RESUMEN
Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.
Asunto(s)
Sistemas de Liberación de Medicamentos , Precursores Enzimáticos/farmacología , Eritrocitos , Fibrinolíticos/farmacología , Profármacos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/farmacología , Trombosis/prevención & control , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Humanos , Ratones , Proteínas Recombinantes/farmacología , Factores de TiempoRESUMEN
Tissue-type plasminogen activator (tPA) is a potent fibrinolytic enzyme used to treat acute coronary artery obstruction. However, tPA has shown limited utility in other disorders caused by thrombotic vascular occlusion, such as pulmonary embolism. We found that tPA caused dose-dependent effects on the contractility of pulmonary arterial rings that may affect its effectiveness as a thrombolytic agent. At low concentrations (1 nM), tPA stimulated pulmonary vascular contraction in response to phenylephrine, whereas at higher concentrations (20 nM) tPA inhibited pulmonary arterial contractility and promoted pulmonary vascular permeability through an interaction between its "docking site" and N-methyl d-aspartate receptor type 1 (NMDA-R1) expressed by pulmonary arteries. A hexapeptide derived from plasminogen activator inhibitor type 1 that blocked the docking site of tPA, but not its catalytic activity, inhibited its interaction with NMDA-R1, abolished inhibition of pulmonary artery contractility, attenuated vascular permeability, and facilitated fibrinolysis in a murine model of pulmonary embolism. Similar outcomes were seen using a tPA variant that lacks the docking site but retains catalytic activity. These data suggest that it is feasible to attenuate the deleterious extrafibrinolytic effects of tPA and improve its benefit:risk profile in the management of pulmonary embolism.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , Activador de Tejido Plasminógeno/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Endotelio Vascular/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fenilefrina/farmacología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiología , Embolia Pulmonar , Ratas , Ratas Sprague-Dawley , Activador de Tejido Plasminógeno/administración & dosificaciónRESUMEN
Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.
Asunto(s)
Eritrocitos/efectos de los fármacos , Fibrinolíticos/farmacología , Activadores Plasminogénicos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/farmacología , Animales , Agregación Eritrocitaria , Eritrocitos/fisiología , Fibrinolíticos/farmacocinética , Glicoforinas/inmunología , Hemólisis , Humanos , Técnicas In Vitro , Venas Yugulares , Ratones , Ratones Endogámicos C57BL , Mutación , Activadores Plasminogénicos/genética , Activadores Plasminogénicos/farmacocinética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Anticuerpos de Cadena Única/genética , Trombosis de la Vena/sangre , Trombosis de la Vena/prevención & controlRESUMEN
BACKGROUND: Cerebrovascular thrombosis is a major source of morbidity and mortality after surgery, but thromboprophylaxis in this setting is limited because of the formidable risk of perioperative bleeding. Thrombolytics (eg, tissue-type plasminogen activator [tPA]) cannot be used prophylactically in this high-risk setting because of their short duration of action and risk of causing hemorrhage and central nervous system damage. We found that coupling tPA to carrier red blood cells (RBCs) prolongs and localizes tPA activity within the bloodstream and converts it into a thromboprophylactic agent, RBC/tPA. To evaluate the utility of this new approach for preventing cerebrovascular thrombosis, we examined the effect of RBC/tPA in animal models of cerebrovascular thromboembolism and ischemia. METHODS AND RESULTS: Preformed fibrin microemboli were injected into the middle carotid artery of mice, occluding downstream perfusion and causing severe infarction and 50% mortality within 48 hours. Preinjected RBC/tPA rapidly lysed nascent cerebral thromboemboli, providing rapid, durable reperfusion and reducing morbidity and mortality. These beneficial effects were not achieved by preinjection of tPA, even at a 10-fold higher dose, which increased mortality from 50% to 90% by 10 hours after embolization. RBC/tPA injected 10 minutes after tail amputation to simulate postsurgical hemostasis did not cause bleeding from the wound, whereas soluble tPA caused profuse bleeding. RBC/tPA neither aggravated brain damage caused by focal ischemia in a filament model of middle carotid artery occlusion nor caused postthrombotic hemorrhage in hypertensive rats. CONCLUSIONS: These results suggest a potential RBC/tPA utility as thromboprophylaxis in patients who are at risk for acute cerebrovascular thromboembolism.
