Assuntos
Venenos de Crotalídeos/farmacologia , Integrinas/metabolismo , Metaloendopeptidases/farmacologia , Animais , Ligação Competitiva , Bothrops , Colágeno/metabolismo , Venenos de Crotalídeos/metabolismo , Desintegrinas/metabolismo , Desintegrinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Integrinas/antagonistas & inibidores , Ligantes , Metaloendopeptidases/metabolismo , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Ligação Proteica , Receptores de Colágeno , Especificidade por Substrato , Células Tumorais Cultivadas , Veneno de Bothrops jararacaRESUMO
The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.
Assuntos
Plaquetas/metabolismo , Receptores de Trombina/metabolismo , Serina Endopeptidases/metabolismo , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Plaquetas/efeitos dos fármacos , Células Cultivadas , Humanos , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Trombina/farmacologiaRESUMO
The alpha2beta1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Here we describe the isolation of a novel metalloproteinase/disintegrin, which is a potent inhibitor of the collagen binding to alpha2beta1 integrin. This 55-kDa protein (alternagin) and its disintegrin domain (alternagin-C) were isolated from Bothrops alternatus snake venom. Amino acid sequencing of alternagin-C revealed the disintegrin structure. Alternagin and alternagin-C inhibit collagen I-mediated adhesion of K562-alpha2beta1-transfected cells. The IC50 was 134 and 100 nM for alternagin and alternagin-C, respectively. Neither protein interfered with the adhesion of cells expressing alphaIIbeta3, alpha1beta1, alpha5beta1, alpha4beta1 alphavbeta3, and alpha9beta1 integrins to other ligands such as fibrinogen, fibronectin, and collagen IV. Alternagin and alternagin-C also mediated the adhesion of the K562-alpha2beta1-transfected cells. Our results show that the disintegrin-like domain of alternagin is responsible for its ability to inhibit collagen binding to alpha2beta1 integrin.
Assuntos
Adesão Celular/efeitos dos fármacos , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/farmacologia , Desintegrinas/farmacologia , Integrinas/antagonistas & inibidores , Metaloendopeptidases/farmacologia , Sequência de Aminoácidos , Animais , Bothrops , Células CHO , Colágeno/metabolismo , Cricetinae , Desintegrinas/química , Humanos , Integrinas/genética , Células K562 , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Colágeno , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Plaquetas/fisiologia , Ativação de Neutrófilo , Peptídeos/fisiologia , Fator Plaquetário 4/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Primers do DNA/química , Heparina/farmacologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Elastase Pancreática/metabolismo , Toxina Pertussis , Receptores de Interleucina/fisiologia , Receptores de Interleucina-8A , Proteínas Recombinantes de Fusão , Proteínas Recombinantes , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia , beta-TromboglobulinaRESUMO
Aprotinin reduces blood loss after cardiac operations and decreases the bleeding time. The mechanism of action of aprotinin that produces these effects is not clear. During simulated extracorporeal circulation the contact and complement systems, platelets, and neutrophils are activated. We investigated the effect of aprotinin on kallikrein-C1-inhibitor complex and C1-C1-inhibitor complex formation, neutrophil degranulation, and platelet release and aggregation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated at 37 degrees C for 2 hours in a spiral coil membrane oxygenator-roller pump perfusion circuit. Changes in platelet count, leukocyte count, platelet response to adenosine diphosphate, and plasma levels of beta-thromboglobulin, kallikrein-C1-inhibitor complexes, C1-C1-inhibitor complexes, and neutrophil elastase were measured before and at 5, 30, 60, and 120 minutes of recirculation at 0, 0.015, 0.03, 0.06, and 0.12 mg/ml doses of aprotinin. Platelet counts decreased to 36% +/- 12% of control values at 5 minutes and increased to 56% +/- 13% at 120 minutes without aprotinin. Aprotinin did not affect platelet counts, but it did prevent the decrease in sensitivity of platelets to adenosine diphosphate and it attenuated beta-thromboglobulin release. In the absence of aprotinin, kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively to 0.53 +/- 0.14 U/ml and 2.38 +/- 0.33 U/ml, respectively, at 120 minutes. Kallikrein-C1-inhibitor complexes were completely inhibited and C1-C1-inhibitor complexes were partially inhibited at aprotinin concentrations of 0.03 mg/ml or greater. Release of neutrophil elastase was partially but not completely inhibited at the highest dose of aprotinin and was 50% inhibited at a dose of 0.03 mg/ml. Because activation of the fibrinolytic system does not occur in this system, the changes were independent of the inhibition of plasmin. We conclude that aprotinin in high doses completely inhibited kallikrein-induced activation of neutrophils and partially inhibited complement-induced activation. Aprotinin did not directly affect platelet adhesion or aggregation, but it indirectly preserved platelet sensitivity to agonists and also attenuated release of alpha-granule contents. The data indicate that in the presence of aprotinin platelet function was partially preserved, kallikrein production was totally inhibited, complement activation was partially inhibited, and neutrophil release was partially inhibited, thus attenuating the "whole body inflammatory response" associated with cardiopulmonary bypass.
