RESUMEN
Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Asunto(s)
Vasos Sanguíneos/enzimología , Vasos Sanguíneos/crecimiento & desarrollo , Fosfatidato Fosfatasa/metabolismo , Animales , Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/patología , Humanos , Lisofosfolípidos/metabolismo , Fosfatidato Fosfatasa/química , Receptores de Lisoesfingolípidos/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. OBJECTIVES AND METHODS: We used P2Y1 knockout mice to address the role of P2Y12 in Akt phosphorylation in response to thrombin receptors in platelets. RESULTS: Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr³°8 and Ser47³ in P2Y12-deficient platelets. AYPGKF-induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y12 -independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin ß3 deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y12-deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. CONCLUSIONS: Our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.
Asunto(s)
Plaquetas/citología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Plaquetas/metabolismo , Células CHO , Quelantes/farmacología , Cricetinae , Cricetulus , ADN Complementario/metabolismo , Humanos , Ratones , Ratones Noqueados , Péptidos/química , Fosforilación , Receptores Purinérgicos P2Y12/genética , Proteínas Recombinantes/química , Serina/química , Trombina/químicaRESUMEN
Although their central role is in the prevention of bleeding, platelets probably contribute to diverse processes that extend beyond hemostasis and thrombosis. For example, platelets can recruit leukocytes and progenitor cells to sites of vascular injury and inflammation; they release proinflammatory and anti-inflammatory and angiogenic factors and microparticles into the circulation; and they spur thrombin generation. Data from animal models suggest that these functions may contribute to atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury, and transplant rejection. This article represents an integrated summary of presentations given at the Fourth Annual Platelet Colloquium in January 2009. The process of and factors mediating platelet-platelet and platelet-leukocyte interactions in inflammatory and immune responses are discussed, with the roles of P-selectin, chemokines and Src family kinases being highlighted. Also discussed are specific disorders characterized by local or systemic platelet activation, including coronary artery restenosis after percutaneous intervention, alloantibody-mediated transplant rejection, wound healing, and heparin-induced thrombocytopenia.
Asunto(s)
Plaquetas/fisiología , Plaquetas/química , Plaquetas/patología , Comunicación Celular , Enfermedad/etiología , Humanos , Inmunidad , InflamaciónRESUMEN
Lysophosphatidic acid (LPA), its sphingolipid homolog sphingosine 1-phosphate (S1P) and several other related molecules constitute a family of bioactive lipid phosphoric acids that function as receptor-active mediators with roles in cell growth, differentiation, inflammation, immunomodulation, apoptosis and development. LPA and S1P are present in physiologically relevant concentrations in the circulation. In isolated cell culture systems or animal models, these lipids exert a range of effects that suggest that S1P and LPA could play important roles in maintaining normal vascular homeostasis and in vascular injury responses. LPA and S1P act on a series of G protein-coupled receptors, and LPA may also be an endogenous regulator of PPARgamma activity. In this review, we discuss potential roles for lysolipid signaling in the vasculature and mechanisms by which these bioactive lipids could contribute to cardiovascular disease.
Asunto(s)
Vasos Sanguíneos/fisiología , Lisofosfolípidos/fisiología , Enfermedades Cardiovasculares/etiología , Homeostasis , Humanos , Esfingosina/análogos & derivados , Esfingosina/fisiologíaRESUMEN
Murine blood coagulation factors and function are quite similar to those of humans. Because of this similarity and the adaptability of mice to genetic manipulation, murine coagulation factors and inhibitors have been extensively studied. These studies have provided significant insights into human hemostasis. They have also provided useful experimental models for evaluation of the pathophysiology and treatment of thrombosis. This review contains recommendations for obtaining, processing and assaying mouse blood hemostatic components, and it summarizes the extensive literature on murine coagulation factor structure and function, assays and genetic alteration. It is intended to be a convenient reference source for investigators of hemostasis and thrombosis.
Asunto(s)
Modelos Animales de Enfermedad , Animales , Coagulación Sanguínea , Fibrinógeno/genética , Hemostasis/genética , Humanos , Ratones , Modelos Biológicos , Modelos Genéticos , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Trombosis/genéticaRESUMEN
The components and functions of the murine fibrinolytic system are quite similar to those of humans. Because of these similarities and the adaptability of mice to genetic manipulation, murine fibrinolysis has been studied extensively. These studies have yielded important information regarding the function of the several components of fibrinolysis. This review presents information on the structure, function and assay of mouse fibrinolytic parameters and it discusses the results of the extensive studies of genetically modified mice. It is intended to be a convenient reference resource for investigators of fibrinolysis.
