RESUMO
Exposing blood to an artificial surface results in a systemic inflammatory response, including cytokine release and complement activation. We studied the artificial surface-induced inflammation in human whole blood using an extensive panel of inflammatory mediators including proinflammatory cytokines, chemokines and growth-factors and investigated the role of the complement system in the induction of this response. Using multiplex technology, 27 different inflammatory mediators were measured after circulating blood for 4 hours in polyvinyl chloride tubing. The C3 inhibitor compstatin was used to block complement activation. A significant (p < 0.05) increase in 14 of the 27 mediators was induced by the surface, of which 7 were chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, RANTES, eotaxin and IP-10) and 5 were growth-factors (G-CSF, GM-CSF, VEGF, PDGF and FGF). The traditional proinflammatory cytokines like IL-1beta, TNFalpha and IL-6 were not induced, although IL-6, as well as IL-15 and IL-17 increased if the surface was coated with highly bioincompatible laminaran. Inhibition of complement activation with compstatin significantly (p < 0.05) reduced the formation of 12 of the 14 mediators. For 10 of the 12 mediators, the inhibition was by 2/3 or more, for the remaining two the inhibition was more moderate. A highly biocompatible heparin-coated PVC surface was used as negative control and completely abolished the whole inflammatory response. The artificial surface PVC markedly induced a broad spectrum of chemokines and growth-factors, which was largely dependent on activation of complement.
Assuntos
Quimiocinas/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Inflamação/induzido quimicamente , Peptídeos Cíclicos/farmacologia , Cloreto de Polivinila/farmacologia , Anticoagulantes/farmacologia , Quimiocinas/sangue , Complemento C3/antagonistas & inibidores , Complemento C3/efeitos dos fármacos , Citocinas/sangue , Glucanos , Heparina/farmacologia , Humanos , Polissacarídeos/farmacologiaRESUMO
Platelet compatibility after coating an artificial material with functionally active heparin was investigated. Blood was circulated in uncoated or heparin coated PVC tubing. In one hour platelet counts decreased from 155 (113-184)x10(9)/l to 124 (100-148)x10(9)/l with uncoated compared to 164 (132-192)x10(9)/l with heparin coated tubing (intergroup p = 0.02). Beta-thromboglobulin increased from 116 (80-148) microg/l to 1039 (757-1298) microg/l with uncoated and to 352 (229-638) microg/l with heparin coated tubing (intergroup p = 0.005). Platelet counts and beta-thromboglobulin correlated closely with complement activation. Solid-phase enzyme immunoassay demonstrated substantial deposition of CD42a/GPIbIX and CD61/GPIIIa on uncoated, but not on heparin coated tubing (intergroup p<0.0005). Scanning electron microscopy demonstrated activated platelets and aggregates on uncoated in contrast to heparin coated tubing, where scattered, unactivated platelets were found. Changes in P-selectin and microparticles were minor. In conclusion, this heparin surface substantially improved platelet compatibility. Markers of choice for in vitro evaluation were platelet counts, beta-thromboglobulin and platelet deposition.
Assuntos
Materiais Biocompatíveis , Plaquetas/fisiologia , Heparina , Antígenos CD/sangue , Biomarcadores/sangue , Plaquetas/imunologia , Plaquetas/ultraestrutura , Complemento C3/metabolismo , Feminino , Humanos , Técnicas In Vitro , Masculino , Teste de Materiais , Microscopia Eletrônica de Varredura , Ativação Plaquetária/fisiologia , Contagem de Plaquetas , Cloreto de Polivinila , Propriedades de Superfície , beta-Tromboglobulina/metabolismoRESUMO
The aim of the present investigation was to clarify whether immobilized heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound alpha-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound alpha-FXIIa was slightly slower than with purified components. Nevertheless, neither beta-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound alpha-FXIIa within 1 second. In the liquid phase, beta-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the beta-FXIIa inhibition rate and prevent FXI activation in plasma, although beta-FXIIa and FXIa-AT complex formation occurred. It is concluded that surface-immobilized heparin, unlike heparin in solution, effectively inhibits the initial contact activation enzymes by an antithrombin-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway.
