RESUMEN
Although the survival rate for early detected cancers is high, once a cancer metastasizes to bone, it is incurable. Interestingly, patients without visible metastases display abnormal bone formation and resorption, suggesting a link between primary cancers and the bone microenvironment prior to metastasis, and this link likely facilitates preparation of the pre-metastatic niche. We hypothesized that communication with the primary tumor would result in bone remodeling alterations, and that platelets could facilitate this communication. By using three tumor models, we demonstrate that primary tumor growth stimulates bone formation measured by microcomputed tomography. Further, platelet depletion prevented tumor-induced bone formation, highlighting the importance of platelets in the communication between tumors and the bone microenvironment. Finally, we determine that platelets sequester a variety of tumor-derived proteins, TGF-ß1 and MMP-1 in particular, which regulate bone formation. Thus, our data reveal that platelets function as mediators of tumor-bone communication prior to metastasis.
Asunto(s)
Plaquetas/metabolismo , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias/patología , Fosfatasa Alcalina/sangre , Fosfatasa Alcalina/metabolismo , Animales , Neoplasias Óseas/diagnóstico por imagen , Huesos/diagnóstico por imagen , Huesos/metabolismo , Huesos/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Osteocalcina/sangre , Osteocalcina/metabolismo , Osteogénesis , Radiografía , Factor de Crecimiento Transformador beta1/metabolismo , Trasplante HeterólogoRESUMEN
BACKGROUND: Activation of Akt and increased expression of integrin ß(3) are the two most important changes that have been linked to the attainment of metastatic potential by prostate cancer cells. However, a direct link between Akt activity and inside-out activation of integrin ß(3) in mediating prostate cancer cell metastatic properties is not established. METHODS: Using functional and biochemical approaches, we examined the role of Akt1 in the affinity modulation of integrin ß(3) in prostate cancer cells. RESULTS: Although expression of murine TRAMP and human PC3 cells with constitutively active Akt1 (CA-Akt1) enhanced their affinity for integrin α(v)ß(3) specific ligands and motility on various extracellular matrix proteins, the reverse was observed with the expression of dominant-negative Akt1 (DN-Akt1). Although enhanced motility and transendothelial migration of CA-Akt1-expressing cells were blunted by co-expression with DN-integrin ß(3) or upon pre-treatment with integrin ß(3)-blocking antibodies (LM 609), impaired motility and transendothelial migration of DN-Akt1-expressing cells were rescued by pre-treatment of prostate cancer cells with integrin ß(3)-activating antibodies, AP7.4. CONCLUSION: Our data is the first to demonstrate a link between Akt1 activity and affinity modulation of integrin ß(3) in the regulation of prostate cancer cell motility, transendothelial migration and chemotaxis to metastatic stimuli.
Asunto(s)
Movimiento Celular , Quimiotaxis , Integrina beta3/metabolismo , Micrometástasis de Neoplasia , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Línea Celular Tumoral , Endotelio/metabolismo , Humanos , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/patología , Transducción de SeñalRESUMEN
BACKGROUND: Kindlin-3 is a novel integrin activator in hematopoietic cells, and its deficiency leads to immune problems and severe bleeding, known as leukocyte adhesion deficiency III (LAD-III). Our current understanding of Kindlin-3 function primarily relies on analysis of animal models or cell lines. OBJECTIVES: To understand the functions of Kindlin-3 in human primary blood cells. PATIENTS/METHODS: We analyzed primary and immortalized hematopoietic cells obtained from a new LAD-III patient with immune problems, bleeding, a history of anemia, and abnormally shaped red blood cells. RESULTS: The patient's white blood cells (WBCs) and platelets showed defects in agonist-induced integrin activation and botrocetin-induced platelet agglutination. Primary leukocytes from this patient exhibited abnormal activation of ß(1) integrin. Integrin activation defects were responsible for the observed deficiency in the botrocetin-induced platelet response. Analysis of patient genomic DNA revealed a novel mutation in the Kindlin3 gene. The mutation abolished Kindlin-3 expression in primary WBCs and platelets, owing to abnormal splicing. Kindlin-3 is expressed in red blood cells (RBCs), and its deficiency is proposed to lead to abnormally shaped RBCs. Immortalized patient WBCs expressed a truncated form of Kindlin-3 that was not sufficient to support integrin activation. Expression of Kindlin-3 cDNA in immortalized patient WBCs rescued integrin activation defects, whereas overexpression of the truncated form did not. CONCLUSIONS: Kindlin-3 deficiency impairs integrin function, including activation of ß(1) integrin. Abnormalities in glycoprotein Ib-IX function in Kindlin-3-deficient platelets are secondary to integrin defects. The region of Kindlin-3 encoded by exon 11 is crucial for its ability to activate integrins in humans.
