RESUMEN
Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the interaction of heparin, vitronectin, and the complement system. Previous studies demonstrated that heparin diminishes vitronectin inhibition of complement activity. Numerous studies have also demonstrated that heparin exerts a net inhibitory effect on complement. We used two dimensional affinity resolution electrophoresis (2DARE) to examine this apparent paradox. 2DARE allowed simultaneous determination of binding affinity of heparin for vitronectin as well as the M(r) of the heparin species. In the 2DARE experiment, the interaction of heparin with vitronectin caused retardation of the movement of the heparin through the tube gel in the first dimension. The degree of the retardation of movement was used to calculate the approximate K(d) of that interaction. The heparin from the tube gel was then subjected to a second dimension electrophoresis to determine the M(r) of the heparin. 2DARE analysis of the interaction of heparin with vitronectin clearly demonstrated that a sub-population of heparin chains with M(r) > 8000 bound vitronectin with high affinity whereas most high M(r) chains and all lower M(r) chains showed little to no affinity for vitronectin. Our findings are consistent with the hypothesis that a unique binding domain exists in certain heparin chains for vitronectin.
Asunto(s)
Heparina/metabolismo , Vitronectina/metabolismo , Electroforesis en Gel Bidimensional , Humanos , Peso Molecular , Polímeros/metabolismoRESUMEN
We present a 1,600 g infant who underwent successful balloon aortic valvuloplasty from the right carotid artery approach. A simple technique to facilitate access to the left ventricle and expedite the procedure is described. Issues unique to performing balloon aortic valvuloplasty on such a small child are discussed.
Asunto(s)
Estenosis de la Válvula Aórtica/terapia , Cateterismo/métodos , Enfermedades del Prematuro/terapia , Recien Nacido Prematuro , Humanos , Recién Nacido de Bajo Peso , Recién NacidoRESUMEN
Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by interfering with C1 and may exert this activity in vivo.
Asunto(s)
Proteínas Sanguíneas/farmacología , Vía Clásica del Complemento/efectos de los fármacos , Neurotoxinas/farmacología , Peroxidasas/farmacología , Ribonucleasas , Complemento C3/efectos de los fármacos , Complemento C4/efectos de los fármacos , Proteínas en los Gránulos del Eosinófilo , Peroxidasa del Eosinófilo , Neurotoxina Derivada del Eosinófilo , HumanosRESUMEN
Heparin is a polydisperse, highly sulfated polysaccharide consisting of repeating 1-->4 linked uronic acid and glucosamine sugar residues that binds to coagulation proteins, complement proteins, and growth factors to regulate a variety of biological activities. Heparin is best known as an anticoagulant, an activity that results largely from a specific pentasaccharide sequence in heparin that interacts with a unique site in antithrombin III. Little is known about additional structures within heparin that might interact with antithrombin III or the heparin structures that interact with the myriad of other heparin-binding proteins and peptides. Unfractionated glycosaminoglycan heparin that had been prepared from porcine intestinal mucosa was examined for its capacity to bind antithrombin III using a new technique developed to quantitate that interaction. Two-dimensional affinity resolution electrophoresis is a powerful method that allows assessment of unique species of heparin molecules that bind to protein, allowing determination of heparin molecular weight for each protein-binding heparin species as well as the dissociation constant of each interaction. This study provides the first definitive evidence that glycosaminoglycan heparin contains at least three populations of molecules with affinity for antithrombin III. Furthermore, the affinity of each heparin species for antithrombin III appears to vary inversely with the size of the heparin chain, with some smaller oligosaccharides having greater affinity for antithrombin III than larger oligosaccharides.
Asunto(s)
Antitrombina III/metabolismo , Heparina/aislamiento & purificación , Heparina/metabolismo , Animales , Sitios de Unión , Electroforesis en Gel Bidimensional/métodos , Glicosaminoglicanos/química , Heparina/química , Técnicas In Vitro , Mucosa Intestinal/química , Estructura Molecular , PorcinosRESUMEN
Heparin has been shown to inhibit activity of the alternative, classical and terminal pathways of complement by regulating C1, C1 inhibitor, C4 binding protein, C3b, factor H and S-protein. In vivo, heparin inhibits cobra venom factor activation of complement in a dose-related manner in guinea pigs. However, the ability of heparin and of modified heparin to inhibit complement activation in serum has not been examined systematically. The present study compared commercial heparin with a modified heparin that has reduced anticoagulant activity (N-desulfated, N-acetylated heparin) for ability to inhibit cobra venom factor and zymosan-induced complement activation in guinea pig and human serum. Both heparins inhibited cobra venom factor and zymosan-induced consumption of C3 activity in both human and guinea pig serum. In both serum types, commercial heparin was about twice as active as modified heparin on a weight basis for ability to inhibit cobra venom factor-induced complement activation. Both heparins also inhibited zymosan-induced complement activation in human serum. About four times more heparin was required to inhibit cobra venom factor-induced complement activation in guinea pig serum than in human serum while heparin was more than ten times more active in human serum than in guinea pig serum when zymosan was used as the activator of complement. This study suggests that heparin is considerably more effective in regulating complement activity in humans than in guinea pigs, an animal model in which heparin clearly has in vivo capacity to regulate complement activity. These observations represent an important step in the development of new clinically relevant oligosaccharide-derived pharmacologic agents to regulate complement activity.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Venenos Elapídicos/antagonistas & inhibidores , Heparina/farmacología , Zimosan/antagonistas & inhibidores , Animales , Complemento C3/metabolismo , Cobayas , Humanos , Técnicas In VitroRESUMEN
1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared. 2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting. 3. Individual antibodies showed different reactivity toward the three heparin lyases. 4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate. 5. The antibodies can be used to rapidly distinguish between the three heparin lyases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Inmunoglobulina G/inmunología , Polisacárido Liasas/inmunología , Animales , Western Blotting , Flavobacterium/enzimología , Liasa de Heparina , Inmunoensayo , Ratones , Dodecil Sulfato de Sodio/farmacologíaRESUMEN
The M(r) values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of M(r) determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the M(r) values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average M(r), weight-average M(r), and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the M(r) values of heparin and LMW heparin.