Assuntos
Anticoagulantes/uso terapêutico , Antitrombina III/farmacologia , Proteínas Inativadoras do Complemento 1/farmacologia , Fator XII/efeitos dos fármacos , Fator XIIa/efeitos dos fármacos , Heparina/uso terapêutico , Adsorção , Humanos , Valores de ReferênciaRESUMO
This is an in vitro study of the effects of doxorubicin on heparin immobilized on polyvinyl chloride (PVC) tubing. Doxorubicin contains an amino group that binds up to 16 heparin molecules, forming insoluble complexes if they are added to the same infusion. Three systems were tested: doxorubicin in perfusing blood, cerebrospinal fluid, and 0.9% sodium chloride (NaCl). The antithrombogenicity of immobilized heparin is impaired on exposure to doxorubicin. However, the reaction is reversible provided the PVC tubing system is thoroughly washed. Heparinized tubing perfused for 12 hours in blood with doxorubicin (0.027 mg/mL) decreased the activity of the immobilized heparin to 6.0% compared with 43% of that exposed to blood only. Exposure to doxorubicin (0.27 mg/mL) for 15 minutes in NaCl decreased the activity to 3% compared with that of NaCl only. Continuous washing for 10 minutes (8 mL/min) resulted in regained activity. This indicated a reversible reaction between immobilized heparin and doxorubicin. Cyclophosphamide, netilmicin, and gentamicin did not affect the antithrombogenicity of heparin in any solution.
Assuntos
Materiais Biocompatíveis , Cateterismo Venoso Central/instrumentação , Doxorrubicina , Eritrócitos/fisiologia , Fibrinolíticos , Heparina , Cloreto de Polivinila , Líquido Cefalorraquidiano , Ciclofosfamida , Humanos , Microscopia Eletrônica de Varredura , Netilmicina , Perfusão , PlasmaRESUMO
End-point immobilization of heparin to artificial materials gives rise to a surface that prevents triggering of the plasma contact activation system and, presumably as a result thereof, generally has thrombo-resistant properties. The present investigation was undertaken to determine what density of immobilized heparin molecules expressing functionally intact antithrombin binding sites is required to achieve these blood compatible properties. Six different heparin surfaces were prepared on polyethylene tubing and studied in contact with human plasma. The content of bound heparin was the same on all surfaces while the densities of antithrombin binding sites ranged from 1 to 28 pmol/cm2. The surfaces expressing 4 pmol/cm2 or more of specific antithrombin binding sites generated no measurable enzymatic activity in contact with plasma, either on the exposed surfaces or in the plasma phases. Below this level, the degree of activation gradually increased with decreasing densities, and in parallel the thrombo-resistant properties deteriorated. Addition of heparin to the plasma phase reduced the capacity of the heparin surfaces to bind antithrombin, leading to a diminished ability of the surfaces to prevent contact activation. This finding supports the hypothesis that antithrombin is the critical coagulation inhibitor for the suppression of contact activation on end-point immobilized heparin.
Assuntos
Anticoagulantes , Antitrombina III , Materiais Biocompatíveis , Heparina , Plasma/fisiologia , Trombose/prevenção & controle , Anticoagulantes/farmacologia , Sítios de Ligação , Materiais Biocompatíveis/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Heparina/farmacologia , Humanos , Técnicas In Vitro , Teste de Materiais , Plasma/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
An in vitro technique was developed for assessment of the biocompatibility of materials for use in clinical applications. Artificial materials were exposed to blood, and the resulting complement activation was quantified both in the plasma and on the material surface by enzyme immunoassays based on monoclonal antibodies specific for neoepitopes exposed in complement activation products. Several materials were evaluated, and the effect of surface modifications, including end-point immobilized heparin, was studied. The results revealed widely varying complement activation properties of the different materials and confirmed that heparin markedly improves biocompatibility. The present method is superior to analysis limited to either the fluid phase or solid phase since certain materials adsorb activation products (exemplified by Tecoflex) whereas others do not although activation was evident from fluid-phase assay (silicone). Furthermore, direct determination of activation-specific neoepitopes on the surface represents an improvement compared with previously described methods for detection of adsorbed components.