Asunto(s)
Fibrinógeno/metabolismo , Fibrinolíticos/química , Activadores Plasminogénicos/fisiología , Plasminógeno/fisiología , Animales , Modelos Animales de Enfermedad , Fibrinógeno/química , Fibrinólisis , Hemostasis , Humanos , Cinética , Ratones , Modelos Biológicos , Modelos Genéticos , Plasminógeno/genética , Activadores Plasminogénicos/genética , Especificidad de la EspecieAsunto(s)
Angiopatías Diabéticas/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , PPAR gamma/agonistas , Trombosis/tratamiento farmacológico , Angiopatías Diabéticas/etiología , Angiopatías Diabéticas/prevención & control , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Pioglitazona , Activación Plaquetaria , Tiazolidinedionas/uso terapéutico , Trombosis/etiología , Trombosis/prevención & controlRESUMEN
The beta3-integrin family consists of alphaIIbbeta3 (also known as glycoprotein IIb/IIIa) and alpha(v)beta3. alphaIIbbeta3 is found on platelets and megakaryocytes and has an essential role in hemostasis. alpha(v)beta3 has a broader distribution, and it functions in angiogenesis, neointimal formation after vascular injury, and leukocyte trafficking. There are important interactions between thrombin and beta3-integrins relative to both "inside-out" (integrin activation) and "outside-in" (modification of cellular events by ligand binding to integrins) signaling. Thrombin, by binding to G protein-coupled, protease-activated receptors, is a potent activator of alphaIIbbeta3. Conversely, outside-in signaling through alphaIIbbeta3 amplifies events initiated by thrombin and is necessary for full platelet spreading, platelet aggregation, granule secretion, and the formation of a stable platelet thrombus. In smooth muscle cells, alpha(v)beta3-integrins influence various responses to thrombin, including proliferation, c-Jun NH2-terminal kinase-1 activation, and focal adhesion formation. Other interactions between beta3-integrins and thrombin include beta3-integrin promotion of the generation of thrombin by localizing prothrombin to cellular surfaces and/or enhancing the formation of procoagulant microparticles and the requirement of beta3-integrin function for platelet-dependent clot retraction. In summary, there is increasing evidence that interactions between beta3-integrins and thrombin play important roles in the regulation of hemostatic and vascular functions.
Asunto(s)
Plaquetas/metabolismo , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Integrina beta3/metabolismo , Trombina/fisiología , Animales , Coagulación Sanguínea/fisiología , Humanos , Activación Plaquetaria/fisiología , Transducción de Señal/fisiologíaAsunto(s)
Activación Plaquetaria , Adhesividad Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria , Factor de von Willebrand/fisiología , Humanos , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Agregación de Receptores , Transducción de Señal , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P-selectin and its leukocyte ligand PSGL-1; subsequently the interaction is strengthened by activation of alphaMbeta2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated alphaMbeta2 to its platelet counterreceptor(s). OBJECTIVES: Because mouse models are being used to define the role of PMN-platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets. METHODS: Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry. RESULTS: In time course experiments, 90 +/- 1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (+/- SEM) number of platelets per positive PMN was 8.4 +/- 1.5. A monoclonal antibody to P-selectin reduced the percentage of PMNs with attached platelets to 16 +/- 2.4% (P = 0.001), and only 8 +/- 5% of PMNs interacted with platelets from P-selectin-/- mice. In contrast, monoclonal antibodies to PSGL-1, beta2-integrin, and alphaIIbbeta3 had much less or no effect on the production of mixed cell aggregates. To better identify a secondary contribution of beta2-integrins, P-selectin interactions were disrupted by briefly adding 5 mm EGTA to already-formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70 +/- 3.5% (P = 0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5 +/- 1.7). The combination of brief EGTA treatment and a monoclonal antibody to beta2-integrin lowered the percentage of PMN with attached platelets to 50 +/- 7% and reduced the number of platelets attached per positive PMN to 3.6 +/- 0.7 (P = 0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN-platelet adhesion, while the inactive analog PP3 was ineffective. CONCLUSIONS: These results confirm that P-selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from beta2-integrins and Src family kinases.
