RESUMO
Apolipoprotein (apo) E4 is the major known genetic risk factor for Alzheimer's disease (AD). We have shown in vitro and in vivo that apoE4 preferentially undergoes aberrant cleavage in neurons, yielding neurotoxic C-terminal-truncated fragments. To study the effect of these fragments on amyloid-ß (Aß) clearance/deposition and their potential synergy with Aß in eliciting neuronal and behavioral deficits, we cross-bred transgenic mice expressing apoE3, apoE4, or apoE4(Δ272-299) with mice expressing human amyloid protein precursor (APP) harboring familial AD mutations (hAPP(FAD)). At 6-8 mo of age, hAPP(FAD) mice expressing apoE3 or apoE4 had lower levels of hippocampal Aß (94% and 89%, respectively) and less Aß deposition (89% and 87%) than hAPP(FAD) mice without apoE, whereas hAPP(FAD) mice expressing mouse apoE had higher Aß levels. Thus, human apoE stimulates Aß clearance, but mouse apoE does not. Expression of apoE4(Δ272-299) reduced total Aß levels by only 63% and Aß deposition by 46% compared with hAPP(FAD) mice without apoE. Unlike apoE3 and apoE4, the C-terminal-truncated apoE4 bound poorly with Aß peptides, leading to decreased Aß clearance and increased Aß deposition. Despite their lower levels of Aß and Aß deposition, hAPP(FAD)/apoE4(Δ272-299) mice accumulated pathogenic Aß oligomers and displayed neuronal and behavioral deficits similar to or more severe than those in hAPP(FAD) mice. Thus, the C-terminal-truncated apoE4 fragment inefficiently clears Aß peptides and acts in concert with low levels of Aß to elicit neuronal and behavioral deficits in mice.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E4/fisiologia , Comportamento Animal , Neurônios/fisiologia , Animais , Apolipoproteína E4/metabolismo , Humanos , Camundongos , Ligação ProteicaRESUMO
Frontotemporal lobar degeneration with motor neuron disease (FTLD-MND) is characterized by neuronal cytoplasmic inclusions containing TDP-43. Apolipoprotein E4 (apoE4), derived from the apoE ϵ4 allele, enhances brain atrophy in FTLD through unknown mechanisms. Here, we studied two siblings with C9ORF72-linked familial FTLD-MND, an apoE ϵ4 homozygote and an apoE ϵ3 homozygote. The apoE ϵ4 homozygote had more cognitive-behavioral symptoms, fronto-insulo-temporal atrophy, and apoE fragments and aggregates in the anterior cingulate cortex. ApoE formed complexes with TDP-43 that were more abundant in the apoE ϵ4 homozygote. Although differences seen in a sibling pair could arise due to chance, these findings raise the possibility that apoE4 exacerbates brain pathology in FTLD through formation of neurotoxic apoE fragments and interactions with TDP-43.
Assuntos
Apolipoproteínas E/genética , Proteínas de Ligação a DNA/genética , Degeneração Lobar Frontotemporal/genética , Doença dos Neurônios Motores/genética , Feminino , Degeneração Lobar Frontotemporal/complicações , Degeneração Lobar Frontotemporal/diagnóstico , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Doença dos Neurônios Motores/complicações , Doença dos Neurônios Motores/diagnóstico , Testes Neuropsicológicos , Escalas de Graduação Psiquiátrica , IrmãosRESUMO
Apolipoprotein E4 (apoE4) is the major genetic risk factor for Alzheimer's disease. However, the underlying mechanisms are unclear. We found that female apoE4 knock-in (KI) mice had an age-dependent decrease in hilar GABAergic interneurons that correlated with the extent of learning and memory deficits, as determined in the Morris water maze, in aged mice. Treating apoE4-KI mice with daily peritoneal injections of the GABA(A) receptor potentiator pentobarbital at 20 mg/kg for 4 weeks rescued the learning and memory deficits. In neurotoxic apoE4 fragment transgenic mice, hilar GABAergic interneuron loss was even more pronounced and also correlated with the extent of learning and memory deficits. Neurodegeneration and tauopathy occurred earliest in hilar interneurons in apoE4 fragment transgenic mice; eliminating endogenous Tau prevented hilar GABAergic interneuron loss and the learning and memory deficits. The GABA(A) receptor antagonist picrotoxin abolished this rescue, while pentobarbital rescued learning deficits in the presence of endogenous Tau. Thus, apoE4 causes age- and Tau-dependent impairment of hilar GABAergic interneurons, leading to learning and memory deficits in mice. Consequently, reducing Tau and enhancing GABA signaling are potential strategies to treat or prevent apoE4-related Alzheimer's disease.