Assuntos
Materiais Biocompatíveis/farmacologia , Ativação do Complemento/efeitos dos fármacos , Epitopos/sangue , Heparina/metabolismo , Membranas Artificiais , Complemento C3b/efeitos dos fármacos , Complemento C5a/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/efeitos dos fármacos , Heparina/farmacologia , Humanos , Látex/farmacologia , Modelos Biológicos , Poliuretanos/farmacologia , Cloreto de Polivinila/farmacologia , Silicones/farmacologia , Propriedades de SuperfícieRESUMO
The uptake and activation of FXII from blood plasma was studied in small-diameter polyethylene tubing, surface-modified by end-point immobilization of heparin. Two preparations of heparin were used to modify the contact-activating properties of the plastic tubing: unfractionated, functionally active heparin and low-affinity heparin, lacking the specific antithrombin-binding sequence and virtually devoid of anticoagulant activity. The uptakes of FXII on the two heparin surfaces were similar. No activated FXII could be demonstrated on the unfractionated heparin surface, whereas on the low-affinity heparin surface nearly all FXII underwent spontaneous activation. The suppression of FXII activation on the unfractionated heparin surface was investigated by using plasma depleted of antithrombin, complement C1 esterase inhibitor, or both. The removal of antithrombin resulted in extensive activation of FXII, whereas the depletion of C1 esterase inhibitor had only a minor effect. Experiments with recalcified plasma showed rapid clot formation during exposure to the low-affinity heparin surface. After depletion of antithrombin, but not complement C1 esterase inhibitor, the recalcified plasma clotted in contact with the unfractionated heparin surface as well. We conclude that antithrombin and the antithrombin-binding sequence in the surface-immobilized heparin are essential for the prevention of surface activation of FXII and triggering of the intrinsic coagulation system.
Assuntos
Antitrombinas/química , Heparina/química , Animais , Cálcio/sangue , Bovinos , Proteínas Inativadoras do Complemento 1/farmacologia , Fator XII/química , Fator Xa/química , Humanos , Ligação Proteica , Propriedades de Superfície , Trombina/química , Tempo de Coagulação do Sangue TotalRESUMO
Efforts to improve the blood compatibility of artificial materials have involved coating the surfaces with heparin. However, the anticoagulation activity of heparin is based on its binding to antithrombin, and if the specific structure involved in this interaction is compromised by the coating procedure, then the activity is lost. Surface modification with end-point-immobilized heparin has been found to be successful in inhibiting coagulation factors and minimizing complement activation.
Assuntos
Equipamentos e Provisões/normas , Heparina/farmacologia , Antitrombina III/metabolismo , Coagulação Sanguínea , Ativação do Complemento , Equipamentos e Provisões/efeitos adversos , Heparina/química , Heparina/metabolismo , Humanos , Teste de Materiais , Ligação Proteica , Propriedades de SuperfícieRESUMO
A well established technique to improve blood compatibility of artificial materials for use in the circulation is to coat the surface with heparin. The present report describes the antithrombin mediated inhibition of thrombin and factor Xa by surfaces modified with end point immobilized heparin. The reaction was followed by conventional chromogenic substrate based enzyme assays as well as by immunological measurement of the enzyme inhibitor (thrombin-antithrombin) complex formation. Both enzymes were rapidly inactivated by heparin surfaces after selective presaturation with antithrombin on the immobilized high affinity heparin molecules. The thrombin inhibitory capacity was enhanced when both high and low affinity heparin were preadsorbed with inhibitor. The main part of the thrombin-antithrombin complex formed remained bound to the surface, however, without functionally blocking the activity of the high affinity sequence of the immobilized heparin. Aliquots of recalcified plasma were slowly rotated in loops of heparinized tubing to investigate whether the main thromboresistant function of the surface was exerted at the level of thrombin or by inactivation of preceding enzymes. After 1 h no visible clotting occurred and only trace amounts of thrombin (0.07 IU/ml), measured as thrombin-antithrombin complexes, had been formed. In non-heparinized loops and in the test tube plasma clotted after 20 min. The thrombin generation when clotting occurred was in the order of 10 IU/ml. It is concluded that the immobilized heparin mediates inhibition of the coagulation cascade prior to prothrombin activation.
Assuntos
Antitrombinas/metabolismo , Materiais Biocompatíveis , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa , Heparina/farmacologia , Polietilenos , Trombina/antagonistas & inibidores , Adsorção , Antitrombina III/análise , Compostos Cromogênicos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Heparina/metabolismo , Humanos , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/análise , Ligação Proteica , Propriedades de Superfície , Trombina/metabolismo , Trombose/prevenção & controleRESUMO
Two different heparin surfaces, structurally closely related and of similar negative charge characteristics, were compared with regard to adsorption and activation of coagulation Factor XII (FXII). One surface was prepared by immobilization of unfractioned heparin, which yielded a surface containing both heparin molecules with high and with low affinity for antithrombin (unfractioned [UF] heparin surface). The other surface consisted of a fraction of heparin molecules with low affinity for antithrombin (LA heparin surface) and essentially devoid of antithrombin-binding as well as anticoagulant activity. Both surfaces adsorbed FXII from plasma to a similar extent, and essentially the same quantities of bound factor could be recovered from the surfaces. The two heparin surfaces, however, differed markedly with regard to activation of the adsorbed FXII. On the LA heparin surface, a major portion of the surface-bound FXII was recovered in its enzymatically active form (FXIIa), but only trace amounts of the FXII taken up by the UF heparin surface had undergone activation. When FXII-deficient plasma was used instead of normal plasma, no surface-associated enzyme activity could be recovered on either surface. The presence of free standard heparin or low molecular weight heparin in the plasma exposed to the LA heparin surface did not prevent conversion of FXII to FXIIa.
