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1.
Biochem Biophys Res Commun ; 712-713: 149946, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38643717

RESUMO

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.


Assuntos
Apolipoproteínas A , Plaquetas , Agregação Plaquetária , Trombose , Humanos , Trombose/genética , Trombose/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Polimorfismo Genético , Apoproteína(a)/genética , Apoproteína(a)/metabolismo , Apoproteína(a)/química , Selectina-P/genética , Selectina-P/metabolismo
2.
Blood ; 136(8): 974-988, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32584951

RESUMO

Several adaptor molecules bind to cytoplasmic tails of ß-integrins and facilitate bidirectional signaling, which is critical in thrombosis and hemostasis. Interfering with integrin-adaptor interactions spatially or temporally to inhibit thrombosis without affecting hemostasis is an attractive strategy for the development of safe antithrombotic drugs. We show for the first time that the 14-3-3ζ-c-Src-integrin-ß3 complex is formed during platelet activation. 14-3-3ζ-c-Src interaction is mediated by the -PIRLGLALNFSVFYYE- fragment (PE16) on the 14-3-3ζ and SH2-domain on c-Src, whereas the 14-3-3ζ-integrin-ß3 interaction is mediated by the -ESKVFYLKMKGDYYRYL- fragment (EL17) on the 14-3-3ζ and -KEATSTF- fragment (KF7) on the ß3-integrin cytoplasmic tail. The EL17-motif inhibitor, or KF7 peptide, interferes with the formation of the 14-3-3ζ-c-Src-integrin-ß3 complex and selectively inhibits ß3 outside-in signaling without affecting the integrin-fibrinogen interaction, which suppresses thrombosis without causing significant bleeding. This study characterized a previously unidentified 14-3-3ζ-c-Src-integrin-ß3 complex in platelets and provided a novel strategy for the development of safe and effective antithrombotic treatments.


Assuntos
Proteínas 14-3-3/metabolismo , Integrina beta3/metabolismo , Ativação Plaquetária , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas 14-3-3/genética , Adulto , Animais , Feminino , Células HEK293 , Humanos , Integrina beta3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Ativação Plaquetária/genética , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Transdução de Sinais/fisiologia
3.
Arch Toxicol ; 95(11): 3589-3599, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34519865

RESUMO

Venomous snakebites cause clinical manifestations that range from local to systemic and are considered a significant global health challenge. Persistent or refractory thrombocytopenia has been frequently reported in snakebite patients, especially in cases caused by viperidae snakes. Viper envenomation-induced thrombocytopenia may persist in the absence of significant consumption coagulopathy even after the antivenom treatment, yet the mechanism remains largely unknown. Our study aims to investigate the mechanism and discover novel therapeutic targets for coagulopathy-independent thrombocytopenia caused by viper envenomation. Here we found that patients bitten by Protobothrops mucrosquamatus and Trimeresurus stejnegeri, rather than Naja. atra may develop antivenom-resistant and coagulopathy-independent thrombocytopenia. Crude venoms and the derived C-type lectin-like proteins from these vipers significantly increased platelet surface expression of neuraminidase and platelet desialylation, therefore led to platelet ingestion by both macrophages and hepatocytes in vitro, and drastically decreased peripheral platelet counts in vivo. Our study is the first to demonstrate that desialylation-mediated platelet clearance is a novel mechanism of viper envenomation-induced refractory thrombocytopenia and C-type lectin-like proteins derived from the viper venoms contribute to snake venom-induced thrombocytopenia. The results of this study suggest the inhibition of platelet desialylation as a novel therapeutic strategy against viper venom-induced refractory thrombocytopenia.


Assuntos
Hepatócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Trombocitopenia/etiologia , Venenos de Víboras/toxicidade , Animais , Antivenenos/farmacologia , Plaquetas/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neuraminidase/metabolismo , Mordeduras de Serpentes/complicações , Trombocitopenia/patologia , Venenos de Víboras/química , Viperidae
4.
Blood ; 129(13): 1840-1854, 2017 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-28122739

RESUMO

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.