Asunto(s)
Heparina de Bajo-Peso-Molecular/química , Heparina/química , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida , Heparina de Bajo-Peso-Molecular/análogos & derivados , Datos de Secuencia Molecular , Peso Molecular , Sensibilidad y EspecificidadRESUMEN
Eosinophil granules contain several cationic proteins that mediate tissue damage in allergic disease. The present study examined the capacity and mechanisms by which these cationic proteins regulate activity of the alternative pathway of C. Eosinophil peroxidase and eosinophil cationic protein inhibited formation of cell-bound alternative pathway C3 convertase, causing 50% inhibition of lysis at about 0.19 and 0.75 microgram/10(7) cellular intermediates, respectively. Major basic protein inhibited alternative pathway C3 activity by only 19% at 1.5 micrograms/10(7) cellular intermediates. Eosinophil-derived neurotoxin had no activity on the alternative pathway. The eosinophil granule proteins were examined for the mechanism by which they inhibited alternative pathway activity. Eosinophil peroxidase and major basic protein inhibited fluid phase factor B consumption in a reaction mixture that also contained factors D and C3b, eosinophil-derived neurotoxin had no activity on factor B consumption, and eosinophil cationic protein consumed factor B in the absence of C3b and factor D. Both eosinophil cationic protein and eosinophil peroxidase enhanced the decay of preformed alternative pathway convertase. Lysis of EAC4b,3b cellular intermediates formed to contain a low surface amount of C3b was more inhibited than was lysis of cells formed with a standard amount of C3b on the surface. This suggests that these eosinophil proteins acted predominantly on C3b to regulate alternative pathway activity. We also found that none of the eosinophil granule cationic proteins had any effect on later events after the formation of the C3 convertase. We conclude that although eosinophil-derived neurotoxin (isoelectric pH value (pI) = 8.9) does not regulate alternative pathway activity, the more highly charged eosinophil granule cationic proteins--major basic protein (pI = 10.9), eosinophil cationic protein (pI = 10.8), and eosinophil peroxidase (pI = 10.8)--do share the capacity to regulate C activity and may exert this activity in vivo.
Asunto(s)
Proteínas Sanguíneas/farmacología , Vía Alternativa del Complemento/efectos de los fármacos , Eosinófilos/química , Ribonucleasas , Factor B del Complemento/farmacología , Factor D del Complemento/farmacología , Relación Dosis-Respuesta a Droga , Proteínas en los Gránulos del Eosinófilo , Humanos , Técnicas In Vitro , Peroxidasas/farmacologíaRESUMEN
Heparin regulates C activity in vitro, but has not been examined for this activity in vivo. The present study investigated the ability of commercial heparin and derivatized (N-desulfated, N-acetylated) heparin (Hep-NAc) with greatly diminished anticoagulant activity to inhibit C activation in guinea pigs. Catheters were placed in the right atrium of guinea pigs and kept patent with frequent saline flushes. The next day, heparin, Hep-NAc, or saline was given and 2.5 min later cobra venom factor or saline was given. Blood was drawn at intervals and assayed for total hemolytic C, C3 hemolytic activity, free hemoglobin, and activated partial thromboplastin time. Total hemolytic C and C3 activity decreased less rapidly in heparin- and Hep-NAc-pretreated animals than in non-pretreated animals, indicating that both heparins inhibited C activation. Heparin and Hep-NAc also inhibited cobra venom factor-induced hemolysis. This study demonstrates that commercial heparin and modified heparin inhibit C activation in vivo. This represents an important step in the development of an oligosaccharide drug to regulate C activation.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Heparina/farmacología , Animales , Complemento C3/metabolismo , Ensayo de Actividad Hemolítica de Complemento , Venenos Elapídicos/farmacología , Cobayas , Masculino , Tiempo de Tromboplastina ParcialRESUMEN
We compared eight commercially available, pre-activated affinity chromatography supports for ability to immobilize C3b that would retain functional activity. Pre-activated supports that we studied were: cyanogen bromide activated agarose, N-hydroxysuccinimide activated agarose, Reacti-Gel HW-65, Actigel A aldehyde activated agarose, thiopropyl activated agarose, 1,4-bis(2,3-epoxypropoxy) butane activated agarose, Reacti-Gel GF-2000 and tresyl activated agarose. The amount of C3b immobilized by each support varied from 81% for Actigel A aldehyde activated agarose to only 19% for Reacti-Gel GF-2000. We examined the functional capacity of the C3b immobilized on these various supports to participate in the alternative pathway. Immobilized C3b was mixed with factors D and B of the alternative pathway and examined over time for ability to consume factor B hemolytic activity. C3b immobilized on thiopropyl activated agarose consumed factor B at a rate comparable to unbound fluid phase C3b. C3b immobilized on other supports was less active in participating in factor B consumption. Thus, we have demonstrated the ability to immobilize C3b onto a solid matrix with the immobilized C3b retaining the ability to participate in the alternative pathway. This immobilized C3b can be used to fractionate substances with high C3b binding affinity.