Assuntos
Fator XII/metabolismo , Heparina/farmacologia , Adsorção , Fator XII/efeitos dos fármacos , Fator XIIa/efeitos dos fármacos , Heparina de Baixo Peso Molecular/farmacologia , Humanos , Propriedades de SuperfícieRESUMO
Rheologic factors are likely to influence the balance between thrombotic and antithrombotic forces at the level of the vascular wall. In this study, the effects of flow-velocity/wall shear stress on the interaction of antithrombin (AT) with surface-immobilized heparin were investigated. The binding of AT to low-affinity and high-affinity heparin could be discriminated by measurements at physiological of elevated ionic strength. Under low shear stress conditions, substantial binding of AT to both high- and low-affinity heparin was observed, in relative quantities largely reflecting the proportion of these polysaccharide populations on the surface. With increasing shear stress, the binding to high-affinity sites was relatively constant, while total low-affinity binding decreased. Furthermore, under the highest shear stress (greater than 1,000 N/m2), the binding of AT to low-affinity heparin completely disappeared while binding to the high-affinity fraction persisted. These results were related to values obtained in a mathematical model, describing the theoretical maximum transport of AT from the liquid phase to the surface under the conditions used in the experimental system.
Assuntos
Antitrombinas/metabolismo , Heparina/farmacocinética , Animais , Sítios de Ligação , Velocidade do Fluxo Sanguíneo , Humanos , Técnicas In Vitro , Modelos Cardiovasculares , Reologia , Estresse Mecânico , Propriedades de SuperfícieRESUMO
The degree of biocompatibility of biomaterials can be evaluated using various assay systems detecting activation of the blood cascade systems, leukocytes or platelets. Activation of complement is one mechanism associated with adverse effects observed when bioincompatible materials are used. We present data showing that the terminal complement complex, an indicator of terminal pathway activation, is suitable for evaluation of biocompatibility of biomaterials such as cardiopulmonary bypass devices. Furthermore, our results suggest that bioincompatibility is improved when artificial surfaces are modified with end point attached functionally active heparin.
Assuntos
Ativação do Complemento , Heparina/administração & dosagem , Teste de Materiais , Oxigenadores , Complemento C3/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Sulfato de Dextrana/química , Glucanos , Heparina/química , Humanos , Técnicas In Vitro , Oxigenadores de Membrana , Polietilenos/química , Polissacarídeos/química , Cloreto de Polivinila/químicaRESUMO
A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.
Assuntos
Acetilglucosamina/análise , Glucosamina/análise , Heparina/análise , Polissacarídeos/análise , Sulfatos/análise , Colorimetria/métodos , Desaminação , Concentração de Íons de Hidrogênio , Cinética , Ácido NitrosoRESUMO
The antithrombin-binding region in heparin is a pentasaccharide sequence with the predominant structure GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-IdoA -(2-OSO3)-GlcNSO3(6-OSO3) (where GlcA and IdoA represent D-glucuronic and L-iduronic acid, respectively), in which the 3-O-sulfate residue on the internal glucosaminyl unit is a marker group for this particular region of the polysaccharide molecule. A heparin octasaccharide which contained the above pentasaccharide sequence was N/O-desulfated and re-N-sulfated and was then incubated with adenosine 3'-phosphate 5'-phospho[35S]sulfate in the presence of a microsomal fraction from mouse mastocytoma tissue. Fractionation of the resulting 35S-labeled octasaccharide on antithrombin-Sepharose yielded a high affinity fraction that accounted for approximately 2% of the total incorporated label. Structural analysis of this fraction indicated that the internal glucosamine unit of the pentasaccharide sequence was 3-O-35S-sulfated, whereas both adjacent glucosamine units carried 6-O-[35S]sulfate groups. In contrast, the fractions with low affinity for antithrombin (approximately 98% of incorporated 35S) showed no consistent O-35S sulfation pattern and essentially lacked glucosaminyl 3-O-[35S]sulfate groups. It is suggested that the 3-O-sulfation reaction concludes the formation of the antithrombin-binding region. This proposal was corroborated in a similar experiment using a synthetic pentasaccharide with the structure GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-Id oA (2-OSO3)-GlcNSO3(6-OSO3) as sulfate acceptor. This molecule corresponds to a functional antithrombin-binding region but for the lack of a 3-O-sulfate group at the internal glucosamine unit. The 35S-labeled pentasaccharide recovered after incubation bound with high affinity to antithrombin-Sepharose and contained a 3-O-[35S]sulfate group at the internal glucosamine residue as the only detectable labeled component. The use of this pentasaccharide substrate along with the affinity matrix provides a highly specific assay for the 3-O-sulfotransferase.