Assuntos
Cadeias beta de Integrinas/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Domínio Catalítico , Moléculas de Adesão Celular , Humanos , Camundongos , Proteínas do Tecido Nervoso , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Proteínas Recombinantes , Semaforinas , Trombose/prevenção & controle
5.
Blood ; 126(2): 247-56, 2015 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-26036802

RESUMO

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder characterized by autoantibodies targeting platelet surface proteins, most commonly GPIIbIIIa (αIIbß3 integrin), leading to platelet destruction. Recently, CD8(+) cytotoxic T-lymphocytes (CTLs) targeting platelets and megakaryocytes have also been implicated in thrombocytopenia. Because steroids are the most commonly administered therapy for ITP worldwide, we established both active (immunized splenocyte engraftment) and passive (antibody injection) murine models of steroid treatment. Surprisingly, we found that, in both models, CD8(+) T cells limited the severity of the thrombocytopenia and were required for an efficacious response to steroid therapy. Conversely, CD8(+) T-cell depletion led to more severe thrombocytopenia, whereas CD8(+) T-cell transfusion ameliorated thrombocytopenia. CD8(+) T-regulatory cell (Treg) subsets were detected, and interestingly, dexamethasone (DEX) treatment selectively expanded CD8(+) Tregs while decreasing CTLs. In vitro coculture studies revealed CD8(+) Tregs suppressed CD4(+) and CD19(+) proliferation, platelet-associated immunoglobulin G generation, CTL cytotoxicity, platelet apoptosis, and clearance. Furthermore, we found increased production of anti-inflammatory interleukin-10 in coculture studies and in vivo after steroid treatment. Thus, we uncovered subsets of CD8(+) Tregs and demonstrated their potent immunosuppressive and protective roles in experimentally induced thrombocytopenia. The data further elucidate mechanisms of steroid treatment and suggest therapeutic potential for CD8(+) Tregs in immune thrombocytopenia.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Dexametasona/uso terapêutico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/imunologia , Animais , Plaquetas/imunologia , Linfócitos T CD8-Positivos/transplante , Terapia Combinada , Modelos Animais de Doenças , Imunoterapia Adotiva , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Púrpura Trombocitopênica Idiopática/terapia , Linfócitos T Citotóxicos , Resultado do Tratamento
6.
J Nutr ; 147(10): 1917-1925, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28855423

RESUMO

Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown.Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway.Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 µM) of Cy-3-g. The expression of activated integrin αIIbß3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3-4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot.Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-µM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (-54.02%) and arterial (-22.90%) shear stresses. The 5-µM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: -48.21%, P-selectin: -50.63%), secretion (CD63: -73.89%, CD40L: -43.70%), fibrinogen binding (-56.79%), and aggregation (-17.81%). The 5-µM dose attenuated (P < 0.01) thrombus growth (-66.67%) without prolonging bleeding time in mice. The 50-µM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk-linker for activation of T cells (LAT)-SLP76 (Syk: -39.08%, LAT: -32.25%, SLP76: -40.00%) and the expression of Lyn (-31.89%), Fyn (-36.27%), and phospholipase C-γ2 (-39.08%).Conclusions: Cy-3-g inhibits human platelet activation, aggregation, secretion, and thrombus formation, and downregulates the collagen-GPVI signaling pathway. Supplementation of Cy-3-g may have protective effects against atherothrombosis.