Asunto(s)
Transfusión de Eritrocitos/métodos , Eritrocitos , Trombosis Intracraneal/prevención & control , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/administración & dosificación , Animales , Trastornos Cerebrovasculares/patología , Trastornos Cerebrovasculares/prevención & control , Eritrocitos/citología , Humanos , Trombosis Intracraneal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas SHRRESUMEN
A fibrinolytic agent consisting of a tissue-type plasminogen activator (tPA) coupled to the surface of red blood cells (RBCs) can dissolve nascent clots from within the clot, in a Trojan horse-like strategy, while having minimal effects on preexisting hemostatic clots or extravascular tissue. After intravenous injection, the fibrinolytic activity of RBC-tPA persisted in the bloodstream at least tenfold longer than did that of free tPA. In a model of venous thrombosis induced by intravenously injected fibrin microemboli aggregating in pulmonary vasculature, soluble tPA lysed pulmonary clots lodged before but not after tPA injection, whereas the converse was true for RBC-tPA. Free tPA failed to lyse occlusive carotid thrombosis whether injected before or after vascular trauma, whereas RBC-tPA circulating before, but not injected after, thrombus formation restored blood flow. This RBC-based drug delivery strategy alters the fibrinolytic profile of tPA, permitting prophylactic fibrinolysis.
Asunto(s)
Trombosis de las Arterias Carótidas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Transfusión de Eritrocitos/métodos , Eritrocitos/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Tejido Plasminógeno/metabolismo , Animales , Coagulación Sanguínea/efectos de los fármacos , Fibrinólisis , Fibrinolíticos/uso terapéutico , Inyecciones Intravenosas , Ratones , Ratas , Resultado del TratamientoRESUMEN
Streptavidin-biotin conjugates of enzymes with carrier antibodies provide a versatile means for targeting selected cellular populations in cell cultures and in vivo. Both specific delivery to cells and proper subcellular addressing of enzyme cargoes are important parameters of targeting. This chapter describes methodologies for evaluating the binding and internalization of labeled conjugates directed to endothelial surface adhesion molecules in cell cultures using anti-intercellular adhesion molecule/catalase or antiplatelet endothelial cell adhesion molecule/catalase conjugates as examples. It also describes protocols for characterization of biodistribution and pulmonary targeting of radiolabeled conjugates in rats using anti-intercellular adhesion molecule/tPA conjugates as an example. The experimental procedures, results, and notes provided may help in investigations of vascular immunotargeting of reporter, experimental, diagnostic, or therapeutic enzymes to endothelial and, perhaps, other cell types, both in vitro and in vivo.
Asunto(s)
Anticuerpos/química , Endotelio/metabolismo , Enzimas/química , Inmunoconjugados/metabolismo , Animales , Anticuerpos/análisis , Anticuerpos/metabolismo , Sistemas de Liberación de Medicamentos , Enzimas/análisis , Enzimas/metabolismo , Inmunoconjugados/análisis , Inmunoconjugados/química , Pulmón/metabolismo , Microscopía Fluorescente , Especificidad de Órganos , Radioinmunodetección/métodos , RatasRESUMEN
Coupling plasminogen activators to carrier red blood cells (RBC) prolongs their life-time in the circulation and restricts extravascular side effects, thereby allowing their utility for short-term thromboprophylaxis. Unlike constitutively active plasminogen activators, single chain urokinase plasminogen activator (scuPA) is activated by plasmin proteolysis or binding to its receptor, uPAR. In this study we conjugated recombinant soluble uPAR (suPAR) to rat RBC, forming RBC/suPAR complex. RBC carrying suPAR circulated in rats similarly to naïve RBC and markedly prolonged the circulation time of suPAR. RBC/suPAR carrying approximately 3x10(4) suPAR molecules per RBC specifically bound up to 2x10(4) molecules of scuPA, retained approximately 75% of scuPA-binding capacity after circulation in rats and markedly altered the functional profile of bound scuPA. RBC carrying directly conjugated scuPA adhered to endothelial cells, while showing no appreciable fibrinolytic activity. In contrast, RBC/suPAR loaded with scuPA did not exhibit increased adhesion to endothelium, while effectively dissolving fibrin clots. This molecular design, capitalizing on unique biological features of the interaction of scuPA with its receptor, provides a promising modality to deliver a pro-drug for prevention of thrombosis.