Asunto(s)
Síndrome de Deficiencia de Adhesión del Leucocito/fisiopatología , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/inmunología , Western Blotting , Línea Celular , Niño , Femenino , Citometría de Flujo , Humanos , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , ARN Mensajero/genéticaRESUMEN
The management of pain and morbidity due to the spreading and growth of cancer within bone remains to be a paramount problem in clinical care. Cancer cells actively transform bone, however, the molecular requirements and mechanisms of this process remain unclear. This study shows that functional modulation of the alphavbeta3 integrin receptor in prostate cancer cells is required for progression within bone and determines tumor-induced bone tissue transformation. Using histology and quantitative microCT analysis, we show that alphavbeta3 integrin is required not only for tumor growth within the bone but for tumor-induced bone gain, a response resembling bone lesions in prostate cancer patients. Expression of normal, fully functional alphavbeta3 enabled tumor growth in bone (incidence: 4/4), whereas alphavbeta3 (-), inactive or constitutively active mutants of alphavbeta3 did not (incidence: 0/4, 0/6 and 1/7, respectively) within a 35-day-period. This response appeared to be bone-specific in comparison to the subcutis where tumor incidence was greater than 60% for all groups. Interestingly, bone residing prostate cancer cells expressing normal or dis-regulated alphavbeta3 (either inactive of constitutively active), but not those lacking beta3 promoted bone gain or afforded protection from bone loss in the presence or absence of histologically detectable tumor 35 days following implantation. As bone is replete with ligands for beta3 integrin, we next demonstrated that alphavbeta3 integrin activation on tumor cells is essential for the recognition of key bone-specific matrix proteins. As a result, prostate cancer cells expressing fully functional but not dis-regulated alphavbeta3 integrin are able to control their own adherence and migration to bone matrix, functions that facilitate tumor growth and control bone lesion development.
Asunto(s)
Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Remodelación Ósea/fisiología , Huesos/patología , Integrina alfaVbeta3/fisiología , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Integrina alfaVbeta3/biosíntesis , Integrina alfaVbeta3/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de la Próstata/patología , Vitronectina/metabolismoRESUMEN
The purpose of the study was to evaluate safety, effects on platelet aggregation and pharmacokinetics of F(ab')(2) fragments of anti-glycoprotein (GP) IIb-IIIa murine monoclonal antibody FRaMon (F(ab')(2) FRaMon) upon its intravenous administration in patients undergoing high-risk coronary angioplasty. Patients were treated before angioplasty with F(ab')(2) FRaMon at 0.2 mg/kg (n = 17) and 0.25 mg/kg (n = 12) bolus or with abciximab at 0.25 mg/kg bolus + 12 h infusion at 0.125 microg/kg per min (n = 29). F(ab')(2) FRaMon at both doses decreased platelet aggregation induced by 20 microM ADP to <10, <20, <40 and <70% of the predrug level at 1, 12, 24 and 72 h after injection, respectively. No significant differences were observed between F(ab')(2) FRaMon and abciximab antiaggregatory effects. In none of the patients did F(ab')(2) FRaMon cause allergic reactions, major bleedings or deep thrombocytopenia. Antibodies against F(ab')(2) FRaMon were detected in one patient. Free F(ab')(2) FRaMon was cleared from plasma within 12 h, while platelet-bound preparation occupied >95, 70-80 and 40-50% of GP IIb-IIIa at 1 and 12-24 h and 3 days after injection, respectively. Thrombotic complications within the first month after angioplasty in groups treated with F(ab')(2) FRaMon and abciximab were observed in one and two patients, respectively. The data obtained have shown that F(ab')(2) FRaMon at bolus administration to patients undergoing coronary angioplasty caused no serious side effects and at comparative dosage inhibited platelet aggregation with the same efficacy as abciximab at bolus + infusion administration.