Assuntos
Plaquetas/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Plantas Comestíveis/química , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombose/prevenção & controle , Proteínas Adaptadoras de Transdução de Sinal/sangue , Adulto , Idoso , Animais , Antocianinas/farmacologia , Antocianinas/uso terapêutico , Antígenos CD/sangue , Aterosclerose/sangue , Aterosclerose/dietoterapia , Aterosclerose/etiologia , Colágeno/sangue , Feminino , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Hemostasia/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Selectina-P/sangue , Fosfoproteínas/sangue , Extratos Vegetais/uso terapêutico , Ativação Plaquetária/efeitos dos fármacos , Transdução de Sinais , Trombose/sangue , Trombose/etiologia
7.
Thromb J ; 14(Suppl 1): 29, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27766055

RESUMO

Platelets are central mediators of thrombosis and hemostasis. At the site of vascular injury, platelet accumulation (i.e. adhesion and aggregation) constitutes the first wave of hemostasis. Blood coagulation, initiated by the coagulation cascades, is the second wave of thrombin generation and enhance phosphatidylserine exposure, can markedly potentiate cell-based thrombin generation and enhance blood coagulation. Recently, deposition of plasma fibronectin and other proteins onto the injured vessel wall has been identified as a new "protein wave of hemostasis" that occurs prior to platelet accumulation (i.e. the classical first wave of hemostasis). These three waves of hemostasis, in the event of atherosclerotic plaque rupture, may turn pathogenic, and cause uncontrolled vessel occlusion and thrombotic disorders (e.g. heart attack and stroke). Current anti-platelet therapies have significantly reduced cardiovascular mortality, however, on-treatment thrombotic events, thrombocytopenia, and bleeding complications are still major concerns that continue to motivate innovation and drive therapeutic advances. Emerging evidence has brought platelet adhesion molecules back into the spotlight as targets for the development of novel anti-thrombotic agents. These potential antiplatelet targets mainly include the platelet receptors glycoprotein (GP) Ib-IX-V complex, ß3 integrins (αIIb subunit and PSI domain of ß3 subunit) and GPVI. Numerous efforts have been made aiming to balance the efficacy of inhibiting thrombosis without compromising hemostasis. This mini-review will update the mechanisms of thrombosis and the current state of antiplatelet therapies, and will focus on platelet adhesion molecules and the novel anti-thrombotic therapies that target them.

8.
J Thromb Haemost ; 21(5): 1274-1288, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36732162

RESUMO

BACKGROUND: Platelet GPIbα-von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb-VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. OBJECTIVE: The objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. METHODS: Natural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα-VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX-induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. RESULTS: We found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbß3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF-mediated platelet aggregation, spreading, and thrombus growth at high shear. CONCLUSION: Fucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb-VWF interaction after fucoidan is hydrolyzed.


Assuntos
Trombose , Fator de von Willebrand , Humanos , Animais , Camundongos , Fator de von Willebrand/metabolismo , Plaquetas/metabolismo , Agregação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Polissacarídeos/farmacologia , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Trombose/metabolismo , Fibrinogênio/metabolismo , Ligação Proteica
9.
Research (Wash D C) ; 6: 0124, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37223472

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 virus is an ongoing global health burden. Severe cases of COVID-19 and the rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are both associated with thrombosis and thrombocytopenia; however, the underlying mechanisms remain inadequately understood. Both infection and vaccination utilize the spike protein receptor-binding domain (RBD) of SARS-CoV-2. We found that intravenous injection of recombinant RBD caused significant platelet clearance in mice. Further investigation revealed the RBD could bind platelets, cause platelet activation, and potentiate platelet aggregation, which was exacerbated in the Delta and Kappa variants. The RBD-platelet interaction was partially dependent on the ß3 integrin as binding was significantly reduced in ß3-/- mice. Furthermore, RBD binding to human and mouse platelets was significantly reduced with related αIIbß3 antagonists and mutation of the RGD (arginine-glycine-aspartate) integrin binding motif to RGE (arginine-glycine-glutamate). We developed anti-RBD polyclonal and several monoclonal antibodies (mAbs) and identified 4F2 and 4H12 for their potent dual inhibition of RBD-induced platelet activation, aggregation, and clearance in vivo, and SARS-CoV-2 infection and replication in Vero E6 cells. Our data show that the RBD can bind platelets partially though αIIbß3 and induce platelet activation and clearance, which may contribute to thrombosis and thrombocytopenia observed in COVID-19 and VITT. Our newly developed mAbs 4F2 and 4H12 have potential not only for diagnosis of SARS-CoV-2 virus antigen but also importantly for therapy against COVID-19.