Asunto(s)
Portadores de Fármacos , Transfusión de Eritrocitos , Eritrocitos/metabolismo , Fibrinolíticos/farmacología , Profármacos/farmacología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/sangre , Terapia Trombolítica , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Biotinilación , Adhesión Celular , Células Cultivadas , Química Farmacéutica , Composición de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fibrinólisis/efectos de los fármacos , Fibrinolíticos/sangre , Fibrinolíticos/química , Humanos , Profármacos/química , Profármacos/metabolismo , Unión Proteica , Ratas , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Proteínas Recombinantes/sangre , Activador de Plasminógeno de Tipo Uroquinasa/sangre , Activador de Plasminógeno de Tipo Uroquinasa/químicaRESUMEN
A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti-PECAM-1 antibody single-chain variable fragment (anti-PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature.(1) To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/low-molecular-weight (lmw)-scuPA with a thrombin activation site, generating anti-PECAM scFv/uPA-T that (1) is latent and activated by thrombin instead of plasmin; (2) binds to PECAM-1; (3) does not consume plasma fibrinogen; (4) accumulates in mouse lungs after intravenous injection; and (5) resists PA inhibitor PAI-1 until activated by thrombin. In mouse models of pulmonary thrombosis caused by thromboplastin and ischemia-reperfusion (I/R), scFv/uPA-T provided more potent thromboprophylaxis and greater lung protection than plasmin-sensitive scFv/uPA. Endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time- and site-specific regulation of proteolytic reactions that can be modulated for therapeutic benefit.
Asunto(s)
Endotelio Vascular/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/fisiología , Circulación Pulmonar/fisiología , Trombina/fisiología , Terapia Trombolítica , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Animales , Endotelio Vascular/efectos de los fármacos , Fibrinógeno/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Distribución Tisular , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Coupling tissue-type plasminogen activator (tPA) to carrier red blood cells (RBC) prolongs its intravascular life span and permits its use for thromboprophylaxis. Here, we studied the susceptibility of RBC/tPA to PA inhibitors including plasminogen activator inhibitor-1 (PAI-1) that constrain its activity and may reduce the duration of its effect. Despite lesser spatial and diffusional limitations, soluble tPA was far less effective than RBC/tPA in dissolving clots formed in vitro from blood of wild-type (WT) mice (40 versus 80% lysis at equal doses of tPA). Furthermore, after i.v. injection, soluble tPA lost activity faster in transgenic mice expressing a high level of PAI-1 than in WT mice, whereas the activity of RBC/tPA was unaffected. PAI-1 inactivated soluble tPA at equimolar ratios in vitro, but it had no effect on the amidolytic or fibrinolytic activity of RBC/tPA. RBC/tPA was also more resistant than soluble tPA to in vitro inhibition by other serpins (alpha2-macroglobulin and alpha1-antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis.
