RESUMO
Glycoprotein (GP)VI, that binds collagen, together with GPIb-IX-V which binds vonWillebrand factor, forms an adheso-signalling complex on platelets that initiates thrombus formation in haemostasis and thrombosis. In this study, we show that two snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of GPVI by a mechanism that involves activation of endogenous platelet metalloproteinases. Alborhagin is a viper venom metalloproteinase from Trimeresurus albolabris, while crotarhagin is a previously undescribed toxin from the rattlesnake Crotalus horridus horridus ( approximately 60-kDa non-reduced and reduced). Like alborhagin, crotarhagin induces aggregation in human platelet-rich plasma (maximal activity, approximately 0.3 microg/ml). Aggregation of washed platelets was inhibited by soluble GPVI ectodomain expressed as an Fc-fusion protein, confirming crotarhagin targeted GPVI. Treating washed platelets with crotarhagin or alborhagin resulted in time-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble GPVI fragment in supernatants. Crotarhagin also induced shedding in GPVItransfected RBL-2H3 cells. Crotarhagin-induced shedding was metalloproteinase-dependent (inhibited by EDTA), but also blocked by inhibitors of GPVI signalling (Src kinase inhibitors, PP1 or PP2, or Syk inhibitor, piceatannol), indicating shedding required GPVI-dependent platelet activation. Together, the data suggest that the rattlesnake metalloproteinase, crotarhagin, and the viper toxin alborhagin, induce GPVI shedding by a mechanism involving activation of endogenous platelet metalloproteinases rather than direct cleavage of GPVI.
Assuntos
Plaquetas/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Metaloendopeptidases/toxicidade , Metaloproteases/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Receptores de Colágeno/efeitos dos fármacos , Venenos de Víboras/toxicidade , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Ativação Enzimática , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fragmentos de Peptídeos/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Receptores de Colágeno/química , Receptores de Colágeno/genética , Receptores de Colágeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Quinase Syk , Fatores de Tempo , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.
Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Lectinas Tipo C/química , Agregação Plaquetária/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anti-Infecciosos/isolamento & purificação , Venenos de Crotalídeos/isolamento & purificação , Crotalus/fisiologia , Integrinas/fisiologia , Lectinas Tipo C/isolamento & purificação , Dados de Sequência Molecular , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores de Colágeno/efeitos dos fármacosRESUMO
The leflunomide metabolite analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK) which plays an important role in platelet physiology by regulating the glycoprotein GPVI-FcRgamma-coupled collagen receptor signaling pathway. At low micromolar concentrations, LFM-A13 inhibited collagen-induced ultrastructural changes indicative of activation. LFM-A13 inhibited collagen (but not thrombin, TRAP-6, or ADP)-induced platelet aggregation in a concentration-dependent fashion with an IC50 value of 2.8 microM. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 1 to 100 mg/kg. At nontoxic dose levels, LFM-A13 prolonged the tail bleeding times of mice and improved event-free survival in two mouse models of agonist-induced invariably fatal pulmonary thromboembolism. To our knowledge, LFM-A13 is the first anti-thrombotic agent which prevents platelet aggregation by inhibiting BTK.