RESUMEN
Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, alpha(IIb)beta(3). In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant alpha(IIb)beta(3) may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable alpha(IIb)beta(3), findings consistent with type II GT. Genotyping of LD revealed 2 novel beta(3) mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among beta integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Probeta(3) cotransfected with wild-type alpha(IIb) into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Probeta(3) formed a complex with endogenous a(v) and retracted fibrin clots similarly to wild-type beta(3). The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for alpha(IIb)beta(3) to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding beta(3) Leu262 may maintain beta(3) in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.
Asunto(s)
Antígenos CD/fisiología , Fibrina/metabolismo , Fibrinógeno/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Mutación Puntual , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Células COS , Línea Celular , Preescolar , Femenino , Humanos , Integrina beta3 , Riñón , Leucina , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Linaje , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Reacción en Cadena de la Polimerasa , Prolina , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Trombastenia/genética , Transfección , XenopusRESUMEN
Previous studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human alpha-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Envejecimiento/sangre , Recién Nacido/sangre , Activación Plaquetaria/efectos de los fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Adenosina Difosfato/farmacología , Adulto , Epinefrina/farmacología , Femenino , Citometría de Flujo , Humanos , Masculino , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Trombina/farmacología , Tromboxano A2/análogos & derivados , Tromboxano A2/farmacologíaRESUMEN
To investigate the possibility that thrombin and/or other platelet activators change the platelet surface expression of glycoprotein IV (GPIV, CD36), we used a panel of five GPIV-specific monoclonal antibodies (OKM5, 5F1, FA6-152, 8A6, and F13) directed against different epitopes. All these antibodies bound to resting platelets in a concentration-dependent and saturable manner, as determined by flow cytometry of washed platelets. Thrombin (1 U/mL) induced an approximately twofold increase in the platelet surface binding of each of these monoclonal antibodies. Immunofluorescence microscopy demonstrated an internal pool of GPIV that, after thrombin stimulation, redistributed to the platelet surface. In a whole-blood flow-cytometric assay, alpha-thrombin and the thromboxane A2 analogue U46619 each resulted in an approximately twofold increase in the platelet surface binding of OKM5, whereas ADP had a more modest effect, and collagen and epinephrine had little effect. The activation-induced up-regulation of the platelet OKM5 epitope occurred in vivo as demonstrated by flow cytometric analysis of whole blood emerging from a standardized skin puncture site. In summary, both in vitro and in vivo platelet activation results in increased platelet surface expression of GPIV, as a result of a redistribution of GPIV from an internal pool.
Asunto(s)
Antígenos CD/sangre , Activación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/sangre , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Anticuerpos Monoclonales/sangre , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Antígenos CD36 , Humanos , Lactante , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Receptores de Citoadhesina/metabolismo , Trombina/farmacología , Tromboxano A2/análogos & derivados , Tromboxano A2/farmacología , Vasoconstrictores/farmacologíaRESUMEN
Thrombin decreases the platelet surface expression of the glycoprotein (GP) Ib-IX complex. To determine whether this effect is reversible, flow cytometric studies were performed with GPIb-IX-specific monoclonal antibodies. In both whole blood and washed platelet systems, incubation of platelets with thrombin or a combination of adenosine diphosphate and epinephrine resulted in a maximal decrease of the platelet surface expression of GPIb-IX within 5 minutes, after which there was a time-dependent return of the platelet surface GPIb-IX complex, which was maximal by 60 minutes. Exposure of the same platelets to additional exogenous thrombin resulted in a second decrease in platelet surface GPIb-IX, followed by a second reconstitution of platelet surface GPIb-IX. Throughout these experiments there was no measurable release from the platelets of glycocalicin (a proteolytic fragment of GPIb). Experiments in which platelets were preincubated with a biotinylated GPIb-specific MoAb showed that the GPIb molecules that returned to the platelet surface were the same molecules that had been translocated to the intraplatelet pool. The GPIb molecules that returned to the platelet surface were functionally competent to bind von Willebrand factor, as determined by ristocetin-induced platelet agglutination and ristocetin-induced binding of exogenous von Willebrand factor. Inhibitors of protein kinase C and myosin light-chain kinase enhanced the reexpression of platelet surface GPIb. In summary, the activation-induced decrease in the platelet surface expression of the GPIb-IX complex is reversible. Inactivation of protein kinase C and myosin light-chain kinase are important mechanisms in the reexpression of the platelet surface GPIb-IX complex.