10.
Blood ; 116(18): 3660-8, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-20647570

RESUMO

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined ß3 integrin-deficient and FcRn-deficient (ß3(-/-)FcRn(-/-)) mice. We found that ß3(-/-)FcRn(-/-) mice are immunoresponsive to ß3(+/+)FcRn(-/-) platelets. The generated antibodies were ß3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult ß3(+/+)FcRn(-/-) mice. FNIT was observed when immunized ß3(-/-)FcRn(+/+) females were bred with ß3(+/+)FcRn(+/+) males, while no FNIT occurred in ß3(-/-)FcRn(-/-) females bred with ß3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.


Assuntos
Feto/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Integrina beta3/imunologia , Receptores Fc/imunologia , Trombocitopenia Neonatal Aloimune/imunologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Plaquetas/imunologia , Regulação para Baixo , Feminino , Feto/fisiopatologia , Deleção de Genes , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulinas Intravenosas/uso terapêutico , Integrina beta3/genética , Masculino , Troca Materno-Fetal , Camundongos , Modelos Animais , Placenta/metabolismo , Gravidez , Transporte Proteico , Receptores Fc/genética , Trombocitopenia Neonatal Aloimune/tratamento farmacológico
11.
Blood ; 115(6): 1247-53, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20007808

RESUMO

Immune thrombocytopenia (ITP) is a bleeding disorder characterized by antibody-opsonized platelets being prematurely destroyed in the spleen, although some patients with ITP may have a cell-mediated form of thrombocytopenia. Although several animal models of ITP have been developed, few mimic primary chronic ITP nor have any shown cell-mediated platelet destruction. To create this type of model, splenocytes from CD61 knockout mice immunized against CD61(+) platelets were transferred into severe combined immunodeficient (SCID) (CD61(+)) mouse recipients, and their platelet counts and phenotypes were observed. As few as 5 x 10(4) splenocytes induced a significant thrombocytopenia and bleeding mortality (80%) in recipients within 3 weeks after transfer. Depletion of lymphocyte subsets before transfer showed that the splenocyte's ability to induce thrombocytopenia and bleeding completely depended on CD4(+) T helper cells and that both CD19(+) B cell (antibody)- and CD8(+) T cell (cell)-mediated effector mechanisms were responsible. Treatment of the SCID mouse recipients with intravenous gamma-globulins raised platelet counts and completely prevented bleeding mortality induced by antibody-mediated effector mechanisms but did not affect cell-mediated disease. This novel model not only shows both antibody- and cell-mediated ITP and bleeding but also suggests that these 2 effector mechanisms have a differential response to therapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Imunoglobulinas Intravenosas/administração & dosagem , Púrpura Trombocitopênica Idiopática/imunologia , Púrpura Trombocitopênica Idiopática/terapia , Animais , Antígenos CD19/imunologia , Plaquetas/imunologia , Feminino , Citometria de Fluxo , Integrina beta3/imunologia , Depleção Linfocítica , Megacariócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/patologia , Baço/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia
12.
Transfusion ; 52(7): 1446-57, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22251227

RESUMO

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal or neonatal platelets (PLTs). Results from our recent large screening study suggest that the pathophysiology of FNAIT is more similar to hemolytic disease of the fetus and newborn (HDFN) than previously thought. Immunization against HPA-1a might therefore be preventable by a prophylactic regimen of inducing antibody-mediated immune suppression (AMIS), which has been documented to be a useful prophylaxis against HDFN. This preclinical proof-of-concept study investigated whether passive administration of anti-ß3 integrin could induce AMIS and thereby prevent clinical complications of FNAIT. STUDY DESIGN AND METHODS: A murine model of FNAIT using ß3 integrin (GPIIIa)-deficient (ß3-/-) mice was employed for this study. AMIS in ß3-/- mice was induced by intravenous administration of human anti-HPA-1a immunoglobulin G or murine anti-ß3 antisera given as prophylaxis after transfusion of HPA-1a-positive human PLTs or murine wild-type PLTs, respectively. RESULTS: AMIS against both human and murine PLT antigens was induced using this prophylactic approach, reducing the amount of maternal PLT antibodies by up to 90%. Neonatal PLT counts were significantly increased and pregnancy outcome was improved in a dose-dependent manner. The incidence of intracranial hemorrhage, miscarriage, and dead-born pups in mice receiving high-dose prophylaxis was reduced to that of normal controls. We also observed that the severity of thrombocytopenia inversely correlated with birth weight. CONCLUSION: This work conceptually proves that prophylactic administration of PLT antibodies induces AMIS and prevents poor pregnancy outcome in FNAIT.