Asunto(s)
Angioplastia Coronaria con Balón/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Abciximab , Animales , Anticuerpos/sangre , Anticuerpos Monoclonales/efectos adversos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/efectos adversos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacocinética , Recuento de Plaquetas , Reoperación , Medición de Riesgo , SeguridadRESUMEN
AIM: To study the effects of F(ab')2 fragments of the monoclonal antibody (monAB) FRaMon against glycoproteins (GP) IIb-IIIa on platelet aggregating activity in vitro and after injection to healthy volunteers. MATERIAL AND METHODS: In vitro experiments were performed to study effects of F(ab')2 and Fab fragments of FRaMon on platelet aggregation (PA) and 14C-serotonin secretion and characteristics of 125I-labelled F(ab')2 and Fab FRaMon binding to platelets. In vivo effects of F(ab')2 FRaMon (preparation FRAMON) were studied upon its bolus i.v. injection to 10 healthy volunteers at the doses of 0.025-0.2 mg/kg. PA was registered before and 1, 6, 12, 24 hours and 3 and 12-15 days after FRAMON injection. FRAMON binding to platelets in the vascular bed was evaluated by inhibition of 125I-FRAMON in vitro binding to platelets obtained from volunteers. Development of antibodies against FRAMON was evaluated by ELISA two weeks after FRAMON injection. RESULTS: In vitro F(ab')2 FRaMon completely blocked PA induced by ADP and thrombin at the concentrations < 4 and < 7.5 mcg/ml, respectively, and revealed higher inhibitory capacity than Fab FRaMon. F(ab')2 FRamon also inhibited 14C-serotonin secretion from ADP-activated platelets. F(ab')2 FRamon interacted with two GP IIb-IIIa molecules on one platelet and bound to platelets more tightly than Fab FRaMon. F(ab')2 FRaMon (preparation FRAMON) bolus i.v. injection to healthy volunteers at the doses of 0.025-0.2 mg/kg did not induce any signs of individual intolerance, including allergic reactions, bleeding and thrombocytopenia. FRAMON at 0.2 mg/kg almost completely inhibited ADP-induced PA of volunteer's platelets 1 h after injection and by more than 70% at 12 h and by more than 50% at 24 h after injection. PA ability recovered to normal 3 days after injection. Antibodies against FRAMON were detected in 1 out of 10 volunteers. CONCLUSION: F(ab')2 fragments of monAB FRaMon effectively inhibited aggregating ability both in vitro and after injection to healthy volunteers and could be suggested as a basis for development of a new GP IIb-IIIa antagonist.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Animales , Humanos , Técnicas In Vitro , RatonesRESUMEN
Because of its major role in regulating platelet functions and its prominence on the cell surface, integrin alphaIIbbeta3 has been the subject of intensive investigations. Such studies have provided substantial insights into its structure-function relationships and have led to the development of anti-thrombotic drugs that target the receptor. Nevertheless, recent findings have indicated that our understanding of the structure and function of alphaIIbbeta3 remains inadequate. This article addresses two aspects of still evolving alphaIIbbeta3 function: 1) the interface between alphaIIbbeta3 and the blood coagulation system, resulting from interaction of prothrombin with the receptor; and 2) the molecular basis for recognition of the RGD and the fibrinogen gamma-chain peptide ligands by alphaIIbbeta3. As illustrated by these two examples, there is still much to be learned about alphaIIbbeta3 if we are to fully appreciate its functions and its potential as a therapeutic target.
