RESUMO
Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.
Assuntos
Coagulação Sanguínea/fisiologia , Leishmania/metabolismo , Fosfatidilserinas/metabolismo , Psychodidae/parasitologia , Saliva/metabolismo , Animais , Anticoagulantes/metabolismo , Cisteína Endopeptidases , Fator V/antagonistas & inibidores , Fator X/antagonistas & inibidores , Fator Xa , Inibidores do Fator Xa , Humanos , Insetos Vetores/parasitologia , Proteínas de Neoplasias/antagonistas & inibidores , Tempo de Tromboplastina Parcial , Fosfatidilcolinas/metabolismo , Psychodidae/metabolismo , Trombina/antagonistas & inibidores , Extratos de Tecidos/metabolismoRESUMO
Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.
Assuntos
Animais , Humanos , Coagulação Sanguínea/fisiologia , Leishmania/metabolismo , Fosfatidilserinas/metabolismo , Psychodidae/parasitologia , Saliva/metabolismo , Anticoagulantes/metabolismo , Cisteína Endopeptidases , Fator V/antagonistas & inibidores , Fator X/antagonistas & inibidores , Fator Xa/antagonistas & inibidores , Insetos Vetores/parasitologia , Proteínas de Neoplasias/antagonistas & inibidores , Tempo de Tromboplastina Parcial , Fosfatidilcolinas/metabolismo , Psychodidae/metabolismo , Trombina/antagonistas & inibidores , Extratos de Tecidos/metabolismoRESUMO
A produção de microvesículas (MVs) por células tumorais tem sido implicada emuma variedade de processos biológicos, incluindo o estabelecimento de estadoshipercoaguláveis associados ao câncer. Ao mesmo tempo, sabe-se que fatoreshemostáticos desempenham um papel crucial em diversos aspectos da biologiatumoral. Em particular, a expressão da proteína iniciadora da cascata de coagulação Fator Tecidual (FT) tem sido frequentemente correlacionada com o perfil de agressividade de células neoplásicas. Nesse contexto, examinamos a produção deMVs por diferentes linhagens celulares tumorais, assim como a presença de FTnessas estruturas, investigando seu papel no estabelecimento de estados pró-trombóticos e em outros processos celulares importantes para a biologia do tumor. Primeiramente, caracterizamos as propriedades pró-coagulantes e pró-trombóticas de MVs produzidas pela linhagem de melanoma Tm1, tal como comparada à sua linhagem parental de melanócitos não-tumorigênicos (melan-a). Células tumorais exibiram uma taxa de produção de MVs cerca de duas vezes maior do que a observada para melanócitos. MVs de melanoma, por sua vez, apresentaram uma atividade pró-coagulante mais pronunciada, assim como níveis elevados de FT emsua superfície. Esses resultados se refletiram em um maior potencial pró-trombótico in vivo, o qual foi dependente de FT. Análises do plasma de camundongos com melanoma demonstraram a presença de MVs tumorais pró-coagulantes, positivaspara FT, na circulação desses animais. Uma vez que FT pode ser incorporado emMVs tumorais, as quais são capazes de transferir seu conteúdo membranar eintravesicular entre diferentes células, analisamos então o compartilhamento de MVs contendo FT entre linhagens celulares de câncer de mama com diferentes perfis de agressividade. Como esperado, células MDA-MB-231, altamente invasivas e metástaticas, apresentaram maiores níveis de FT funcional em sua superfície, quando comparadas à linhagem celular...
Shedding of microvesicles (MVs) by cancer cells is implicated in a variety ofbiological effects, including the establishment of cancer-associated hypercoagulable states. At the same time, it is known that hemostatic factors play a critical role in diverse aspects of tumor biology. In particular, the expression of the clotting initiatorprotein tissue factor (TF) has been frequently correlated with cancer cellaggressiveness. In this context, we examined MVs production by different tumor cell lines, as well as the TF content of these structures, and investigated its role on the establishment of prothromboti c states and other cellular processes related to tumor biology. First, we characterized the procoagulant and prothrombotic properties of MVs produced by the melanoma cell line Tm1 as compared to its parental nontumourigenicmelanocyte-derived cell line (melan-a). Tumour cells exhibited a twofoldhigher rate of MVs production as compared to melan-a. Melanoma MVsdisplayed a more pronounced procoagulant activity and elevated levels of surface TF. These results were reflected in a higher prothrombotic potential in vivo, which was TF-dependent. Analysis of plasma obtained from melanoma-bearing miceshowed the presence of TF-positive procoagulant tumor-derived MVs at thecirculation of these animals. Since TF was shown to be incorporated into tumorderived MVs, which may transfer their membrane and intravesicular cargo between different cells, we then analyzed the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. As expected, the highly invasive and metastatic MDA-MB-231 cells displayed higher levels of functional TF on their surface when compared to the less aggressive cell line MCF-7. Accordingly,MVs derived from MDA-MB-231 cells exhibited a higher procoagulant potential, as well as a greater ability to promote the assembly of the TF-dependent extrinsic tenase complex (TF/Factor VIIa/Factor X)...