Assuntos
Antígenos de Plaquetas Humanas/imunologia , Incompatibilidade de Grupos Sanguíneos/prevenção & controle , Doenças Fetais/prevenção & controle , Imunoglobulina G/farmacologia , Integrina beta3/imunologia , Isoanticorpos/farmacologia , Troca Materno-Fetal/imunologia , Trombocitopenia Neonatal Aloimune/prevenção & controle , Animais , Incompatibilidade de Grupos Sanguíneos/genética , Incompatibilidade de Grupos Sanguíneos/imunologia , Incompatibilidade de Grupos Sanguíneos/patologia , Modelos Animais de Doenças , Feminino , Doenças Fetais/genética , Doenças Fetais/imunologia , Doenças Fetais/patologia , Humanos , Imunoglobulina G/imunologia , Recém-Nascido , Integrina beta3/genética , Isoanticorpos/imunologia , Masculino , Gravidez , Trombocitopenia Neonatal Aloimune/genética , Trombocitopenia Neonatal Aloimune/imunologia , Trombocitopenia Neonatal Aloimune/patologia
13.
Am J Hematol ; 87(2): 206-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22139961

RESUMO

Immune thrombocytopenia (ITP) is characterized by platelet clearance mediated primarily by autoantibodies against the platelet GPIIbIIIa and/or GPIbα. Steroid therapy is a first-line treatment for ITP. However, some patients are refractory to this therapy and currently no method can predict which patients will respond. To evaluate whether steroids are equally efficacious in treating patients with ITP caused by anti-GPIIbIIIa versus anti-GPIbα antibodies, we performed a retrospective study on 176 newly diagnosed patients with acute ITP who had severe bleeding symptoms and were admitted as resident patients to the hospital. The patients were treated first with intravenous administration of high-dose dexamethasone (DXM), followed by oral administration of prednisone. Response to therapy was observed in a majority of patients with antibodies specific for GPIIbIIIa (31/43) or without detectable antibodies against either GPIIbIIIa or GPIbα (36/45). In contrast, the steroid response was significantly lower in patients with anti-GPIbα antibodies (9/34) or with antibodies against both GPIbα and GPIIbIIIa (16/54). The preliminary findings of this study suggest that in future prospective clinical trials including corticosteroids, the anti-GPIbα, and -GPIIbIIIa status should be assessed in order to test its potential relevance in deciding future treatments.


Assuntos
Autoanticorpos/imunologia , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Glicoproteínas de Membrana/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Prednisona/uso terapêutico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Plaquetas/patologia , Dexametasona/farmacologia , Esquema de Medicação , Monitoramento de Medicamentos , Feminino , Glucocorticoides/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Complexo Glicoproteico GPIb-IX de Plaquetas , Prednisona/farmacologia , Púrpura Trombocitopênica Idiopática/imunologia , Púrpura Trombocitopênica Idiopática/patologia , Estudos Retrospectivos , Resultado do Tratamento
14.
Blood ; 114(2): 425-36, 2009 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-19332769

RESUMO

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg(-/-)) mice, and engagement of the C-terminus of the fibrinogen gamma chain with beta3 integrin was required for this process. Furthermore, Fg(-/-) platelets significantly increased P-selectin expression following transfusion into beta3 integrin-deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.


