RESUMO
The platelet glycoprotein (GP) Ib-IX-V complex mediates the attachment of platelets to the blood vessel wall by binding von Willebrand factor (VWF), an interaction that also transmits signals for platelet activation and aggregation. Because the complex is extensively palmitoylated, a modification known to target proteins to lipid rafts, we investigated the role of raft localization in GP Ib-IX-V functions. In unstimulated platelets, a minor portion of the complex localized to Triton-insoluble raft fractions; this portion increased three to sixfold with platelet activation by VWF. Raft-associated GP Ib-IX-V was selectively palmitoylated, with GP Ib-IX-V-associated palmitate increasing in the raft fraction on VWF-mediated activation. The raft fraction was also the site of association between GP Ib-IX-V and the Fc receptor FcgammaRIIA. The importance of this association was demonstrated by the ability of the FcgammaRIIA antibody IV.3 to inhibit shear-induced platelet aggregation. Disruption of rafts by depleting membrane cholesterol impaired several GP Ib-IX-V-dependent platelet fractions: aggregation to VWF under static conditions and under shear stress, tyrosine phosphorylation, and adhesion to a VWF surface. Partial restoration of membrane cholesterol content partially restored shear-induced platelet aggregation and tyrosine phosphorylation. Thus, localization of the GP Ib-IX-V complex within rafts is crucial for both platelet adhesion and postadhesion signaling.
Assuntos
Metabolismo dos Lipídeos , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Colesterol/metabolismo , Humanos , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores de IgG/metabolismoRESUMO
Peptidases play a vital and often highly specific role in the physiological and pathological generation and termination of peptide hormone signals. The thermolysin-like family of metalloendopeptidases involved in the extracellular processing of neuroendocrine and cardiovascular peptides are of particular significance, reflecting both their specificity for particular peptide substrates and their utility as therapeutic targets. Although the functions of the membrane-bound members of this family, such as angiotensin-converting enzyme and neutral endopeptidase, are well established, a role for the predominantly soluble family members in peptide metabolism is only just emerging. This review will focus on the biochemistry, cell biology, and physiology of the soluble metalloendopeptidases EC 3.4.24.15 (thimet oligopeptidase) and EC 3.4.24.16 (neurolysin), as well as presenting evidence that both peptidases play an important role in such diverse functions as reproduction, nociception, and cardiovascular homeostasis.
Assuntos
Metaloendopeptidases/fisiologia , Sistemas Neurossecretores , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sistema Cardiovascular/enzimologia , Imuno-Histoquímica , Metaloendopeptidases/análise , Metaloendopeptidases/química , Modelos Moleculares , Dados de Sequência Molecular , Sistemas Neurossecretores/enzimologia , Alinhamento de Sequência , Solubilidade , Especificidade por Substrato , Distribuição TecidualRESUMO
Sulfatide (galactocylceramide-3'-sulfate), a cell surface glycosphingolipid interacts with several cell adhesion molecules including fibrinogen, von Willebrand factor (VWF), P-selectin, thrombospondin (TSP) and laminin, which are involved in haemostasis. We have used a sulfatide-specific single-chain fragment variable (scFv) antibody probe PA38 and sulfatide-deficient mice to investigate the role of membrane sulfatide in platelet function. PA38 bound to platelets and binding increased following platelet activation. Sulfatide was localized as a large cluster towards the center of the platelet surface when examined in a confocal microscope. PA38 (20 microg/ml) inhibited the adhesion of activated platelets to fibrinogen, VWF, P-selectin, TSP1 and laminin by 30%, 30%, 75%, 20% and 35%, respectively, compared to a control scFv (p < 0.05). Furthermore, PA38 inhibited collagen, ADP, thrombin and ristocetin-induced platelet aggregation in PRP by 25%, 30%, 18% and 20%, respectively, compared to the control scFv (p < 0.05). In a PFA-100 platelet function assay, PA38 prolonged the occlusion time by 25% (p < 0.05). Under flow PA38 decreased the thrombus formation on collagen by 31%, (p < 0.01). Sulfatide-deficient mice displayed an extended lag-phase in collagen-induced platelet aggregation compared to wild type (p < 0.05), though in-vivo haemostasis did not differ significantly. Thus, this study provides new evidence for a role for membrane sulfatide in platelet function.