Asunto(s)
Antígenos CD18/fisiología , Selectina-P/fisiología , Adhesividad Plaquetaria , Familia-src Quinasas/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Plaquetas/citología , Ácido Edético/farmacología , Citometría de Flujo , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/fisiología , Selectina-P/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Trombosis/etiología , Familia-src Quinasas/metabolismoRESUMEN
Alphavbeta3-integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because alpha-thrombin contributes to neointimal formation, we examined the hypothesis that alphavbeta3-integrins influence alpha-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed alphavbeta3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to alpha-thrombin were partially inhibited by anti-beta3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that alpha-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by alphavbeta3 antagonists. Beta3-integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. Alpha-thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). Alphavbeta3-integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from beta3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, alphavbeta3-integrins play an important role in alpha-thrombin-induced proliferation and focal adhesion formation in RASMC.
Asunto(s)
Adhesiones Focales/efectos de los fármacos , Integrina alfaVbeta3/antagonistas & inhibidores , Integrina alfaVbeta3/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Trombina/farmacología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Adhesiones Focales/fisiología , Integrina alfaVbeta3/metabolismo , Miocitos del Músculo Liso/fisiología , Ratas , Ratas Sprague-Dawley , Trombina/antagonistas & inhibidoresRESUMEN
Murine models employing genetically altered mice have the potential to provide important new information about the hemostatic system, but before such data can be extrapolated to humans it is necessary to define the similarities and differences between murine and human hemostasis. After establishing the similarities of murine fibrinogen to human fibrinogen in its pattern of proteolysis in response to plasmin and its cross-linking by factor XIIIa, we studied a new hamster monoclonal antibody (mAb) 7E9 that reacts with the gamma chain of mouse fibrinogen. This antibody inhibits platelet adhesion to fibrinogen, platelet-mediated clot retraction, platelet aggregation, and FXIIIa-mediated cross-linking of fibrin; it also facilitates tissue plasminogen activator (tPA)-mediated lysis of fibrin formed either in the absence or presence of platelets. These data provide evidence that the C-terminus of mouse fibrinogen gamma chain, like that of human fibrinogen, is involved in fibrinogen binding to platelets and FXIIIa-mediated cross-linking of fibrin. Our data raise the possibility that a therapeutic agent that targets the C-terminus of the gamma chain in human fibrinogen might have broad antithrombotic and profibrinolytic effects.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Plaquetas/fisiología , Factor XIIIa/fisiología , Fibrina/metabolismo , Fibrinógeno/fisiología , Fibrinólisis/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Retracción del Coagulo , Cricetinae , Fibrinógeno/antagonistas & inhibidores , Fibrinógeno/inmunología , Fibrinolisina/metabolismo , Humanos , Ratones , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Unión Proteica , Activador de Tejido Plasminógeno/fisiologíaRESUMEN
Information is lacking regarding dynamic platelet accumulation at the site of the occluded middle cerebral artery (MCA) and the relationship between platelet aggregation in downstream cerebral microvessels and loss of perfusion and vascular integrity of these microvessels. In the present study, we employed a model of embolic MCA occlusion in the rat to simultaneously measure temporal and spatial profiles of platelet accumulation at the site of the embolus occluding the MCA and within downstream cerebral microvessels. We also measured the integrity of microvessels and matrix metalloproteinase (MMP) activity in ischemic brain. Rats (n=36) were subjected to embolic MCA occlusion. Immunohistochemistry was used to detect microvascular integrity, plasminogen activator inhibitor 1 (PAI-1) and the deposition of fibrin. SDS-PAGE zymography was used to measure MMP2 and MMP9 activities. Accumulation of platelets and increases in PAI-1 immunoreactivity at the site of the embolus occluding the MCA were detected 1 h (n=7) and 4 h (n=7) after ischemia, respectively, and numbers of GPIIb/IIIa immunoreactive downstream cerebral microvessels increased significantly (209+/-59; n=7; P<0.05) 4 h after ischemia, suggesting dynamic platelet aggregation. A significant (n=7; P<0.01) diffuse loss of type IV collagen immunoreactivity in microvessels was temporally associated with platelet GPIIb/IIIa immunoreactivity within the vessels. Triple immunostaining revealed that microvessels containing platelet aggregates exhibited loss of type IV collagen immunoreactivity and both intra- and extra-vascular fibrin deposition, suggesting that intravascular platelet aggregation is associated with decreases in the integrity of the microvascular basal lamina and blood-brain barrier leakage. A significant increase (P<0.05) in MMP9 was detected at 4 h (n=3) and 24 h (n=3) after ischemia but levels of MMP2 were not significantly changed in ischemic brain. Our data suggest that dynamic platelet aggregation in ischemic brain may contribute to time-dependent resistance to fibrinolysis. In addition, platelet deposition and increased MMP9 coincided with degradation of type IV collagen and loss of vascular integrity. These data suggest an important role for post-occlusive distal platelet deposition in the pathophysiology of stroke.