Assuntos
Fenômenos Biológicos , NeoplasiasRESUMO
Microvesículas (MVs) são fragmentos de membrana liberados a partir dasuperfície de diversos tipos celulares, após processos como ativação celular eapoptose, e sua formação está intimamente ligada à perda de assimetriafosfolipídica de membrana, especialmente à exposição de fosfatidilserina (PS).Embora os efeitos biológicos de MVs sejam extremamente variados, acredita-se que a produção de MVs desempenha uma importante função na biologia tumoral, com possível envolvimento na modulação de estados imunossupressivos e pró-trombóticos. Nesse contexto, procuramos esclarecer o papel de microvesículas produzidas pelo melanoma no estabelecimento do tumor maligno. Dessa forma, observamos que células B16F10, uma linhagem de melanoma altamente metastática, produz, in vitro, grandes quantidades de MVs expondo PS. Tais MVs foram capazes de estimular, in vitro, a produção de TGF-β1 um fatorimunorregulatório com grande importância no processo de progressão maligna por macrófagos, assim como aumentar o potencial metastático in vivo de células B16F10. Ambos os efeitos foram dependentes de PS, sendo revertidos por anexina V. Interessantemente, MVs de melanoma induziram ainda metástasespulmonares após inóculo de células B16F10 em camundongos BALB/c,normalmente resistentes a essa linhagem celular. Além disso, buscamoscaracterizar as propriedades pró-coagulantes de MVs produzidas pela linhagem de melanoma Tm1, tal como comparada à sua linhagem parental de melanócitos não-tumorigênicos (melan-A). Apesar da taxa de produção de MVs ser consideravelmente maior na linhagem celular tumoral, a exposição de PS foi bastante semelhante em MVs derivadas de ambas as linhagens melan-A e Tm1. Isso se refletiu na capacidade dessas estruturas em promover a formação do complexo pró-coagulante protrombinase, um processo dependente da presença de superfícies aniônicas ricas em PS. Como esperado, ambas as MVs permitiram a ativação de protrombina em níveis similares...
Microvesicles (MVs) are membrane fragments that can be shed from the cellsurface of various cell types under conditions such as cell activation andapoptosis, being this phenomenon closely associated with loss of phospholipid asymmetry, especially to phosphatidylserine (PS) exposure. Despite the variety of biological effects related to MVs, it is supposed that MVs production play an important role in tumor biology and might modulate immunosupressive and prothrombotic states. Within this context, we attempted to elucidate thecontribution of melanoma-derived MVs to the establishment of malignant tumor.Herein we observed that B16F10 cells, a highly metastatic melanoma cell line,produce large quantities of PS-containing microvesicles in vitro. Tumor MVs increased TGF-β1 production by cultured macrophages and, in vivo, enhanced the metastatic potential of B16F10 cells in C57BL/6 mice, both effects being reversed by annexin V. Most strikingly, MVs induced melanoma metastasis in BALB/c mice, which are normally resistant to this tumor cell line. Furthermore, we characterizedthe procoagulant properties of MVs produced by the tumorigenic melanoma cell line Tm1, as compared to its counterpart non-tumorigenic melanocyte-derived cell line, melan-A. Although the rate of MVs production was considerably higher in the tumorigenic cell line, PS exposure was nearly identical in MVs from both melan-Aand Tm-1. This result is reflected by the ability of these structures to assembly the procoagulant complex prothrombinase, a process that is dependent on the presence of PS-rich anionic membranes. As expected, both MVs supported prothrombin activation at similar rates. On the other hand, melanoma-derived MVs shortened the coagulation time of murine plasma in a lower concentration range than melan-A MVs, suggesting the presence of higher amounts of the clotting initiator, Tissue Factor. In support of this observation, Tissue Factor was detected on the surface...