Assuntos
Antitrombinas/metabolismo , Heparina/biossíntese , Sulfatos/metabolismo , Animais , Sítios de Ligação , Configuração de Carboidratos , Linhagem Celular , Sarcoma de Mastócitos/enzimologia , Camundongos , Microssomos/enzimologia , Oligossacarídeos/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Sulfurtransferases/metabolismoRESUMO
The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.
Assuntos
Heparina/biossíntese , Polissacarídeos/metabolismo , Animais , Sistema Livre de Células , Fenômenos Químicos , Química , Cromatografia em Gel , Glucuronatos/metabolismo , Ácido Glucurônico , Substâncias Macromoleculares , Microssomos/metabolismo , Nucleotídeos/farmacologia , Octoxinol , Polietilenoglicóis/farmacologia , Trítio , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
The mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depolymerized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 and 11 picomoles/cm2, respectively. Thus the estimated density of biologically active high and low affinity heparin respectively would be 40 and 90 ng/cm2. The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. It was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromised AT binding sequences.
Assuntos
Antitrombina III/metabolismo , Heparina/metabolismo , Inibidores de Serina Proteinase , Animais , Fator Xa , Humanos , Técnicas In Vitro , Teste de Materiais , Propriedades de Superfície , Trombose/prevenção & controleRESUMO
Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.
Assuntos
Butiratos/farmacologia , Heparina/biossíntese , Sarcoma de Mastócitos/metabolismo , Amidoidrolases/metabolismo , Animais , Ácido Butírico , Linhagem Celular , Sistema Livre de Células , Heparina/isolamento & purificação , Camundongos , Camundongos Endogâmicos , Microssomos/enzimologia , Radioisótopos de EnxofreRESUMO
Oligosaccharides with different affinities for antithrombin were isolated following partial deaminative cleavage of pig mucosal heparin with nitrous acid. The smallest high-affinity component obtained was previously identified as an octasaccharide with the predominant structure: (Formula: see text). The interaction of this octasaccharide, and of deca- and dodecasaccharides containing the same octasaccharide sequence, with antithrombin was studied by spectroscopic techniques. The near-ultraviolet difference spectra, circular dichroism spectra, and fluorescence enhancements induced by adding these oligosaccharides to antithrombin differed only slightly from the corresponding parameters measured in the presence of undegraded high-affinity heparin. Moreover, the binding constants obtained for the oligosaccharides and for high-affinity heparin were similar (1.0-2.9 X 10(7) M-1 at I = 0.3). In contrast, two hexasaccharides corresponding to units 1-6 and 3-8, respectively, of the above sequence showed about a 1000-fold lower affinity for antithrombin, and also induced considerably different spectral perturbations in antithrombin. Since the 1-6 hexasaccharide contains a reducing-terminal anhydromannose residue instead of the N-sulfated glucosamine unit 6 of the intact sequence, these results strongly support our previous conclusion that the N-sulfate group at position 6 is essential to the interaction with antithrombin. The low affinity of the hexasaccharide 3-8 provides further evidence that a pentasaccharide sequence 2-6 constitutes the actual antithrombin-binding region in the heparin molecule. Structural analysis of the various oligosaccharides revealed natural variants with an N-sulfate group substituted for the N-acetyl group at position 2. The preponderance of N-acetyl over N-sulfate groups at this position may be rationalized in terms of the mechanism of heparin biosynthesis, assuming that the D-gluco configuration of unit 3 is an essential feature of the antithrombin-binding region.
Assuntos
Antitrombinas , Heparina , Animais , Boroidretos , Sequência de Carboidratos , Fenômenos Químicos , Química , Heparina/isolamento & purificação , Mucosa Intestinal , Oligossacarídeos/isolamento & purificação , Relação Estrutura-Atividade , SuínosRESUMO
A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Km of 74 mg polysaccharide/liter was determined, which corresponds to 2.1 X 10(-4) M, expressed as concentration of uronic acid; Vmax was 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3'-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mM), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mM. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry.