Scaled interfacial activity of proteins at a hydrophobic solid/aqueous-buffer interface

Contact-angle goniometry confirms that interfacial energetics of protein adsorption to the hydrophobic solid/aqueous-buffer (solid-liquid, SL) surface is not fundamentally different than adsorption to the aqueous-buffer/air (liquid-vapor, LV) interface measured by pendant-drop tensiometry. Adsorption isotherms of 9 globular blood proteins with molecular weight (MW) spanning from 10 to 1000 kDa on methyl-terminated self-assembled monolayer surfaces demonstrate that (i) proteins are weak surfactants, reducing contact angles by no more than about 15 degrees at maximum solution concentrations ( approximately 10 mg/mL); (ii) the corresponding dynamic range of spreading pressure Pi(a) < 20 mN/m; and (iii) the maximum spreading pressure Pi(max) (a) for these diverse proteins falls within a relatively narrow 5 mN/m band. As with adsorption to the LV interface, we find that concentration scaling substantially alters perception of protein interfacial activity measured by Pi(a). Proteins appear more similar than dissimilar on a weight/volume basis whereas molarity scaling reveals a systematic ordering by MW, suggesting that adsorption is substantially driven by solution concentration rather than diversity in protein amphilicity. Scaling as a ratio-to-physiological-concentration demonstrates that certain proteins exhibit Pi(max)(a) at-and-well-below physiological concentration whereas others require substantially higher solution concentration to attain Pi(max)(a). Important among this latter category of proteins is blood factor XII, assumed by the classical biochemical mechanism of plasma coagulation to be highly surface active, even in the presence of overwhelming concentrations of other blood constituents such as albumin and immunoglobulin that are shown by this work to be among the class of highly surface-active proteins at physiologic concentration. The overarching interpretation of this work is that water plays a dominant, controlling role in the adsorption of globular-blood proteins to hydrophobic surfaces and that energetics of hydration control the amount of protein adsorbed to poorly water-wettable biomaterials.     

Identification of plasma proteins adsorbed to hemodialyzers during clinical use

An investigation has been made of the protein layers formed on hemodialysis membranes during clinical use. Dialyzers having membranes of polymethylmethacrylate (PMMA), Cuprophane, cellulose acetate (CA), and saponified cellulose ester (SCE) were examined. Immediately following dialysis the dialyzers were washed free of blood and the membranes eluted with 2% SDS. The eluates were examined by SDS-PAGE followed by protein immunoblotting. Antisera to 16 common plasma proteins were used to probe for the presence of these proteins in the eluates. Most of the proteins tested for were found in the different eluates, suggesting that the protein layers are extremely complex. The protein compositions were qualitatively different on the different membranes. Except for HMWK the contact phase clotting factors were present in very small amounts and were largely activated. The clear presence of HMWK and the relatively small amounts of fibrinogen provide support for the occurrence of the Vroman effect. Fibrinogen was found to be degraded and this may be related to the observation that plasminogen was activated to plasmin. Complement C3 was an abundant component of all eluates. It was degraded to small fragments in a way which could not be related to complement activation. Many of the other proteins, particularly those of high molecular weight, were extensively degraded. It is speculated that this heretofore unremarked phenomenon may be due to the action of enzymes released by cell damage.     

Identification of proteins adsorbed from human plasma to glass bead columns: plasmin-induced degradation of adsorbed fibrinogen

In a bead column experiment, attempts have been made to identify the proteins adsorbed from plasma onto a glass surface. Proteins adsorbed after a 3-h contact time were eluted sequentially by 1 M tris buffer and SDS. Polyacrylamide gel electrophoresis of eluted proteins showed a multiplicity of components, and not all of these could be identified. Positive identifications were made by immunodiffusion against specific antibodies, band positions on electrophoresis gels, and location of radioactivity in gels when specific radiolabeled proteins were added to plasma. Proteins found were albumin, IgG, fibrinogen, plasminogen, and fibrinogen degradation products (FDP). A major component with an apparent molecular weight of 25,000 remains unidentified. It is unrelated to albumin, IgG, fibrinogen, factor XII, or plasminogen. Adsorbed fibrinogen was less degraded when experiments were performed with plasmas deficient in either plasminogen or factor XII. It is therefore concluded that FDP are formed by activation of adsorbed plasminogen, as was found previously for purified fibrinogen containing a trace of plasminogen. At least part of this activation is potentiated by the contact activation phase of plasma coagulation, in particular activated factor XII.     