Asunto(s)
Eritrocitos/metabolismo , Glicocálix/fisiología , Activador de Tejido Plasminógeno/metabolismo , Animales , Coagulación Sanguínea/fisiología , Fibrinólisis/fisiología , Glucosa/farmacología , Técnicas In Vitro , Radioisótopos de Yodo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Inhibidor 1 de Activador Plasminogénico/fisiología , Serpinas/sangre , Serpinas/química , Serpinas/metabolismo , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/genéticaRESUMEN
The objective of this study was to quantitatively assess changes in cell adhesion molecule (CAM) expression on the pulmonary endothelial surface during hyperoxia and to assess the functional significance of those changes on cellular trafficking and development of oxygen-induced lung injury. Mice were placed in >95% O(2) for 0-72 h, and pulmonary injury and neutrophil (PMN) sequestration were assessed. Specific pulmonary CAM expression was quantified with a dual-radiolabeled MAb technique. To test the role of CAMs in PMN trafficking during hyperoxia, blocking MAbs to murine P-selectin, ICAM-1, or platelet-endothelial cell adhesion molecule-1 (PECAM-1) were injected in wild-type mice. Mice genetically deficient in these CAMs and PMN-depleted mice were also evaluated. PMN sequestration occurred within 8 h of hyperoxia, although alveolar emigration occurred later (between 48 and 72 h), coincident with rapid escalation of the lung injury. Hyperoxia significantly increased pulmonary uptake of radiolabeled antibodies to P-selectin, ICAM-1, and PECAM-1, reflecting an increase in their level on pulmonary endothelium and possibly sequestered blood cells. Although both anti-PECAM-1 and anti-ICAM-1 antibodies suppressed PMN alveolar influx in wild-type mice, only mice genetically deficient in PECAM-1 showed PMN influx suppression. Neither CAM blockade, nor genetic deficiency, nor PMN depletion attenuated lung injury. We conclude that early pulmonary PMN retention during hyperoxia is not temporally associated with an increase in endothelial CAMs; however, subsequent PMN emigration into the alveolar space may be supported by PECAM-1 and ICAM-1. Blocking PMN recruitment did not prevent lung injury, supporting dissociation between PMN infiltration and lung injury during hyperoxia in mice.
Asunto(s)
Movimiento Celular/inmunología , Hiperoxia/inmunología , Neutrófilos/inmunología , Alveolos Pulmonares/inmunología , Síndrome de Dificultad Respiratoria/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Hiperoxia/patología , Inmunoglobulina G/farmacología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Radioisótopos de Yodo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutropenia/inmunología , Neutropenia/patología , Neutrófilos/patología , Oxígeno/farmacología , Selectina-P/genética , Selectina-P/inmunología , Selectina-P/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Alveolos Pulmonares/patología , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
Plasminogen activators (PAs) are not used for thromboprophylaxis due to rapid clearance, bleeding, and extravascular toxicity. We describe a novel strategy that overcomes these limitations. We conjugated tissue-type PA (tPA) to a monoclonal antibody (mAb) against complement receptor type 1 (CR1) expressed primarily on human RBCs. Anti-CR1/tPA conjugate, but not control conjugate (mIgG/tPA), bound to human RBCs (1.2 x 10(3) tPA molecules/cell at saturation), endowing them with fibrinolytic activity. In vitro, RBC-bound anti-CR1/tPA caused 90% clot lysis versus 20% by naive RBCs. In vivo, more than 40% of anti-CR1/(125)I-tPA remained within the circulation ( approximately 90% bound to RBCs) 3 hours after injection in transgenic mice expressing human CR1 (TgN-hCR1) versus less than 10% in wild-type (WT) mice, without RBC damage; approximately 90% of mIgG/(125)I-tPA was cleared from the circulation within 30 minutes in both WT and TgN-hCR1 mice. Anti-CR1/tPA accelerated lysis of pulmonary emboli and prevented stable occlusive carotid arterial thrombi from forming after injection in TgN-hCR1 mice, but not in WT mice, whereas soluble tPA and mIgG/tPA were ineffective. Anti-CR1/tPA caused 20-fold less rebleeding in TgN-hCR1 mice than the same dose of tPA. CR1-directed immunotargeting of PAs to circulating RBCs provides a safe and practical means to deliver fibrinolytics for thromboprophylaxis in settings characterized by a high imminent risk of thrombosis.