Asunto(s)
Plaquetas/metabolismo , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/análisis , Receptores de Superficie Celular/análisis , Adulto , Humanos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombina/farmacología , Factor de von Willebrand/metabolismoRESUMEN
A hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22 degrees C and 37 degrees C. the forearm skin temperature was maintained at temperatures between 22 degrees C and 37 degrees C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB2 (the stable metabolite of TXA2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37 degrees C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB2 generation, and the bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hipotermia Inducida , Activación Plaquetaria/fisiología , Adulto , Secuencia de Aminoácidos , Tiempo de Sangría , Regulación hacia Abajo , Femenino , Humanos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Selectina-P , Agregación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/análisis , Glicoproteínas de Membrana Plaquetaria/sangre , Receptores de Superficie Celular/análisis , Tromboxano B2/biosíntesis , Regulación hacia ArribaRESUMEN
BACKGROUND: It has been hypothesized that platelets are activated, or made more activatible, by strenuous exercise and that these changes may play a role in the genesis of exercise-induced coronary ischemia. Previous studies have yielded conflicting results but have used assays (eg, platelet aggregation, plasma platelet factor 4, and plasma beta-thromboglobulin) that are subject to methodological problems. METHODS AND RESULTS: In the present study, a whole blood flow cytometric method was used to study the platelet activation state and reactivity of 12 physically active and 12 sedentary individuals before and after standardized treadmill exercise testing. The peptide gly-pro-arg-pro (GPRP) was included in this assay to prevent fibrin polymerization and platelet aggregation, thus allowing the measurement of the reactivity to thrombin of individual platelets in the physiological milieu of whole blood. A panel of fluorescent-labeled monoclonal antibodies was used to monitor activation-dependent platelet surface changes: downregulation of glycoprotein (GP) Ib (6D1) and upregulation of GMP-140 (S12), the GPIIb-IIIa complex (PAC1), and GPIV (OKM5). In samples obtained before exercise, platelets not exposed to thrombin showed no evidence of in vitro activation. In the sedentary subjects, exercise caused a consistent and significant augmentation of the platelet activation state and reactivity as judged by the binding of 6D1 in the presence of thrombin 0.05 U/mL (P < .001), 0.005 U/mL (P = .001), and 0 U/mL (P = .004) and by the binding of OKM5 in the presence of thrombin 0.05 U/mL (P < .001), 0.005 U/mL (P = .029), and 0 U/mL (P = .035). Exercise increased the binding of PAC1 at only a single thrombin concentration (0.005 U/mL, P = .027) and did not alter the binding of S12 at any thrombin concentration. In contrast, in the physically active subjects, exercise failed to cause a consistent alteration in either platelet activation state or platelet reactivity. No significant differences were found between the 12 male and 12 female volunteers. CONCLUSIONS: Strenuous exercise in sedentary subjects but not physically active subjects resulted in both platelet activation and platelet hyperreactivity. These changes were more readily detected with monoclonal antibodies directed against GPIb (6D1) and, to a lesser extent, GPIV (OKM5) rather than those directed against the GPIIb-IIIa complex (PAC1) and GMP-140 (S12). Platelet activation by thrombin, generally regarded as the most physiologically important agonist, can be studied in whole blood in a clinical setting through the use of the peptide GPRP.
Asunto(s)
Ejercicio Físico/fisiología , Activación Plaquetaria/fisiología , Adulto , Sitios de Unión de Anticuerpos , Plaquetas/inmunología , Prueba de Esfuerzo , Femenino , Citometría de Flujo , Humanos , Masculino , Pruebas de Función PlaquetariaRESUMEN
The use of cardiopulmonary bypass (CPB) during cardiac surgery is associated with a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time. However, the nature of the putative platelet function defect is controversial. In this study, blood was analyzed at 10 time points before, during, and after CPB. We used a whole-blood flow cytometric assay to study platelet surface glycoproteins in (1) peripheral blood, (2) peripheral blood activated in vitro by either phorbol myristate acetate, the thromboxane (TX)A2 analog U46619, or a combination of adenosine diphosphate and epinephrine, and (3) the blood emerging from a bleeding-time wound (shed blood). Activation-dependent changes were detected by monoclonal antibodies directed against the glycoprotein (GP)Ib-IX and GPIIb-IIIa complexes and P-selectin. In addition, we measured plasma glycocalicin (a proteolytic fragment of GPIb) and shed-blood TXB2 (a stable breakdown product of TXA2). In shed blood emerging from a bleeding-time wound, the usual time-dependent increase in platelet surface P-selectin was absent during CPB, but returned to normal within 2 hours. This abnormality paralleled both the CPB-induced prolongation of the bleeding time and a CPB-induced marked reduction in shed-blood TXB2 generation. In contrast, there was no loss of platelet reactivity to in vitro agonists during or after CPB. In peripheral blood, platelet surface P-selectin was negligible at every time point, demonstrating that CPB resulted in a minimal number of circulating degranulated platelets. CPB did not change the platelet surface expression of GPIb in peripheral blood, as determined by the platelet binding of a panel of monoclonal antibodies, ristocetin-induced binding of von Willebrand factor, and a lack of increase in plasma glycocalicin. CPB did not change the platelet surface expression of the GPIIb-IIIa complex in peripheral blood, as determined by the platelet binding of fibrinogen and a panel of monoclonal antibodies. In summary, CPB resulted in (1) markedly deficient platelet reactivity in response to an in vivo wound, (2) normal platelet reactivity in vitro, (3) no loss of the platelet surface GPIb-IX and GPIIb-IIIa complexes, and (4) a minimal number of circulating degranulated platelets. These data suggest that the "platelet function defect" of CPB is not a defect intrinsic to the platelet, but is an extrinsic defect such as an in vivo lack of availability of platelet agonists. The near universal use of heparin during CPB is likely to contribute substantially to this defect via its inhibition of thrombin, the preeminent platelet activator.