Assuntos
Plaquetas/metabolismo , Membrana Celular/metabolismo , Fibrinogênio/metabolismo , Espaço Intracelular/metabolismo , Selectina-P/metabolismo , Adulto , Animais , Plaquetas/ultraestrutura , Fibrinogênio/genética , Humanos , Integrina beta3/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
15.
J Tradit Chin Med ; 29(1): 60-3, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19514191

RESUMO

OBJECTIVE: To probe into the mechanisms of electroacupuncture (EA) at Shangjuxu (ST 37) for treatment of the ulcerative colitis (UC). METHODS: Forty male Wistar rats were randomly divided into 4 groups: a normal group, a model group, a Shangjuxu group and a non-acupoint group, 10 rats in each group. The UC rat model was made with enema of trinitro-benzenesulfonic acid (TNBSA), and the changes of interleukin-1beta (IL-1beta) and interleukin-4 (IL-4) contents after EA at Shangjuxu (ST 37) were observed. RESULTS: EA at Shangjuxu (ST 37) could significantly decrease the IL-1beta content and increase the IL-4 content in the colic tissues of the UC rats with significant differences as compared with the model group and the non-acupoint group (P<0.05 or P<0.01). CONCLUSION: The mechanisms of EA at Shangjuxu (ST 37) for treatment of the UC rats is possibly related with the decrease of IL-1beta, a inflammation-promoting cytokine, and the increase of IL-4, a anti-inflammatory cytokine.


Assuntos
Pontos de Acupuntura , Colite Ulcerativa/imunologia , Colite Ulcerativa/terapia , Eletroacupuntura , Interleucina-1beta/imunologia , Interleucina-4/imunologia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar
16.
Nat Commun ; 9(1): 3608, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30190457

RESUMO

Platelet αIIbß3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet ß3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbß3 integrin, and the direct apoA-IV-αIIbß3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbß3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbß3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.


Assuntos
Apolipoproteínas A/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/metabolismo , Adulto , Animais , Apolipoproteínas A/genética , Apolipoproteínas A/farmacologia , Ácido Aspártico/metabolismo , Sítios de Ligação , Ritmo Circadiano/fisiologia , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Período Pós-Prandial , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Trombose/tratamento farmacológico
17.
Semin Fetal Neonatal Med ; 21(1): 19-27, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26810319

RESUMO

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is an alloimmune disorder resulting from platelet opsonization by maternal antibodies that destroy fetal platelets. The major risk of FNAIT is severe bleeding, particularly intracranial hemorrhage. Miscarriage has also been reported but the incidence requires further study. Analogous to adult autoimmune thrombocytopenia (ITP), the major target antigen in FNAIT is the platelet membrane glycoprotein (GP)IIbIIIa. FNAIT caused by antibodies against platelet GPIbα or other antigens has also been reported, but the reported incidence of the anti-GPIbα-mediated FNAIT is far lower than in ITP. To date, the maternal immune response to fetal platelet antigens is still not well understood and it is unclear why bleeding is more severe in FNAIT than in ITP. In this review, we introduce the pathogenesis of FNAIT, particularly those new discoveries from animal models, and discuss possible improvements for the diagnosis, therapy, and prevention of this devastating disease.


Assuntos
Trombocitopenia Neonatal Aloimune , Animais , Modelos Animais de Doenças , Feminino , Humanos , Recém-Nascido , Assistência Perinatal/métodos , Gravidez , Diagnóstico Pré-Natal , Trombocitopenia Neonatal Aloimune/diagnóstico , Trombocitopenia Neonatal Aloimune/etiologia , Trombocitopenia Neonatal Aloimune/imunologia , Trombocitopenia Neonatal Aloimune/terapia
18.
Genes Dis ; 2(2): 173-185, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28345015