The measurement of blood density to investigate protein deposition at the blood/hollow fiber membrane interface during ultrafiltration

Concentration polarization and secondary membrane formation change the membrane hydraulic permeability of capillary filters during hemofiltration reducing initial value filtration rates up to 50%. This leads to a significant loss of filter efficiency which must be taken into consideration when designing filters for long-term application such as in implantable artificial kidneys. By measuring blood density uninterrupted over a period of time using the mechanical oscillatory technique it is possible to follow the dynamics of protein deposition at the interface between the blood and capillary walls. The resulting picture of the deposition behaviour can lead to a better understanding of time-dependent filtration where flow and pressure conditions change. Protein deposition (Pt) with polysulfone membranes in relation to the effective capillary surface was in the range of 2.2. mg/(h x cm2) according to a logarithmic function (Pt = a + b x log(t)).     

Analysis of proteins adsorbed to glass from human plasma using immunoblotting methods

Glass beads were contacted with heparinized human plasma and the adsorbed proteins eluted sequentially with 1 M tris buffer, pH 7.4, and 2% SDS. The proteins in the eluates were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting procedures using antisera to 16 common plasma proteins. All sixteen proteins were found to be present in the eluates. The immunoblots provided evidence of contact phase activation although the role of the surface could not be ascertained since some contact activation was apparent in the starting plasma. Evidence was also obtained for the occurrence of complement activation and the fibrinogen Vroman effect on the glass bead surface. Extensive degradation of high molecular weight proteins observed previously in eluates from clinically used hemodialyzers was not observed in the present in vitro plasma experiments, thus supporting the hypothesis that such degradation is due to enzymes liberated in the in vivo experiments by blood cell damage.     

Influence of blood rheology on intrarenal blood flow distribution.

This study examined the effects of hematocrit and colloid-induced changes in blood rheology on renal blood flow (RBF) distribution. Blood viscosity, measured by coneplate viscometry, was raised 65% in anesthetized dogs either by increasing hematocrit or giving isoncotic dextran 500. Blood volume was kept constant and arterial pressure was unchanged. An increase in hematocrit caused either no change in RBF or a slight increase at each cortical level (zones 1-4). However, dextran hyperviscosity caused decreases in all four zones, the most pronounced in zone 1 (8.03-5.19 ml min(-1) g(-1)) and the least in zone 4 (1.63-1.46 ml min(-1) g(-1)). These data suggest either that increased hematocrit has less effect on "apparent viscosity" of blood within the kidney than does plasma colloid or that increased hematocrit causes renal vasodilatation while colloid hyperviscosity does not cause it. Since GFR remained constant in both types of hyperviscosity, it is possible that GFR is the autoregulated variable and the observed changes in RBF distribution and vascular resistance resulted from the changes in afferent and efferent arteriolar resistance required to preserve GFR.     

Analysis of proteins adsorbed to glass from human plasma using immunoblotting methods

Glass beads were contacted with heparinized human plasma and the adsorbed proteins eluted sequentially with 1 M tris buffer, pH 7.4, and 2% SDS. The proteins in the eluates were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting procedures using antisera to 16 common plasma proteins. All sixteen proteins were found to be present in the eluates. The immunoblots provided evidence of contact phase activation although the role of the surface could not be ascertained since some contact activation was apparent in the starting plasma. Evidence was also obtained for the occurrence of complement activation and the fibrinogen Vroman effect on the glass bead surface. Extensive degradation of high molecular weight proteins observed previously in eluates from clinically used hemodialyzers [3] was not observed in the present in vitro plasma experiments, thus supporting the hypothesis that such degradation is due to enzymes liberated in the in vivo experiments by blood cell damage.     

