RESUMEN
Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A (EDA) containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep) in the blood of rheumatoid arthritis (RA) patients. In cryogelation EDA(+)FN cross-links to form an interaggregate of cryogel with Hep. In the present study, we determined the recognition structure of Hep for EDA(+)FN by using oligo- and desulfonated-Hep. The affinity constant (KA) (1.2 x 10(8) per M) of oligo-Hep for EDA(+)FN did not change with a decrease in number-average molecular weight (4.9 x 10(4)-->6.0 x 10(3)). The KA-value of desulfonated-Hep for EDA(+)FN decreased from 3.2 x 10(8) to 1.0 x 10(7) per M with a decrease in the sulfonation ratio (7.0-->4.3%). We also determined the recognition structure of EDA(+)FN for Hep by an inhibition experiment on the heparin binding domain II (HepII) in EDA(+)FN with the synthetic peptides, Arg-Arg-Ala-Arg (RRAR), Asp-Gln-Ala-Arg (DNAR), Ile-Lys-Tyr-Glu-Lys (IKYEK), and Gly-Arg-Lys-Lys-Try (GRKKT). The GRKKT sequence clearly inhibited bonding between EDA(+)FN and Heps containing oligo- and desulfonated-Hep. The amount of cryogel formed in the RA-patient model plasma corresponded to the EDA(+)FN concentration in cryogel (36.7%) normalized by the EDA(+)FN concentration in plasma. When GRKKT was added to plasma, the EDA(+)FN concentration fell to 10.5%. These results demonstrated that inhibition of cryogelation in plasma could progress to a novel treatment for RA.
Asunto(s)
Fibronectinas/química , Heparina/química , Secuencia de Aminoácidos , Arginina/química , Artritis Reumatoide/metabolismo , Proteínas Sanguíneas/química , Secuencia de Carbohidratos , Criogeles , Congelación , Geles/química , Glicina/química , Hidrogeles , Concentración de Iones de Hidrógeno , Cinética , Lisina/química , Modelos Estadísticos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Treonina/química , Factores de TiempoRESUMEN
Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin, exhibit abnormally high concentrations of extra domain A containing fibronectin [EDA(+)FN] in their plasma. The selective removal of EDA(+)FN from patient blood is therefore of potential therapeutic benefit. Gellan-sulfate is a candidate ligand for the removal of EDA(+)FN due to its high affinity for FN. In this study, we prepare a novel adsorber for the direct removal of EDA(+)FN from patient blood. The adsorber has both a plasma separation function and EDA(+)FN trapping zones, and is prepared by cross-linking gellan-sulfate with epichlorohydrine. The ratio of gellan-sulfate to gellan in the adsorber is 48%. The surface and internal structure of gellan beads were observed by a range of microscopic techniques, and the beads were found to have a dilayer structure, consisting of a porous outer layer and an underlying gellan-sulfate phase as the adsorber. The affinity constants of the gellan-sulfate beads for EDA(+)FN were almost the same in blood as in buffer because the porous gellan coating acts to separate plasma from the cellular fraction of the blood. The removal rate of plasma proteins and blood cells from mock RA blood was measured for coated and uncoated gellan-sulfate beads. Removal rates were 30-32% for EDA(+)FN, 6-10% for fibrinogen, 10-14% for antithrombin III, 8% for C3, 4-7% for C4, and 0% for albumin. The removal rates of uncoated beads were 11% for white blood cells, 0% for red blood cells and 33% for platelets, whereas removal rates of 0% for white blood cells, 0% for red blood cells and 20% for platelets were achieved for coated beads. The coating effectively inhibits the adsorption of white blood cells and platelets. Existing problems with direct adsorbers, including selectivity and plasma separation, have been solved by this material.
Asunto(s)
Artritis Reumatoide/sangre , Eliminación de Componentes Sanguíneos/métodos , Fibronectinas/sangre , Adsorción , Humanos , Polisacáridos/química , Polisacáridos Bacterianos/química , Estructura Terciaria de Proteína , Ésteres del Ácido Sulfúrico/químicaRESUMEN
Cryogel is a physical gel formed by heterophilic aggregation of extra domain A containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep), which are found in high concentrations in the blood of patients suffering from rheumatoid arthritis. In this study, we clarify the specific interactions between cryogel components in terms of the affinity constant (K(A)), obtained by surface plasmon resonance (SPR). It is found that Fbg self-interactions occur at lower temperatures, and that K(A) of Fbg-Hep changes with temperature. Specifically, K(A) (2.0 x 10(8) [M(-1)]) of Fbg-Hep at 5 degrees C increases significantly from that (1.0x10(7) [M(-1)]) at 40 degrees C. K(A) of EDA(+)FN-Hep increases with temperature, by approximately 100-fold between 40 degrees C (K(A)=10(12) [M(-1)]) and 20 degrees C (K(A)=10(10) [M(-1)]). Although K(A) of the FN fragments of Hep-binding domain containing an EDA region [EDA(+)HBD(+)] and Hep increases with temperatures above 30 degrees C, K(A)s of HBD(+)-Hep and EDA(+)-Hep are not temperature-dependent. Therefore, EDA(+)HBD(+), formed as a special structure for high Hep affinity, exhibits temperature-dependent interaction with Hep. These results suggest that the main role of EDA(+)FN in cryogelation is to support the interaction with Hep.
Asunto(s)
Proteínas Sanguíneas/química , Fibronectinas/química , Geles/química , Plasma/química , Sitios de Unión , Criogeles , Fibronectinas/metabolismo , Heparina/química , Humanos , Hidrogeles , Nefelometría y Turbidimetría , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
A new concept for cell-hybrid biomaterial is proposed in which human unbilical vein endothelial cells (HUVEC) are adhered to an immobilized gellan sulfate (GS) surface. Extra domain A containing fibronectin (EDA(+)FN) released from HUVEC is necessary for cell adhesion and multiplication. The material design in this study is based on these self-released cell adhesion proteins. The interaction between GS and EDA(+)FN was evaluated using the affinity constant (KA); the value obtained was 1.03x10(8) (M(-1)). These results suggest that the adhesion of HUVEC to GS may be supported by the adhesion of EDA(+)FN to GS. We also found that this new material adheres to HUVEC, allowing the reintroduction of EDA(+)FN, which is self-produced by the cell. This material is relatively easy to produce, not requiring the usual coating of adhesion proteins in pretreatment.
Asunto(s)
Fibronectinas/metabolismo , Polisacáridos Bacterianos/metabolismo , Sulfatos/metabolismo , Secuencia de Carbohidratos , Adhesión Celular/fisiología , División Celular , Células Cultivadas , Endotelio Vascular/citología , Humanos , Polisacáridos/química , Polisacáridos/metabolismo , Polisacáridos Bacterianos/química , Sulfatos/químicaRESUMEN
In the development of cell-hybrid biomaterials, the functional activity of cells depends on the selective binding of cells to artificial ligands on the biomaterials. The extracellular matrix (ECM) is the most important ligand for cell activity. ECM is known to contain collagen, one of whose constituents is gelatin. Although natural gelatin has good cell attachment properties, the melting point of gelatin hydrogel is lower than body temperature. Thus, non-chemically cross-linked gelatin hydrogel is not a biomaterial that is used for prostheses. In the present study, we report the preparation of acyl-gelatin hydrogels with high melting point (>37 degrees C) and high affinity for hydrophobic surfaces for easy handling for transportation and adhesion activities on the hydrophobic surfaces. In addition, the doubling time of endothelial cells on the coated cell culture plate was faster than that of natural gelatin owing to the higher adhesion activity of acyl-gelatin. The results clearly demonstrated that the acyl-gelatin acted as an interface that enabled cell adhesion to artificial materials surfaces.