Asunto(s)
Inhibidores de Agregación Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Animales , Humanos , Oligopéptidos/metabolismo , Péptidos Cíclicos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/farmacología , Unión Proteica , Protrombina/efectos de los fármacosRESUMEN
Many similarities exist in the cellular responses elicited by VEGF and governed by integrins. Here, we identify a basis for these interrelationships: VEGF activates integrins. VEGF enhanced cell adhesion, migration, soluble ligand binding, and adenovirus gene transfer mediated by alphavbeta3 and also activated other integrins, alphavbeta5, alpha5beta1, and alpha2beta1, involved in angiogenesis. Certain tumor cells exhibited high spontaneous adhesion and migration, which were attributable to a VEGF-dependent autocrine/paracrine activation of integrins. This activation was mediated by the VEGFR2 receptor and regulated via phosphatidylinositol-3-kinase, Akt, and the PTEN signaling axis. Thus, integrin activation provides a mechanism for VEGF to induce a broad spectrum of cellular responses.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Integrinas/fisiología , Linfocinas/farmacología , Adenoviridae , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Humanos , Melanoma , Neovascularización Fisiológica , Isoformas de Proteínas/farmacología , Receptores de Colágeno , Receptores de Fibronectina/fisiología , Receptores de Vitronectina/fisiología , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Vitronectina/fisiologíaAsunto(s)
Antígenos de Plaqueta Humana/genética , Enfermedades Cardiovasculares/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Polimorfismo Genético , Isoformas de Proteínas/genética , Alelos , Sustitución de Aminoácidos , Antígenos de Plaqueta Humana/química , Enfermedades Cardiovasculares/epidemiología , Moléculas de Adhesión Celular/metabolismo , Retracción del Coagulo , Fibrinógeno/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Predisposición Genética a la Enfermedad , Humanos , Masculino , Fosforilación , Agregación Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Mutación Puntual , Isoformas de Proteínas/química , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Factores de RiesgoRESUMEN
alpha(V)beta(3), a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of alpha(V)beta(3), its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for alpha(V)beta(3) in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of alpha(V)beta(3) activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn(2+) markedly enhanced alpha(V)beta(3)-dependent adhesion to BSP. alpha(V)beta(3)-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that alpha(V)beta(3) activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by alpha(V)beta(3)-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of alpha(V)beta(3) can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of alpha(V)beta(3) on neoplastic cells may contribute to tumor growth and metastatic potential.
Asunto(s)
Adhesión Celular/fisiología , Quimiotaxis/fisiología , Endotelio Vascular/fisiología , Linfocitos/fisiología , Osteoblastos/fisiología , Receptores de Vitronectina/fisiología , Sialoglicoproteínas/fisiología , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Endotelio Vascular/citología , Humanos , Sialoproteína de Unión a Integrina , Linfocitos/citología , Manganeso/farmacología , Melanoma , Oligopéptidos/farmacología , Osteoblastos/citología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Venas UmbilicalesRESUMEN
Regardless of the event that stimulates the aggregation of platelets, the receptor alpha(IIb)beta3--one of a family of adhesion receptors known as integrins--has a key role in the process. The past decade has seen the publication of 10 phase III (randomised) clinical trials of four members of a new class of antiplatelet drugs, the GPIIb-IIIa blockers, targeted at this important receptor. Three (abciximab, eptifibatide, and tirofiban) are licensed for human use. 10 other GbIIb-IIIa blockers are in phase II or III human studies. In all 10 placebo-controlled trials, done in the clinical settings of percutaneous coronary intervention or acute coronary syndrome in patients on aspirin, the endpoints favoured the active drug, with a risk reduction for death or non-fatal myocardial infarction of about 21% overall. With attention to heparin dose the risk of bleeding is not a major concern with these agents. The GPIIb-IIIa blockers are taking the clinician and patient out of the era of aspirin monotherapy when platelet inhibition is required.
