RESUMO
Platelets are increasingly recognized to play a role in the complications of Streptococcus pneumoniae infections. S. pneumoniae expresses neuraminidases, which may alter glycans on the platelet surface. In the present study, we investigated the capability of pneumococcal neuraminidase A (NanA) to remove sialic acid (desialylation) from the platelet surface, the consequences for the platelet activation status and reactivity, and the ability of neuraminidase inhibitors to prevent these effects. Our results show that soluble NanA induces platelet desialylation. Whereas desialylation itself did not induce platelet activation (P-selectin expression and platelet fibrinogen binding), platelets became hyperreactive to ex vivo stimulation by ADP and cross-linked collagen-related peptide (CRP-XL). Platelet aggregation with leukocytes also increased. These processes were dependent on the ADP pathway, as inhibitors of the pathway (apyrase and ticagrelor) abrogated platelet hyperreactivity. Inhibition of NanA-induced platelet desialylation by neuraminidase inhibitors (e.g., oseltamivir acid) also prevented the platelet effects of NanA. Collectively, our findings show that soluble NanA can desialylate platelets, leading to platelet hyperreactivity, which can be prevented by neuraminidase inhibitors.
Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Infecções Pneumocócicas/sangue , Streptococcus pneumoniae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Neuraminidase/genética , Agregação Plaquetária , Infecções Pneumocócicas/metabolismo , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genéticaRESUMO
Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein ß2GP1 (ß2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Hemostasia , Fosfolipídeos/metabolismo , Ativação Plaquetária , Adulto , Idoso , Animais , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/enzimologia , Coagulação Sanguínea , Membrana Celular/ultraestrutura , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/análise , Ácidos Hidroxieicosatetraenoicos/metabolismo , Lipoxigenases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Teóricos , Fosfolipídeos/análise , Trombose Venosa/sangue , Trombose Venosa/induzido quimicamente , Trombose Venosa/enzimologia , beta 2-Glicoproteína I/metabolismoRESUMO
Using a novel porcine model of intravenous Streptococcus pneumoniae infection, we showed that invasive pneumococcal infections induce marked platelet activation and hyperreactivity. This may contribute to the vascular complications seen in pneumococcal infection.
Assuntos
Plaquetas/microbiologia , Ativação Plaquetária , Infecções Pneumocócicas/sangue , Infecções Pneumocócicas/complicações , Streptococcus pneumoniae/fisiologia , Animais , Plaquetas/patologia , Modelos Animais de Doenças , Humanos , Infecções Pneumocócicas/patologia , Pneumonia Pneumocócica/sangue , Pneumonia Pneumocócica/complicações , Pneumonia Pneumocócica/patologia , Streptococcus pneumoniae/isolamento & purificação , SuínosRESUMO
Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APC's signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3beta (GSK3beta), which was blocked by the PI3K inhibitor LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3beta, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent K(d), approximately 30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APC's ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3beta, thereby contributing to APC's beneficial effects on cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lipoproteínas/metabolismo , Fatores de Coagulação Sanguínea , Humanos , Proteínas Relacionadas a Receptor de LDL , Monócitos/citologia , Fosforilação , Ligação Proteica , Receptores de Superfície Celular , Transdução de Sinais , Células U937RESUMO
The solid-phase synthesis of cyclic RGD peptides containing either one or two furanoid sugar amino acids (SAAs) is reported. Using a cyclization-cleavage approach five peptides were successfully assembled and consecutively tested on their ability to bind to the integrin receptors alpha(v)beta(3) and alpha(IIb)beta(3). The cyclic tetrapeptide c[RGD-SAA] (1) showed the most promising activity in an inhibition assay with an IC(50) of 1.49 microM for the alpha(v)beta(3) receptor and 384 nM for the alpha(IIb)beta(3) receptor.