Asunto(s)
Coagulación Sanguínea/fisiología , Plaquetas/fisiología , Isquemia Encefálica/enzimología , Encéfalo/enzimología , Infarto de la Arteria Cerebral Media/fisiopatología , Metaloproteinasas de la Matriz/metabolismo , Microcirculación/fisiopatología , Animales , Plaquetas/citología , Plaquetas/ultraestructura , Barrera Hematoencefálica/fisiología , Encéfalo/patología , Encéfalo/ultraestructura , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Circulación Cerebrovascular/fisiología , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Técnicas In Vitro , Infarto de la Arteria Cerebral Media/enzimología , Infarto de la Arteria Cerebral Media/patología , Laminina/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Microcirculación/patología , Microcirculación/ultraestructura , Microscopía Electrónica , Dinámicas no Lineales , Inhibidor 1 de Activador Plasminogénico/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Platelet integrin alpha IIb beta 3 (GPIIb/IIIa) plays a central role in the initiation of arterial thrombosis, but its contribution to disseminated microvascular thrombosis is less well defined. Therefore, wild-type mice (beta 3(+/+)), beta 3-integrin-deficient mice (beta 3(-/-)), and wild-type mice treated with a hamster monoclonal antibody (1B5) that blocks murine alpha IIb beta 3 function were tested in models of large-vessel and microvascular thrombosis. In the large-vessel model, ferric chloride was used to injure the carotid artery, and the time to thrombosis was measured. In beta 3(+/+) mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the beta 3(-/-) mice tested (P <.001). Fab and F(ab')(2) fragments of 1B5 increased the median time to occlusion. To initiate systemic intravascular thrombosis, prothrombotic agents were administered intravenously, and platelet thrombus formation was monitored by the decrease in circulating platelet count. Three minutes after the injection of adenosine diphosphate (ADP), collagen + epinephrine, or tissue factor, the platelet counts in beta 3(+/+) mice decreased by 289, 424, and 429 x 10(3)/microL, respectively. beta 3(-/-) mice and wild-type mice pretreated with 1B5 Fab (1 mg/kg, IP) were nearly completely protected from the effects of ADP. In contrast, beta 3(-/-) mice were only partially protected from the effects of collagen + epinephrine and minimally protected from the effects of tissue factor. In all cases, less fibrin became deposited in the lungs of beta 3(-/-) mice than in wild-type mice. These results suggest that though alpha IIb beta 3 plays a dominant role in large-vessel thrombosis, it plays a variable role in systemic intravascular thrombosis. (Blood. 2001;98:1055-1062)
Asunto(s)
Antígenos CD/farmacología , Glicoproteínas de Membrana Plaquetaria/deficiencia , Glicoproteínas de Membrana Plaquetaria/farmacología , Trombosis/etiología , Animales , Anticuerpos Monoclonales/farmacología , Plaquetas/química , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/patología , Cloruros , Modelos Animales de Enfermedad , Femenino , Compuestos Férricos , Fibrina/metabolismo , Inmunohistoquímica , Integrina beta3 , Masculino , Ratones , Ratones Noqueados , Microcirculación , Microscopía Electrónica , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Receptores de Vitronectina , Trombosis/patologíaRESUMEN
BACKGROUND: Intimal hyperplasia contributes to restenosis after percutaneous vascular interventions. Both beta(3)-integrins, alpha(V)beta(3) and alpha(IIb)beta(3) (glycoprotein IIb/IIIa), and leukocytes have been implicated in neointimal formation, based in part on the results obtained using antagonists to 1 or both receptors in animal models. METHODS AND RESULTS: The responses in wild-type mice, beta(3)-integrin-deficient mice, and P-selectin-deficient mice were studied in a model of transluminal endothelial injury of the femoral artery. At 4 weeks, beta(3)-integrin-deficient mice were not protected from developing intimal hyperplasia, whereas P-selectin-deficient mice were protected. Within 1 hour of injury, several layers of platelets deposited on the arteries of wild-type mice and a single layer of platelets deposited on the vessels of beta(3)-integrin-deficient mice; in both cases, leukocytes were recruited to the platelet layer. In P-selectin-deficient mice, the platelet layer was less compact and extended further into the lumen but did not recruit leukocytes. CONCLUSIONS: In a model of transluminal arterial injury, absence of early leukocyte recruitment and not deficiency of beta(3)-integrins correlated with a reduction in neointimal formation. Blockade of P-selectins may be an effective therapeutic strategy to decrease restenosis after percutaneous vascular interventions.