RESUMO

Platelets play critical roles in hemostasis and thrombosis. Emerging evidence indicates that they are versatile cells and also involved in many other physiological processes and disease states. Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening bleeding disorder caused by fetal platelet destruction by maternal alloantibodies developed during pregnancy. Gene polymorphisms cause platelet surface protein incompatibilities between mother and fetus, and ultimately lead to maternal alloimmunization. FNAIT is the most common cause of intracranial hemorrhage in full-term infants and can also lead to intrauterine growth retardation and miscarriage. Proper diagnosis, prevention and treatment of FNAIT is challenging due to insufficient knowledge of the disease and a lack of routine screening as well as its frequent occurrence in first pregnancies. Given the ethical difficulties in performing basic research on human fetuses and neonates, animal models are essential to improve our understanding of the pathogenesis and treatment of FNAIT. The aim of this review is to provide an overview on platelets, hemostasis and thrombocytopenia with a focus on the advancements made in FNAIT by utilizing animal models.

19.
J Clin Invest ; 125(4): 1545-56, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25774504

RESUMO

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against ß3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-ß3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-ß3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.


Assuntos
Antígenos de Plaquetas Humanas/imunologia , Autoantígenos/imunologia , Plaquetas/imunologia , Imunidade Materno-Adquirida , Imunoglobulina G/imunologia , Imunoglobulinas Intravenosas/uso terapêutico , Integrina beta3/imunologia , Hemorragias Intracranianas/etiologia , Neovascularização Patológica/etiologia , Trombocitopenia Neonatal Aloimune/imunologia , Animais , Especificidade de Anticorpos , Apoptose , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Modelos Animais de Doenças , Feminino , Sangue Fetal/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Soros Imunes/toxicidade , Integrina beta3/genética , Hemorragias Intracranianas/embriologia , Hemorragias Intracranianas/imunologia , Hemorragias Intracranianas/fisiopatologia , Masculino , Troca Materno-Fetal , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Gravidez , Proteínas Proto-Oncogênicas c-akt/fisiologia , Vasos Retinianos/embriologia , Vasos Retinianos/patologia , Trombocitopenia Neonatal Aloimune/embriologia , Trombocitopenia Neonatal Aloimune/prevenção & controle
20.
Thromb Haemost ; 110(6): 1250-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24067944

RESUMO

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies against fetal/neonatal platelets. FNAIT is also linked with miscarriages, although the incidence and mechanisms of fetal death have not been well studied. IntegrinαIIbß3 (GPIIbIIIa) and the GPIbα complex are major glycoproteins expressed on platelets and are also major antigens targeted in autoimmune thrombocytopenia (ITP), but reported cases of anti-GPIb-mediated FNAIT are rare. Bacterial and viral infections have been causally linked with the pathogenesis of immune-mediated thrombocytopenia (ITP); however, it is unknown whether these infections contribute to the severity of FNAIT. Here, immune responses against platelet antigens were examined by transfusing wild-type (WT) mouse platelets into ß3-/- or GPIbα-/- mice. To mimic bacterial or viral infections, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) were injected intraperitoneally following platelet transfusions. The FNAIT model was established by breeding the immunised female mice with WT male mice. We demonstrated for the first time that the platelet GPIbα has lower immunogenicity compared to ß3 integrin. Interestingly, co-stimulation with LPS or Poly I:C markedly enhanced the immune response against platelet GPIbα and caused severe pathology of FNAIT (i.e. miscarriages). LPS or Poly I:C also enhanced the immune response against platelet ß3 integrin. Our data suggest that bacterial and viral infections facilitate the anti-platelet GPIbα response, which may lead to a severe non-classical FNAIT (i.e. miscarriage but not neonatal bleeding) that has not been adequately reported in humans.


Assuntos
Aborto Espontâneo/imunologia , Plaquetas/imunologia , Doenças Fetais/imunologia , Infecções/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombocitopenia Neonatal Aloimune/imunologia , Aborto Espontâneo/etiologia , Animais , Autoantígenos/imunologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Imunidade , Imunização , Infecções/complicações , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Transfusão de Plaquetas , Poli I-C/administração & dosagem , Gravidez
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