Assuntos
Plaquetas/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Região Variável de Imunoglobulina/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Sulfoglicoesfingolipídeos/metabolismo , Animais , Plaquetas/metabolismo , Membrana Celular/metabolismo , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Modelos Animais de Doenças , Hemorreologia , Hemostasia/efeitos dos fármacos , Humanos , Região Variável de Imunoglobulina/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Função Plaquetária , Sulfoglicoesfingolipídeos/imunologia , Trombose/sangue , Trombose/prevenção & controleRESUMO
Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by approximately 50% (P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.
Assuntos
Endotoxinas/intoxicação , Músculo Esquelético/irrigação sanguínea , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Receptor 4 Toll-Like/metabolismo , Trombose Venosa/imunologia , Vênulas/imunologia , Animais , Lipopolissacarídeos/intoxicação , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , Microcirculação/imunologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Trombose Venosa/induzido quimicamente , Vênulas/efeitos dos fármacosRESUMO
Tissue factor (TF) circulates in plasma, largely on monocyte/macrophage-derived microvesicles that can bind activated platelets through a mechanism involving P-selectin glycoprotein ligand-1 (PSGL-1) on the microvesicles and P-selectin on the platelets. We found these microvesicles to be selectively enriched in both TF and PSGL-1, and deficient in CD45, suggesting that they arise from distinct membrane microdomains. We investigated the possibility that microvesicles arise from cholesterol-rich lipid rafts and found that both TF and PSGL-1, but not CD45, localize to lipid rafts in blood monocytes and in the monocytic cell line THP-1. Consistent with a raft origin of TF-bearing microvesicles, their shedding was significantly reduced with depletion of membrane cholesterol. We also evaluated the interaction between TF-bearing microvesicles and platelets. Microvesicles bound only activated platelets, and required PSGL-1 to do so. The microvesicles not only bound the activated platelets, they fused with them, transferring both proteins and lipid to the platelet membrane. Fusion was blocked by either annexin V or an antibody to PSGL-1 and had an important functional consequence: increasing the proteolytic activity of the TF-VIIa complex. These findings suggest a mechanism by which all of the membrane-bound reactions of the coagulation system can be localized to the surface of activated platelets.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Vesículas Citoplasmáticas/metabolismo , Fusão de Membrana/fisiologia , Microdomínios da Membrana/metabolismo , Tromboplastina/fisiologia , Colesterol/sangue , Colesterol/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Fator VIIa/metabolismo , Humanos , Antígenos Comuns de Leucócito/metabolismo , Glicoproteínas de Membrana/metabolismo , Ativação Plaquetária/fisiologia , Tromboplastina/metabolismo , Fatores de TempoRESUMO
Glycoprotein (GP) Ibalpha, a member of the leucine-rich repeat (LRR) protein family, mediates platelet adhesion to immobilized von Willebrand factor (VWF). We investigated the role in VWF binding of charged residues in the LRR region of GP Ibalpha that are conserved in human, canine, and murine proteins. Substitution of His86 with either Ala or Glu resulted in a gain of VWF-binding function as judged by increased VWF binding in the presence of the modulators ristocetin and botrocetin and by enhanced adhesion of Chinese hamster ovary (CHO) cells expressing the mutant GP Ibalpha to immobilized VWF under conditions of flow. This is the first report of a gain-of-function phenotype resulting from mutations in the LRR region of GP Ibalpha. Because His86 is 2 nm away from the region of GP Ibalpha with the largest surface of contact with VWF, the data suggest that the LRRs regulate GP Ibalpha affinity for VWF allosterically.