Asunto(s)
Eritrocitos/metabolismo , Fibrinólisis , Receptores de Complemento/metabolismo , Activador de Tejido Plasminógeno/administración & dosificación , Animales , Fibrinólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/prevención & control , Terapia Trombolítica , Trombosis/prevención & controlRESUMEN
Plasminogen activators (PAs; e.g., tissue-type, tPA) coupled to red blood cells (RBCs) display in vivo features useful for thromboprophylaxis: prolonged circulation, minimal extravasation, and preferential lysis of nascent versus preexisting clots. Yet, factors controlling the activity of RBC-bound PAs in vivo are not defined and may not mirror the profile of soluble PAs. We tested the role of RBC/PA binding to fibrin in fibrinolysis. RBC/tPA and RBC/tPA variant with low fibrin affinity (rPA) bound to and lysed plasminogen-containing fibrin clots in vitro comparably. In contrast, when coinjected in mice with fibrin emboli lodging in pulmonary vasculature, only RBC/tPA accumulated in lungs, which resulted in a more extensive fibrinolysis versus RBC/rPA (p < 0.01). Reconciling this apparent divergence between in vitro and in vivo behaviors, RBC/tPA, but not RBC/rPA perfused over fibrin in vitro at physiological shear stress bound to fibrin clots and caused greater fibrinolysis versus RBC/rPA (p < 0.001). These results indicate that because of high fibrin affinity, RBC/tPA binding to clots endures hemodynamic stress, which enhances fibrinolysis. Behavior of RBC/PAs under hemodynamic pressure is an important predictor of their performance in vivo.
Asunto(s)
Eritrocitos/metabolismo , Fibrina/metabolismo , Fibrinólisis/efectos de los fármacos , Activador de Tejido Plasminógeno/administración & dosificación , Animales , Hemodinámica/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Embolia Pulmonar/tratamiento farmacológico , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Conjugating tissue-type plasminogen activator (tPA) to red blood cells (RBCs) endows it with features useful for thromboprophylaxis. However, the optimal intensity and duration of thromboprophylaxis vary among clinical settings. To assess how the intrinsic properties of a plasminogen activator (PA) affect functions of the corresponding RBC/PA conjugate, we coupled equal amounts of tPA or Retavase (rPA; a variant with an extended circulation time, lower fibrin affinity, and greater susceptibility to PA inhibitors). Conjugation to RBC markedly prolonged the circulation of each PA in rats and mice, without detrimental effects on carrier RBC. The initial blood clearance of RBC/tPA was faster than RBC/rPA, yet it exerted greater fibrinolytic activity, in part due to greater resistance of tPA and RBC/tPA to plasma inhibitors versus rPA and RBC/rPA observed in vitro. Soluble and RBC-coupled tPA and rPA exerted the same amidolytic activity, yet RBC/tPA lysed fibrin clots more effectively than RBC/rPA, notwithstanding comparable fibrinolytic activity of their soluble counterparts. Conjugation to RBC suppressed rPA's ability to be activated by fibrin, whereas the fibrin activation of RBC-coupled tPA was not hindered. Therefore, the functional profile of RBC/PA is influenced by: pharmacokinetic features provided by carrier RBC (e.g., prolonged circulation), intrinsic PA features (e.g., clearance rate, resistance to inhibitors), and changes imposed by conjugation to RBC (e.g., loss of cofactor stimulation). These factors, different from those guiding the design of soluble PA for lysis of existing clots, can be exploited in the rational design of RBC/PA tailored for specific prophylactic indications.