Assuntos
Aprotinina/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Circulação Extracorpórea , Neutrófilos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Aprotinina/administração & dosagem , Ativação do Complemento/efeitos dos fármacos , Proteínas Inativadoras do Complemento 1/metabolismo , Relação Dose-Resposta a Droga , Fibrinólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Calicreínas/metabolismo , Contagem de Leucócitos/efeitos dos fármacos , Elastase de Leucócito , Modelos Cardiovasculares , Elastase Pancreática/sangue , Contagem de Plaquetas/efeitos dos fármacos , beta-Tromboglobulina/análiseRESUMO
Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.
Assuntos
Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Proteínas/farmacologia , Venenos de Víboras/farmacologia , Fator de von Willebrand/antagonistas & inibidores , Animais , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , Proteínas de Transporte , Bovinos , Cromatografia por Troca Iônica , Venenos de Crotalídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Feminino , Hemaglutinação/efeitos dos fármacos , Hemaglutininas/farmacologia , Humanos , Cinética , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos , Peso Molecular , Contagem de Plaquetas/efeitos dos fármacos , Proteínas/isolamento & purificação , Proteínas/toxicidade , Coelhos , RatosRESUMO
The RGD-containing peptides isolated from the venoms of the Viperidae constitute a new class of small cysteine-rich peptides of variable amino acid composition and biological activity (Huang, T.-F., et al. (1987) J. Biol. Chem. 262, 16157-16163; Gan, Z.R., et al. (1988) J. Biol. Chem 263, 19827-19832; Huang, T.-F., et al. (1989) Biochemistry 28, 661-668), which it is proposed by Gould et al. (unpublished data) that we call 'disintegrins'. These peptides bind to the glycoprotein IIb-IIIa receptor on the platelet surface and inhibit aggregation induced by ADP, thrombin, platelet-activating factor and collagen. These peptides are also potent inhibitors of cell adhesion to fibrinogen (Knudsen, K.M., et al. (1988) Exp. Cell Res. 179, 42-49). We report the isolation of two further RGD-peptides from the venoms of Trimeserusus elegans and Trimeserusus albolabris, purified to homogeneity with high yield by a novel, rapid reverse-phase HPLC method. The primary structures of these two peptides were determined to be single polypeptide chains of 73 amino acids. Albolabrin differed from trigramin by eight residues whilst elegantin differed by 22 residues. The molecular mass of albolabrin calculated on the basis of amino acid sequence was 7574 Da and the pI similarly calculated was 4.27. The molecular mass of elegantin was calculated to be 7806 Da and the theoretical pI to be 4.69. RGD is maintained in the same position (51-53 AA) and all 12 cysteines are identical. Our data suggest that the presence of RGD, the conserved secondary and tertiary structure, are essential for the expression of biological activity by these peptides. Both peptides inhibited ADP-induced platelet aggregation. Extended homologies around the RGDS sequences in human von Willebrand Factor and bovine fibrinogen were found with both peptides.