Asunto(s)
Antígenos CD/fisiología , Arteria Femoral/patología , Selectina-P/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Túnica Íntima/patología , Animales , Antígenos CD/genética , Plaquetas/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Arteria Femoral/lesiones , Arteria Femoral/ultraestructura , Hiperplasia , Integrina beta3 , Leucocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Microscopía Electrónica , Selectina-P/genética , Selectina-P/metabolismo , Glicoproteínas de Membrana Plaquetaria/deficiencia , Glicoproteínas de Membrana Plaquetaria/genética , Factores de Tiempo , Túnica Íntima/metabolismoRESUMEN
The alphaIIbeta3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet alphaIIbeta3, utilizing both beta3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine alphaIIbbeta3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of alphaIIbbeta3 (60-80,000 copies/platelet). Like their human counterparts, murine alphaIIb and beta3 exhibit different electrophoretic motilities under nonreducing (aIIb 135k Da; beta3 92k Da) and reducing (aIIb 120k Da; beta3 108k Da) conditions, and the alphaIIbbeta3 complex is dissociated by EDTA at pH 8 and 37 degrees C. Murine beta3 is less susceptible to proteolysis by plasmin than is human beta3. In addition to defective platelet aggregation, mouse platelets lacking alphaIIbbeta3 and alphaVbeta3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, beta3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, beta3-null platelets have altered tyrosine phosphorylation patterns following thrombin- and collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.
Asunto(s)
Ratones/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Animales , Anticuerpos Monoclonales , Antígenos CD/genética , Fibrinolisina/farmacología , Humanos , Integrina beta3 , Ratones Endogámicos C57BL , Ratones Noqueados , Selectina-P/efectos de los fármacos , Selectina-P/metabolismo , Fosforilación/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/genética , Proteínas/metabolismo , Relación Estructura-Actividad , Trombina/farmacologíaRESUMEN
To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Antígenos/inmunología , Plaquetas/inmunología , Hibridomas/inmunología , Glicoproteínas de Membrana , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos CD/genética , Antígenos CD/inmunología , Plaquetas/citología , Plaquetas/metabolismo , Fusión Celular , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Fibrinógeno/inmunología , Hibridomas/citología , Integrina beta1/inmunología , Integrina beta3 , Ratones , Ratones Endogámicos , Ratones Noqueados , Peso Molecular , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/genética , Tetraspanina 29 , TrombinaRESUMEN
Binding sites for both the collagen-like and globular domains of C1q have been described on a variety of cell types. HUVEC were previously shown to express the 60- to 67-kDa receptor recognizing the collagen-like domain of C1q. This study demonstrates the presence of a distinct 28- to 33-kDa HUVEC protein (gC1qR) that interacts with the globular head domain of C1q. Polyclonal Abs raised against the Raji cell gC1qR partially inhibited HUVEC interaction with immobilized C1q and recognized a 28- to 33-kDa protein on Western blots. The Ab also reacted strongly with poly-L-lysine-immobilized, glutaraldehyde-fixed, intact HUVEC in ELISA assays. No significant difference in reactivity was noted if HUVEC were permeabilized with 0.2% Triton X-100. However, unfixed HUVEC grown on gelatin-coated microtiter wells to 80% confluence failed to express significant amounts of gC1qR Ag. Quantitation of HUVEC gC1qR by gel scanning suggested the presence of 5.7 +/- 3.8 x 10(6) molecules/cell (mean +/- SD; n = 4). A quantitative sandwich ELISA procedure, however, detected only 3.7 +/- 0.6 x 10(5) gC1qR molecules/cell (mean +/- SD; n = 4), consistent with previously described gC1qR multimerization. The capacity of endothelial cells to recognize both the collagen-like and globular domains of C1q via distinct binding sites may have implications for the role of C1q in vascular inflammatory and thrombotic lesions.
Asunto(s)
Complemento C1q/metabolismo , Endotelio Vascular/metabolismo , Receptores de Hialuranos , Glicoproteínas de Membrana , Receptores de Complemento/metabolismo , Sitios de Unión , Proteínas Portadoras , Células Cultivadas , Complemento C1q/química , Endotelio Vascular/química , Humanos , Proteínas Mitocondriales , Estructura Terciaria de Proteína , Receptores de Complemento/química , Receptores de Complemento/genética , Proteínas Recombinantes de Fusión/metabolismo , Venas UmbilicalesRESUMEN
The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.