFESP)Protein from plants may act as inhibitors and thus used to assess blood coagulation, thrombosis and inflammation, in order. to determine the role of certain coagulation enzymes in hemostasis. Such inhibitors may be considered for the development of novel antithrombotic drugs. The isolated inhibitors from Enterolobium contortisiliquum (EcTI), Oelonix regia (DrTI), Acacia schweinfurthii (ASTI), and the bifunctional CrataBL protein, a lectin with inhibitory activityextracted from the Crataeva tapia bark, distinctly inhibit proteases, among them, the coagulation enzymes. Our objective was to establish the structure and function relationship of the inhibitors and their effects on thrombus formation. The inhibitors were purified by ion-exchange, molecular exclusion and affinity chromatoqraphies. The homogeneity of the preparations was assessed by SDS-PAGE, reverse phase chromatography and mass spectrometry, depending on the inhibitor. The effect on coagulation was assessed by the partially activated thromboplastin time, TTPa, and thrombin time, TP. The effects on platelet aggregation were investigated in vitro and ex vivo, as well as in the models of arterial thrombosis via injection of rose bengal in mice, and venous thrombosis through vena cava ligation in rats for the estimation of thrombus formation in vivo. DrTI, AsTI and EcTI increased the aPTT in human plasma due to the inhibition of huPK. DrTI differs from AsTI and EcTI by inhibiting FXla, with a Kiapp of 1.3 nM, indicating the dynamic nature of their reactive sites. DrTI, AsTI, EcTI and CrataBL significantly prolonged the time for total carotid artery occiusion in mice compared to the NaCI control. EcTI was also effective in decreasing the venous thrombus (74%, 2 mg/kg). The effects of DrTI, AsTI and EcTI can be attributed to their activities on the contact blood coagulation enzymes and, in the case of DrTI, AsTI and CrataBL, with platelet inhibition, since these proteins inhibited ex-vivo mice platelet aggregation induced by ADP. Although CrataBL has slight inhibitory effect on FXa it has a potent neutralizing effect of heparin activity. In conclusion, the compounds showed antithrombotic action in experimental models of arterial and venous thrombosis, ali interfering in the intrinsic,' but not in the extrinsic coagulation pathway. Only CrataBL was capable of interacting and neutralizing the effect of heparin. As none of the proteins altered the time of bleeding, it is plausible to consider them for therapeutic application, inhibiting thrombus development with a lower hemorrhagic effect. Support by FAPESP, CNPq, CAPES, Ethics Committee N° 179311
- ItemSomente MetadadadosActivity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?(Elsevier Science Bv, 2017) Silva, Roberta N. [UNIFESP]; Oliveira, Lilian C. G. [UNIFESP]; Parise, Carolina B. [UNIFESP]; Oliveira, Juliana R. [UNIFESP]; Severino, Beatrice; Corvino, Angela; di Vaio, Paola; Temussi, Piero A.; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Luiz [UNIFESP]; Juliano, Maria A. [UNIFESP]Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz = ortho-aminobenzoic acid and Q-EDDnp = glutaminyl-N-(2,4dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R down arrow R-ACC and Z-R down arrow R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates. (C) 2017 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosHepatic clearance of tissue-type plasminogen activator and plasma kallikrein in experimental liver fibrosis(Blackwell Munksgaard, 2003-12-01) Nagaoka, Márcia Regina [UNIFESP]; Kouyoumdjian, Maria [UNIFESP]; Borges, Durval Rosa [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)We have previously shown that tissue-type plasminogen activator (tPA) and rat plasma kallikrein (RPK) share a common, but not unique, pathway for liver clearance. Aim:To evaluate the hepatic clearance of both proteases in experimental liver fibrosis. Methods: the hepatic clearance of these proteases was studied in porcine serum-induced liver fibrosis using the isolated and perfused rat liver model. To better interpret the results, we also studied four other experimental groups: the turpentine oil-induced acute-phase response (AP group), AP group followed by GdCl3 administration (AP/Gd group), CCl4-induced cirrhosis (CCl4 group) and normal group. Results: the tPA clearance decreased significantly by both fibrotic and cirrhotic rat livers whereas the RPK clearance was not altered by the fibrotic rat liver. the hepatic clearance of tPA was reduced in the AP and AP/Gd groups; on the other hand, RPK clearance was increased in the AP group and, interestingly, this effect was neutralized by concomitant GdCl3 administration. Conclusions: We observed that tPA and RPK clearances were affected differently by fibrosis as well as by different stimuli of the acute-phase response, despite the fact that they share a common hepatic clearance mechanism in normal livers, and they were equally affected in cirrhosis.
- ItemSomente MetadadadosPlasma kallikrein and tissue-type plasminogen activator compete for a common pathway into the liver(Elsevier B.V., 1996-05-01) Nagaoka, Márcia Regina [UNIFESP]; Kouyoumdjian, Maria [UNIFESP]; Borges, Durval Rosa [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The liver is the main organ used to clear both tissue and plasma kallikreins from the circulation. Rat plasma kallikrein (rPK) is internalized by hepatocytes by a receptor-mediated mechanism. Since plasma kallikrein and tissue-type plasminogen activator (tPA) are both important for fibrinolysis, we now compare characteristics of the clearance of both by the isolated, exsanguinated and perfused rat liver. in preliminary experiments we observed, in vitro, that rPK and tPA neither form a complex with, nor are they substrates for, each other. the characteristics of rPK and tPA clearance are similar, and we observed that 20 and 200 molar excess of tPA in the perfusion medium decreased and abolished the rPK liver clearance, respectively. These results suggest that rPK and tPA share a common pathway for liver clearance.
- ItemSomente MetadadadosPlasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP(Elsevier France-Editions Scientifiques Medicales Elsevier, 2017) Ottaiano, Tatiana F. [UNIFESP]; Andrade, Sheila S. [UNIFESP]; de Oliveira, Cleide [UNIFESP]; Silva, Mariana C. C. [UNIFESP]; Buri, Marcus V. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Girao, Manoel J. B. C. [UNIFESP]; Sampaio, Misako U. [UNIFESP]; Schmaier, Alvin H.; Wlodawer, Alexander; Maffei, Francisco H. A.; Oliva, Maria Luiza V. [UNIFESP]Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein -coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin alpha(IIb)beta(3) through interactions with the KGD/KGE sequence motif in huPK. Integrin alpha(IIb)beta(3) is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS(473), ERK1/2, and p38 MAPK, and to Cat(2+) release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and alpha(IIb)beta(3) (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.