Assuntos
Apolipoproteína E4/metabolismo , Giro Denteado/metabolismo , Interneurônios/fisiologia , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Ácido gama-Aminobutírico/metabolismo , Proteínas tau/metabolismo , Fatores Etários , Análise de Variância , Animais , Apolipoproteína E4/genética , Células Cultivadas , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiopatologia , Eletrofisiologia , Feminino , Imuno-Histoquímica , Interneurônios/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Transgênicos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Pentobarbital/administração & dosagem , Terminações Pré-Sinápticas/fisiologia , Estatísticas não ParamétricasRESUMO
The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.
Assuntos
Apolipoproteína E4/metabolismo , Camundongos Transgênicos , Neurônios , Isoformas de Proteínas/metabolismo , Animais , Apolipoproteína E4/genética , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Ácido Caínico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Isoformas de Proteínas/genéticaRESUMO
Neuronal expression of apolipoprotein (apo) E4 may contribute to the pathogenesis of Alzheimer's disease (AD). In studying how apoE expression is regulated in neurons, we identified a splicing variant of apoE mRNA with intron-3 retention (apoE-I3). ApoE-I3 mRNA was detected in neuronal cell lines and primary neurons, but not in astrocytic cell lines or primary astrocytes, from humans and mice by reverse transcription (RT)-PCR. In both wild-type and human apoE knock-in mice, apoE-I3 was found predominantly in cortical and hippocampal neurons by in situ hybridization. Cell fractionation and quantitative RT-PCR revealed that over 98% of the apoE-I3 mRNA was retained in the nucleus without protein translation. In transfected primary neurons, apoE expression increased dramatically when intron-3 was deleted from a genomic DNA construct and decreased markedly when intron-3 was inserted into a cDNA construct, suggesting that intron-3 retention/splicing controls apoE expression in neurons. In response to excitotoxic challenge, the apoE-I3 mRNA was markedly increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons in mice; apoE mRNA showed the opposite pattern. This apparent precursor-product relationship between apoE-I3 and apoE mRNA was supported by a transcriptional inhibition study. Thus, neuronal expression of apoE is controlled by transcription of apoE-I3 under normal conditions and by processing of apoE-I3 into mature apoE mRNA in response to injury.
Assuntos
Apolipoproteínas E/biossíntese , Apolipoproteínas E/genética , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Íntrons/fisiologia , Neurônios/metabolismo , Splicing de RNA/fisiologia , Animais , Apolipoproteínas E/metabolismo , Linhagem Celular , Córtex Cerebral/citologia , Hipocampo/citologia , Humanos , Hibridização In Situ , Camundongos , Camundongos Mutantes , Processamento Pós-Transcricional do RNA/fisiologiaRESUMO
Platelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury. This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets. However, the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibalpha, the VWF-binding subunit of the GP Ib-IX-V complex. We evaluated the potential interactions of T30923 with GP Ibalpha and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the beta-sheets of the N-terminal region of GP Ibalpha (E14-D18, S39, D63-S64, and D83-S85). Experimentally, T30923 bound GP Ibalpha and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilised VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP Ibalpha-VWF interaction.
Assuntos
Glicoproteínas/química , Oligonucleotídeos/química , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Conformação Molecular , Oligonucleotídeos/farmacologia , Adesividade Plaquetária , Agregação Plaquetária , Ligação Proteica , Estrutura Terciária de Proteína , Estresse Mecânico , Fator de von Willebrand/químicaRESUMO
To study the profile and regulation of apolipoprotein E (apoE) expression in the CNS, we generated mice in which apoE expression can be detected in vivo with unprecedented sensitivity and resolution. cDNA encoding enhanced green fluorescent protein (EGFP) with a stop codon was inserted by gene targeting into the apoE gene locus (EGFPapoE) immediately after the translation initiation site. Insertion of EGFP into one apoE allele provides a real-time location marker of apoE expression in vivo; the remaining allele is sufficient to maintain normal cellular physiology. In heterozygous EGFPapoE mice, EGFP was highly expressed in hepatocytes and peritoneal macrophages. EGFP was also expressed in brain astrocytes; however some astrocytes (approximately 25%) expressed no EGFP, suggesting that a subset of these cells does not express apoE. EGFP was expressed in <10% of microglia after kainic acid treatment, suggesting that microglia are not a major source of brain apoE. Although hippocampal neurons did not express EGFP under normal conditions, kainic acid treatment induced intense expression of EGFP in injured neurons, demonstrating apoE expression in neurons in response to excitotoxic injury. The neuronal expression was confirmed by in situ hybridization of mouse apoE mRNA and by anti-apoE immunostaining. Smooth muscle cells of large blood vessels and cells surrounding small vessels in the CNS also strongly expressed EGFP, as did cells in the choroid plexus. EGFPapoE reporter mice will be useful for studying the regulation of apoE expression in the CNS and might provide insights into the diverse mechanisms of apoE4-related neurodegeneration.