Asunto(s)
Inhibidores de Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Humanos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/uso terapéuticoRESUMEN
Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin alphaVbeta3 on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for alphaVbeta3 on these cells include: (a) prothrombin binds to purified alphaVbeta3 via a RGD recognition specificity; (b) prothrombin supports alphaVbeta3-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via alphaVbeta3. alphaVbeta3-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the alphaVbeta3 receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of alphaVbeta3 could be induced not only by model agonists, PMA and Mn2+, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of alphaVbeta3 on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of alphaVbeta3 to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by alphaVbeta3 may be controlled by the activation state of this integrin.
Asunto(s)
Endotelio Vascular/citología , Protrombina/metabolismo , Receptores de Vitronectina/metabolismo , Adenosina Difosfato/farmacología , Aorta , Calpaína/antagonistas & inhibidores , Calpaína/fisiología , Citocalasina B/farmacología , Fibrinógeno/metabolismo , Hemostasis , Homeostasis , Humanos , Manganeso/farmacología , Oligopéptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacologíaAsunto(s)
Endotelio Vascular/citología , Músculo Liso Vascular/citología , Receptores de Vitronectina/fisiología , Animales , Arterias/lesiones , Bovinos , Adhesión Celular , División Celular , Movimiento Celular , Células Cultivadas , Retracción del Coagulo , Cricetinae , Humanos , Hiperplasia , Ligandos , Neovascularización Fisiológica , Conejos , Receptores de Vitronectina/química , Transducción de Señal/fisiología , Túnica Íntima/patología , Vitronectina/metabolismoRESUMEN
Prothrombin activation is a pivotal event in thrombosis and hemostasis because thrombin can mediate fibrin formation and can activate and aggregate platelets. Platelet aggregation depends upon the binding of adhesive proteins to integrin alphaIIbbeta3 on the platelet surface. In the present study, a novel interface between the blood coagulation system and platelets is demonstrated by showing that 1) prothrombin binds to alphaIIbbeta3 and 2) this interaction accelerates prothrombin activation. Prothrombin bound to purified alphaIIbbeta3 in a specific, saturable, and divalent cation-dependent manner. This interaction was inhibited by certain monoclonal antibodies to alphaIIbbeta3, by the alphaIIbbeta3 ligands fibrinogen and RGD peptides, but not by thrombin or unrelated proteins. Prothrombin also interacted with alphaIIbbeta3 on resting and stimulated platelets as demonstrated by soluble ligand binding and platelet adhesion assays. Activation of prothrombin by Factor Xa alone or Factor Xa-Va was accelerated by alphaIIbbeta3, and this enhancement was blocked by a monoclonal antibody that inhibited prothrombin binding to the receptor. Taken together, these data identify a previously unrecognized linkage between platelets and the blood coagulation system that may have a significant regulatory consequence.
Asunto(s)
Hemostasis , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Protrombina/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Fibrinógeno/farmacología , Humanos , Cinética , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/aislamiento & purificación , Protrombina/aislamiento & purificación , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Platelet glycoprotein (GP) IIb-IIIa complex (alpha IIb beta 3-integrin) changes its conformation upon platelet activation that results in binding of RGD-containing ligands and expression of ligand-induced binding site (LIBS) neoepitopes. Anti-GIIb-IIIa monoclonal antibody (monAB) CRC54 bound to < or = 10% of GPIIb-IIIa on resting platelets but binding was enhanced by the occupation of GPIIb-IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N-terminal residues of GPIIIa and differed from other LIBS epitopes. CRC54 as well as its Fab fragments were able to induce platelet aggregation. CRC54 also stimulated interaction of GPIIb-IIIa with its ligands (fibrinogen and fibronectin) and conformation-dependent antibodies. The results indicated that changes of GPIIb-IIIa conformation, binding of ligands and platelet aggregation could be stimulated via interaction of anti-LIBS antibody with the N-terminal part of GPIIIa.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Plaquetas/fisiología , Antígenos CD36/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Plaquetas/efectos de los fármacos , Antígenos CD36/inmunología , Epítopos/análisis , Fibrinógeno/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Técnicas In Vitro , Cinética , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Conformación ProteicaRESUMEN
Low density lipoproteins (LDL) enhance the entry of Ca2+ and other bivalent cations via receptor-operated channels. The use of a synthetic peptide and antibodies blocking the glycoproteins IIb and IIIa interaction with ligands revealed that fibrinogen receptor antagonists prevent platelet aggregation without any effect on the ability of LDL to increase the cytoplasmic Ca2+ level. These data indicate that glycoproteins IIb-IIIa are not involved in the activation of the second messenger system by LDL but play a role in the proaggregant effect of LDL by providing cell-to-cell interactions.