Asunto(s)
Eritrocitos/metabolismo , Fibrinolíticos/metabolismo , Proteínas Recombinantes/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Fibrina/farmacología , Fibrinólisis , Humanos , Tasa de Depuración Metabólica , Ratones , Inhibidor 1 de Activador Plasminogénico/farmacología , RatasRESUMEN
Extract: When we are wounded, either externally (for instance, when we cut ourselves) or internally (for instance, due to gastric ulcer or brain hemorrhage), blood clots -- sponge-like plugs that are rapidly formed in response to the injury by activated blood platelets and fibrin in a process called coagulation -- prevent profound bleeding. Thus, good or hemostatic clots save our lives. However, under pathological conditions blood clots can also form inside vessels. Such bad or thrombotic clots occlude blood vessels and cause oxygen starvation of vital organs including the brain (stroke), heart (acute myocardial infarction) or lungs (pulmonary embolism). Thrombosis is one of the leading causes of morbidity and mortality from cardiovascular and other disease conditions. Diverse anti-thrombotic means are being developed. For instance, anticoagulants (such as heparin) and platelet inhibitors (such as aspirin) help to prevent formation of clots (blood thinners). Fibrinolytics, known as plasminogen activators (such as tissue-type plasminogen activator, or tPA) dissolve formed clots by degrading the fibrin meshwork. Both types of therapeutics are widely used in medical practice, e.g., for treatment of two forms of ischemic heart disease caused by thrombi in coronary vessels -- acute myocardial infarction and unstable angina.
RESUMEN
Nanotechnologies promise new means for drug delivery. ICAM-1 is a good target for vascular immunotargeting of nanoparticles to the perturbed endothelium, although endothelial cells do not internalize monomeric anti-ICAM-1 antibodies. However, coupling ICAM-1 antibodies to nanoparticles creates multivalent ligands that enter cells via an amiloride-sensitive endocytic pathway that does not require clathrin or caveolin. Fluorescence microscopy revealed that internalized anti-ICAM nanoparticles are retained in a stable form in early endosomes for an unusually long time (1-2 h) and subsequently were degraded following slow transport to lysosomes. Inhibition of lysosome acidification by chloroquine delayed degradation without affecting anti-ICAM trafficking. Also, the microtubule disrupting agent nocodazole delayed degradation by inhibiting anti-ICAM nanoparticle trafficking to lysosomes. Addition of catalase to create anti-ICAM nanoparticles with antioxidant activity did not affect the mechanisms of nanoparticle uptake or trafficking. Intracellular anti-ICAM/catalase nanoparticles were active, because endothelial cells were resistant to H2O2-induced oxidative injury for 1-2 h after nanoparticle uptake. Chloroquine and nocodazole increased the duration of antioxidant protection by decreasing the extent of anti-ICAM/catalase degradation. Therefore, the unique trafficking pathway followed by internalized anti-ICAM nanoparticles seems well suited for targeted delivery of therapeutic enzymes to endothelial cells and may provide a basis for treatment of acute vascular oxidative stress.
Asunto(s)
Catalasa/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Endotelio Vascular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/enzimología , Humanos , Líquido Intracelular/efectos de los fármacos , Nanotecnología/métodos , Estrés Oxidativo/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
Drug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. (125)I-labeled anti-ICAM and a reporter enzyme (beta-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.
Asunto(s)
Endotelio Vascular/fisiología , Fibrinolíticos/farmacología , Molécula 1 de Adhesión Intercelular/fisiología , Pulmón/fisiología , Activador de Tejido Plasminógeno/metabolismo , Análisis de Varianza , Animales , Anticuerpos/metabolismo , Transporte Biológico , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Endotelio Vascular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Hígado/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Masculino , Ratas , Ratas Sprague-Dawley , Bazo/inmunología , Distribución TisularRESUMEN
Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.