Asunto(s)
Trastornos de la Coagulación Sanguínea/etiología , Plaquetas/fisiología , Puente Cardiopulmonar/efectos adversos , Humanos , Técnicas In Vitro , Selectina-P , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombina/farmacología , Regulación hacia ArribaRESUMEN
We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).
Asunto(s)
Anticuerpos Monoclonales/genética , Hibridomas/química , Activadores Plasminogénicos/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Células Clonales , Fibrina/inmunología , Humanos , Immunoblotting , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple , Proteínas de Neoplasias , Activadores Plasminogénicos/inmunología , ARN Mensajero/análisis , ARN de Transferencia/análisis , Transcripción GenéticaRESUMEN
Platelet degranulation has been implicated in the pathophysiology of acute arterial thrombosis, intimal hyperplasia, and atherogenesis. Most previous studies that examined the effect of heparin on platelet function have used platelet aggregometry. These studies have resulted in contradictory data and, by the nature of the assay, reveal no information with regard to platelet degranulation. In contrast, flow cytometry allows accurate quantification of the extent of platelet degranulation by measurement of the platelet surface binding of a GMP-140 specific monoclonal antibody (S12). GMP-140 is only expressed on the platelet surface after platelet alpha granule release. In the present study increasing concentrations of heparin were added to whole blood anticoagulated with sodium citrate. Platelets were activated with a panel of agonists, and the extent of platelet degranulation was quantified by whole blood flow cytometry. Heparin concentrations as high as 100 units/ml were found to suppress platelet alpha granule release induced by either a thromboxane A2 analog (U46619) or a combination of adenosine diphosphate and epinephrine. Heparin suppressed alpha granule release induced by thrombin both in whole blood and in washed platelets. The addition of heparin after platelet activation had no effect on S12 binding. In summary, heparin in high concentrations is a potent inhibitor of platelet degranulation, an action that is unrelated to its effect on the coagulation cascade. Although the heparin concentrations used in this study exceed those used clinically by a factor of 10 or more, future studies of heparin fractions may allow the separation of the anticoagulant and antiplatelet properties of the molecule and allow the administration of an agent that selectively suppresses platelet degranulation without the humoral anticoagulant effect.
Asunto(s)
Plaquetas/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Heparina/farmacología , Adenosina Difosfato/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales , Plaquetas/metabolismo , Plaquetas/fisiología , Degranulación de la Célula/fisiología , Relación Dosis-Respuesta a Droga , Epinefrina/antagonistas & inhibidores , Citometría de Flujo , Humanos , Ratones , Selectina-P , Glicoproteínas de Membrana Plaquetaria/análisis , Endoperóxidos de Prostaglandinas Sintéticos/antagonistas & inhibidores , Trombina/antagonistas & inhibidoresRESUMEN
We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.
Asunto(s)
Coagulación Sanguínea , Factor IXa/metabolismo , Deficiencia del Factor VII/sangre , Factor VIIIa/metabolismo , Factor VII/metabolismo , Factor VIIa/uso terapéutico , Factor X/metabolismo , Factor X/uso terapéutico , Hemofilia A/sangre , Protrombina/metabolismo , Adolescente , Adulto , Anticuerpos Monoclonales , Deficiencia del Factor VII/terapia , Femenino , Hemofilia A/terapia , Humanos , Masculino , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/uso terapéutico , Valores de ReferenciaRESUMEN
In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.