Asunto(s)
Lipoproteínas LDL/fisiología , Activación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Plaquetas/citología , Plaquetas/metabolismo , Calcio/sangre , Cationes Bivalentes , Comunicación Celular , Humanos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Receptores de LDL/fisiología , Sistemas de Mensajero SecundarioRESUMEN
Monoclonal antibody (MAb) CRC81 against P-selectin, a membrane cell adhesion protein of platelets and endothelial cells (EC), has been obtained and characterized. The antibody selectively interacted with the surface of activated platelets and EC due to the redistribution of P-selectin from intracellular organelles onto the surface by activation. MAb CRC81 could recognize not only human but also rabbit and dog P-selectins. MAb CRC81 was used to establish the heterogeneity of human aorta EC with regard to the P-selectin content. Some of the cells in culture did not contain this protein but were nevertheless positively stained for von Willebrand factor, a specific marker for EC which, similar to P-selectin, is localized in Weibel-Palade's bodies. The proportion of P-selectin negative EC increased dramatically in the course of cell passaging: in primary cultures of aortic EC their content was about 15%, while after the 6th passage it exceeded 90%.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Aorta/metabolismo , Plaquetas/inmunología , Endotelio Vascular/inmunología , Selectina-P/metabolismo , Animales , Aorta/citología , Sitios de Unión de Anticuerpos , Células Cultivadas , Perros , Endotelio Vascular/citología , Humanos , Ratones , Ratones Endogámicos BALB C , Selectina-P/inmunología , Pruebas de Precipitina , ConejosAsunto(s)
Arteriosclerosis/patología , Endotelio Vascular/citología , Adulto , Factores de Edad , Anciano , Aorta/citología , Adhesión Celular , División Celular , Células Cultivadas , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Hiperlipidemias/patología , Leucocitos/citología , Masculino , Selectina-P , Glicoproteínas de Membrana Plaquetaria/metabolismo , Reología , Factor de von Willebrand/metabolismoAsunto(s)
Anticuerpos Monoclonales/farmacología , Inhibidores de Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/inmunología , Adenosina Difosfato/farmacología , Animales , Femenino , Fragmentos Fab de Inmunoglobulinas/farmacología , Ratones , Ratones Endogámicos BALB C , Agregación Plaquetaria/efectos de los fármacos , Trombina/farmacologíaRESUMEN
Glycoproteins (GPs) IIb and IIIa form a Ca(2+)-dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb), CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate, (ii) recognized the GPIIIa band in Western blotting of platelet SDS-lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Immunoblotting of chymotryptic digestion products of purified GPIIb-IIIa has shown that CRC54 epitope is located within residues 1-100 at the N-terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca2+ and Mg2+, 2.34 +/- 0.28 x 10(3) molecules per platelet at saturation. The same level of binding was observed without any divalent cations in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04 +/- 0.28 x 10(3) molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific epitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. However, binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using 51Cr-labelled platelets, binding of 125I-CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 +/- 0.48 x 10(3) per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa; (2) the CRC54-reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.