Asunto(s)
Plaquetas/metabolismo , Trombosis/prevención & control , Activador de Plasminógeno de Tipo Uroquinasa/administración & dosificación , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Animales , Antifibrinolíticos/administración & dosificación , Arterias Carótidas/patología , Modelos Animales de Enfermedad , Hemorragia/inducido químicamente , Ratones , Ratones Transgénicos , Transfusión de Plaquetas , Embolia Pulmonar/terapia , Trombosis/terapia , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Therapeutic molecules conjugated with antibodies to the platelet-endothelial cell adhesion molecule-1 (PECAM-1) accumulate in the pulmonary endothelium after i.v. injection in mice. In this study, we characterized PECAM-directed targeting to the lung and heart after local versus systemic intravascular administration in a large animal model, pigs. Radiolabel tracing showed that 1 h post-i.v. injection, 35% of anti-PECAM versus 2.5% of control IgG had accumulated in the lungs. Infusion of anti-PECAM via a catheter placed in the right pulmonary artery (RPA) resulted in a 3-fold elevation of the uptake in the right lower lobe and 2-fold reduction of uptake in the left lobes in the lung. Cardiac uptake of anti-PECAM was negligible after i.v. and RPA infusion. In contrast, delivery with a catheter placed in the right coronary artery (RCA) resulted in a 4-fold elevation of cardiac uptake of anti-PECAM, but not IgG, compared with i.v. injection. To estimate the targeting of an active reporter enzyme, streptavidin-conjugated beta-galactosidase (beta-Gal) was coupled to anti-PECAM or IgG (anti-PECAM/beta-Gal and IgG/beta-Gal) and injected into the RCA. Beta-Gal activity was markedly elevated in the heart and lungs (5- and 25-fold increased, respectively) after injection of anti-PECAM/beta-Gal, but not IgG/beta-Gal. Image analysis confirmed endothelial targeting of anti-PECAM/beta-Gal in the heart and lung. In summary, anti-PECAM antibody conjugates deliver agents to the pulmonary endothelium regardless of injection route, whereas local arterial infusion permits targeting to the cardiac vasculature. This paradigm may be useful for drug targeting to endothelium in lungs, heart, and possibly other organs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Circulación Coronaria/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Circulación Pulmonar/efectos de los fármacos , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/farmacocinética , Biotina/química , Reacciones Cruzadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunohistoquímica , Infusiones Intravenosas , Radioisótopos de Yodo , Marcaje Isotópico , Pulmón/citología , Pulmón/metabolismo , Miocardio/citología , Miocardio/metabolismo , Especificidad de Órganos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Porcinos , Distribución Tisular , beta-Galactosidasa/farmacocinéticaRESUMEN
Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS.
Asunto(s)
Endotelio Vascular , Glucosa Oxidasa/toxicidad , Inmunotoxinas/toxicidad , Estrés Oxidativo , Circulación Pulmonar , Embolia Pulmonar , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Glucosa Oxidasa/inmunología , Humanos , Inmunotoxinas/inmunología , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Circulación Pulmonar/inmunología , Embolia Pulmonar/etiología , Embolia Pulmonar/inmunología , Embolia Pulmonar/metabolismo , Trombomodulina/inmunologíaRESUMEN
Using tracings of (125)I-labeled fibrin(ogen) in rodents, we examined the hypothesis that platelets impede the lysis of pulmonary emboli. (125)I-Microemboli (ME, 3-10 micron diameter) lodged homogeneously throughout the lungs after intravenous injection in both rats and mice (60% of injected dose), caused no lethality, and underwent spontaneous dissolution (50 and 100% within 1 and 5 h, respectively). Although lung homogenates displayed the most intense fibrinolytic activity of all the major organs, dissolution of ME was much slower in isolated perfused lungs (IPL) than was observed in vivo. Addition of rat plasma to the perfusate facilitated ME dissolution in IPL to a greater extent than did addition of tissue-type plasminogen activator alone, suggesting that permeation of the clot by plasminogen is the rate-limited step in lysis. Platelet-containing ME injected in rats lysed much more slowly than did ME formed from fibrin alone. (125)I-Thrombi, formed in the pulmonary vasculature of mice in response to intravascular activation of platelets by injection of collagen and epinephrine, were essentially resistant to spontaneous dissolution. Moreover, injection of the antiplatelet glycoprotein IIb/IIIa antibody 7E3 F(ab')(2) facilitated spontaneous dissolution of pulmonary ME and augmented fibrinolysis by a marginally effective dose of Retavase (10 microg/kg) in rats. These studies show that platelets suppress pulmonary fibrinolysis. The mechanism(s) by which platelets stabilize ME and utility of platelet inhibitors to facilitate their dissolution deserves further study.