Assuntos
Regulação Alostérica , Mutagênese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Fator de von Willebrand/metabolismo , Substituição de Aminoácidos , Adesão Celular , Sequência Conservada , Humanos , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica/genética , Alinhamento de SequênciaRESUMO
Factor XI binds to activated platelets where it is efficiently activated by thrombin. The factor XI receptor is the platelet membrane glycoprotein (GP) Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), a significant fraction of which exists within lipid rafts on stimulated platelets (Shrimpton, C. N., Borthakur, G., Larrucea, S., Cruz, M. A., Dong, J. F., and Lopez, J. A. (2002) J. Exp. Med. 196, 1057-1066). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids implicated in localizing membrane ligands and in cellular signaling. We now show that factor XI was localized to lipid rafts in activated platelets ( approximately 8% of total bound) but not in resting platelets. Optimal binding of factor XI to membrane rafts required prothrombin (and Ca2+) or high molecular weight kininogen (and Zn2+), which are required for factor XI binding to platelets. An antibody to GPIb (SZ-2) that disrupts factor XI binding to the GPIb-IX-V complex also disrupted factor XI-raft association. The isolated recombinant Apple 3 domain of factor XI, which mediates factor XI binding to platelets, also completely displaces factor XI from membrane rafts. To investigate the physiological relevance of the factor XI-raft association, the structural integrity of lipid rafts was disrupted by cholesterol depletion utilizing methyl-beta-cyclodextrin. Cholesterol depletion completely prevented FXI binding to lipid rafts, and initial rates of factor XI activation by thrombin on activated platelets were inhibited >85%. We conclude that factor XI is localized to GPIb in membrane rafts and that this association is important for promoting the activation of factor XI by thrombin on the platelet surface.
Assuntos
Plaquetas/metabolismo , Fator XI/metabolismo , Microdomínios da Membrana/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , beta-Ciclodextrinas , Plaquetas/citologia , Centrifugação com Gradiente de Concentração , Colesterol/metabolismo , Ciclodextrinas/metabolismo , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Humanos , Cinética , Octoxinol/farmacologia , Ativação Plaquetária , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/metabolismo , Sacarose/farmacologia , Temperatura , Trombina/metabolismo , Fatores de TempoRESUMO
Endopeptidase EC 3.4.24.15 (EP 24.15; thimet oligopeptidase) is a soluble metalloendopeptidase implicated in the metabolism of a number of neuropeptides, including neurotensin, gonadotropin-releasing hormone, and opioid peptides. We have shown previously that thiol reducing agents, such as dithiothreitol, activate EP 24.15 by mediating the conversion of inactive multimeric forms to active monomers and that this conversion involves the disruption of intermolecular disulfide bonds involving cysteine residues 246, 248, and 253. We have identified two components of cerebrospinal fluid that activate recombinant EP 24.15, but have no effect on a thiol-independent cysteine mutant form of the enzyme. The low molecular weight (<10 kDa) component co-elutes with glutathione by reversed-phase HPLC, whereas the high molecular weight component (>50 kDa) is sensitive to digestion with trypsin, suggesting it is proteinaceous in nature. These results suggest that EP 24.15 activity in the brain may be modulated by factors released into cerebrospinal fluid.
Assuntos
Líquido Cefalorraquidiano/química , Dissulfetos/metabolismo , Metaloendopeptidases/líquido cefalorraquidiano , Neuropeptídeos/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Precipitação Fracionada , Glutationa/metabolismo , Metaloendopeptidases/metabolismo , Peso Molecular , Ovinos/líquido cefalorraquidiano , Fatores de Tempo , Tripsina/farmacologiaRESUMO
Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (Kd app of approximately 10 nm; Bmax of approximately 1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn2+ or prothrombin and Ca2+. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibalpha subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His1-Glu282) of glycoprotein Ibalpha that contains the leucine-rich repeats of the NH2-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibalpha. This fragment was able to compete with FXI for binding to activated platelets (Ki of 3.125 +/- 0.25 nm) with a potency similar to that of intact glycocalicin (Ki of 3.72 +/- 0.30 nm). However, a synthetic glycoprotein Ibalpha peptide, Asp269-Asp287, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of 125I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10(-5) m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of 125I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibalpha were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibbeta and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibalpha at sites comprising the leucine-rich repeat sequences within the NH2-terminal globular domain that are separate and distinct from the thrombin-binding site.
Assuntos
Fator XI/metabolismo , Leucina/química , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Sequência de Aminoácidos , Sítios de Ligação , Plaquetas/química , Plaquetas/metabolismo , Cálcio/química , Membrana Celular/metabolismo , Cloretos/química , Venenos de Crotalídeos/química , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Metaloendopeptidases/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ativação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Trombina/química , Tirosina/química , Zinco/química , Compostos de Zinco/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.