Assuntos
Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Neurônios/metabolismo , Animais , Apolipoproteínas E/genética , Perfilação da Expressão Gênica , Marcação de Genes/métodos , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Microscopia de Fluorescência/métodosRESUMO
Adhesion of platelets to the exposed extracellular matrix proteins at sites of vascular injury is partly regulated by the local fluid shear stress. Because the Leu33Pro (Pl(A)) polymorphism of integrin beta(3) confers only a modest increase in adhesion under static conditions, we used CHO and 293 cells expressing the Leu33 or Pro33 isoform of beta(3) in flow chamber experiments to test whether shear forces would alter the Pl(A) adhesive phenotype. We found that shear force augmented the Pro33-mediated enhanced adhesion to fibrinogen. This Pro33-dependent enhancement was aspirin-sensitive and was also observed on immobilized von Willebrand factor and cryoprecipitate, but not fibronectin. Thus, shear stress enhances the adhesive phenotype of the Pro33 cells to multiple physiologic substrates.
Assuntos
Adesão Celular/fisiologia , Integrina beta3/fisiologia , Prolina/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Integrina beta3/química , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Apolipoprotein (apo) E, a polymorphic protein with three isoforms (apoE2, apoE3, and apoE4), is essential for lipid homeostasis. Carriers of apoE4 are at higher risk for developing Alzheimer's disease. We have investigated adult neurogenesis in mice with knockout (KO) for apoE or with knockin (KI) alleles for human apoE3 or apoE4, and we report that neurogenesis is reduced in both apoE-KO and apoE4-KI mice. In apoE-KO mice, increased BMP signaling promoted glial differentiation at the expense of neurogenesis. In contrast, in apoE4-KI mice, presynaptic GABAergic input-mediated maturation of newborn neurons was diminished. Tau phosphorylation, an Alzheimer's disease characteristic, and levels of neurotoxic apoE fragments were both elevated in apoE4-KI hippocampal neurons concomitant with decreased GABAergic interneuron survival. Potentiating GABAergic signaling restored neuronal maturation and neurogenesis in apoE4-KI mice to normal levels. These findings suggest that GABAergic signaling can be targeted to mitigate the deleterious effects of apoE4 on neurogenesis.
Assuntos
Células-Tronco Adultas/metabolismo , Doença de Alzheimer/metabolismo , Apolipoproteínas/metabolismo , Neuroglia/metabolismo , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Animais Recém-Nascidos , Apolipoproteínas/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Agonistas GABAérgicos/administração & dosagem , Técnicas de Introdução de Genes , Hipocampo/patologia , Humanos , Interneurônios/metabolismo , Interneurônios/patologia , Camundongos , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas tau/metabolismoRESUMO
Shiga toxin 1 (Stx-1) and Stx-2 produced by enterohemorrhagic Escherichia coli cause the diarrhea-associated hemolytic uremic syndrome (HUS). This type of HUS is characterized by obstruction of the glomeruli and renal microvasculature by platelet-fibrin thrombi, acute renal failure, thrombocytopenia, microvascular hemolytic anemia, and plasma levels of von Willebrand factor (VWF)-cleaving protease (ADAMTS13) activity that are within a broad normal range. We investigated the mechanism of initial platelet accumulation on Stx-stimulated endothelial cells. Stx-1 or Stx-2 (1-10 nM) stimulated the rapid secretion of unusually large (UL) VWF multimeric strings from human umbilical vein endothelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs). Perfused normal human platelets immediately adhered to the secreted ULVWF multimeric strings. Nanomolar concentrations (1-10 nM) of the Shiga toxins were as effective in inducing the formation of ULVWF-platelet strings as millimolar concentrations (0.1-20 mM) of histamine. The rate of ULVWF-platelet string cleavage by plasma or recombinant ADAMTS13 was delayed by 3 to 10 minutes (or longer) in the presence of 10 nM Stx-1 or Stx-2 compared with 20 mM histamine. Stx-induced formation of ULVWF strings, and impairment of ULVWF-platelet string cleavage by ADAMTS13, may promote initial platelet adhesion above glomerular endothelial cells. These processes may contribute to the evolution of glomerular occlusion by platelet and fibrin thrombi in diarrhea-associated HUS.
Assuntos
Proteínas ADAM/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Síndrome Hemolítico-Urêmica/metabolismo , Toxinas Shiga/farmacologia , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/farmacologia , Proteína ADAMTS13 , Antígenos Glicosídicos Associados a Tumores/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Histamina/farmacologia , Humanos , Peso Molecular , Adesividade Plaquetária , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/metabolismoRESUMO
A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.
Assuntos
Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Fluxo Pulsátil , Fator de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Proteínas Sanguíneas/metabolismo , Endotélio Vascular/citologia , Humanos , Técnicas In Vitro , Metaloendopeptidases/química , Mutagênese , Estrutura Terciária de Proteína , Veias Umbilicais/citologiaRESUMO
ADAMTS13 cleaves ultralarge and hyperreactive von Willebrand factor (ULVWF) freshly released from activated endothelial cells to smaller and less active forms. This process may be affected by the amount of ULVWF released and the processing capacity of ADAMTS13, contributing to the development of thrombotic diseases. We examined the effects of inflammatory cytokines on the release and cleavage of ULVWF to evaluate potential links between inflammation and thrombosis. Human umbilical vein endothelial cells were treated with interleukin 6 (IL-6), IL-8, or tumor necrosis factor alpha (TNF-alpha), and the formation of platelet-decorated ULVWF strings was quantitated. IL-8 and TNF-alpha significantly stimulated the release of ULVWF in a dose-dependent manner. IL-6 induced ULVWF release only when it was in complex with the soluble IL-6 receptor. IL-6, but not IL-8 nor TNF-alpha, inhibited the cleavage of ULVWF strings by ADAMTS13 under flowing, but not static, conditions. These results suggest that inflammatory cytokines may stimulate the ULVWF release (IL-8 and TNF-alpha) and inhibit the ULVWF cleavage (IL-6), resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. The findings describe a potential linkage between inflammation and thrombosis that may be of therapeutic importance.
Assuntos
Endotélio Vascular/metabolismo , Interleucina-6/farmacologia , Interleucina-8/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fator de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Bário/química , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Cinética , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Estrutura Quaternária de Proteína , Receptores de Interleucina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Estimulação Química , Fator de Necrose Tumoral alfa/genética , Veias Umbilicais/citologia , Ureia/química , Fator de von Willebrand/química , Fator de von Willebrand/genéticaRESUMO
Thrombotic thrombocytopenic purpura is caused by congenital or acquired deficiency of ADAMTS-13, a metalloprotease that cleaves the endothelium-derived ultra-large multimers of von Willebrand factor (ULVWF). The proteolysis converts hyper-reactive and thrombogenic ULVWF into smaller and less adhesive plasma forms. Activity of ADAMTS-13 is usually measured in a static system under non-physiological conditions that require protein denaturation and prolonged incubation. We have demonstrated previously that ULVWF multimers, upon release from endothelial cells, form platelet-decorated string-like structures that are rapidly cleaved by ADAMTS-13. Here we report the direct interaction between ADAMTS-13 and VWF under both static and flowing conditions. ADAMTS-13-coated beads adhered to both immobilized VWF and ULVWF strings presented by stimulated endothelial cells. These beads adhered to VWF under both venous (2.5 dynes/cm2) and arterial (30 dynes/cm2) shear stresses. We then demonstrated that ADAMTS-13 beads adhered to immobilized recombinant VWF-A1 and -A3 domains, but soluble metalloprotease bound preferentially to the A3 domain, suggesting that the VWF A3 domain may be the primary docking site for the metalloprotease. We suggest that tensile stresses imposed by fluid shear stretch endothelial bound ULVWF multimers to expose binding sites within the A domains for circulating ADAMTS-13. The bound enzyme then cleaves within the A2 domain that lies in close proximity and releases smaller VWF multimers into the plasma. Once released, these cleaved VWF fragments become inaccessible for the metalloprotease to prevent further cleavage.
Assuntos
Endotélio Vascular/metabolismo , Metaloendopeptidases/metabolismo , Fator de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Sítios de Ligação , Plaquetas/metabolismo , Adesão Celular , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Cinética , Mutação , Complexo Glicoproteico GPIb-IX de Plaquetas/isolamento & purificação , Poliestirenos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Veias Umbilicais/citologia , Fator de von Willebrand/químicaRESUMO
Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibalpha, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm(2)) and arterial (20 and 50 dyne/cm(2)) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.
Assuntos
Endotélio Vascular/metabolismo , Metaloendopeptidases/metabolismo , Púrpura Trombocitopênica Trombótica/enzimologia , Fator de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Adulto , Animais , Plaquetas , Células CHO , Estudos de Casos e Controles , Adesão Celular , Cricetinae , Dimerização , Endotélio Vascular/química , Feminino , Humanos , Masculino , Microscopia de Vídeo , Pessoa de Meia-Idade , Perfusão , Ligação Proteica , Estresse Mecânico , Veias UmbilicaisRESUMO
von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin alpha v beta 3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a alpha v beta 3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF-P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.