The effects of surface chemistry and adsorbed proteins on monocyte/macrophage adhesion to chemically modified polystyrene surfaces

Monocytes and macrophages play critical roles in inflammatory responses to implanted biomaterials. Monocyte adhesion may lead to macrophage activation and the foreign body response. We report that surface chemistry, preadsorbed proteins, and adhesion time all play important roles during monocyte adhesion in vitro. The surface chemistry of tissue culture polystyrene (TCPS), polystyrene, Primaria, and ultra low attachment (ULA) used for adhesion studies was characterized by electron spectroscopy for chemical analysis. Fibrinogen adsorption measured by (125)I-labeled fibrinogen was the lowest on ULA, higher on TCPS, and the highest on polystyrene or Primaria. Monocyte adhesion on protein preadsorbed surfaces for 2 h or 1 day was measured with a lactate-dehydrogenase method. Monocyte adhesion decreased over time. The ability of preadsorbed proteins to modulate monocyte adhesion was surface dependent. Adhesion was the lowest on ULA, higher and similar on TCPS or polystyrene, and the highest on Primaria. Monocyte adhesion on plasma or fibrinogen adsorbed surfaces correlated positively and linearly to the amount of adsorbed fibrinogen. Preadsorbed fibronectin, immunoglobulin G, plasma, or serum also promoted adhesion compared with albumin preadsorbed or uncoated surfaces. Overall, biomaterial surface chemistry, the type and amount of adsorbed proteins, and adhesion time all affected monocyte adhesion in vitro.     

Characterization of the tissue proliferated at the blood interface of carbon/ceramic composites

The present study focuses on cell adhesion/differentiation and material stability of surfaces of the three carbon/ceramic composites implanted in intra-atrial position in dogs for 1 year. Before implantation their surface was characterized by scanning electron microscopy. After harvesting, the tissue proliferated on the blood interface was examined by histology, scanning, and transmission electron microscopy, wavelength dispersive and x-ray spectrometry, electrophoretic and enzymatic characterization of glycosaminoglycans (GAGs) which were compared to endocardiac tissue as control samples. One year after implantation, the pattern of GAGs in the newly developed tissue was characterized by: 1) a constant increase of the total GAGs present on all carbon composites, 2) a significant increase of dermatan sulfate (p less than 0.05), 3) a significant increase of chondroitin sulfate (p less than 0.05), 4) a significant decrease of heparan sulfate in Group 1, whereas this GAG fraction was increased in Groups 2 and 3. Cellular surface differentiation towards endothelial-like cells occurred in places particularly in groups 1 and 3, whereas only fibrous tissue was found covering the implants in Group 2. Fibroblastic cells with dense intracellular deposits, which produced emission of Si, Ca, and C energy as well as extracellular lipidic containing inclusions were observed. The macromolecular modifications were associated with 1) the absence of endothelial lining, 2) the migration of carbon and silicon particles, and 3) the occurrence of calcifications and lipidic inclusions. These results suggest that the relative smoothness of these materials could be responsible for the development of a tissue that did not adhere to the biomaterial, indicating that cell adhesion and functional differentiation are in intimal relationship with the physical-chemical structure of the material surface.     

Influence of alpha-1-acid glycoprotein on blood rheology

Hemorheologic effects of alpha-1-acid glycoprotein (AGP) were studied in experiment on 76 noninbred white male rats. AGP was administered i.p. to intact rats twice in a total dose of 300 mg/kg. The effects were evaluated 72 hours after the first injection. It was found that AGP accelerated platelet and erythrocyte aggregation, platelet-erythrocyte cooperation, enhanced adhesion of erythrocytes and agranulocytes to endothelium. On the contrary, adhesion of granulocytes and platelets to endothelium was diminished. Acceleration of platelet aggregation was mediated by stimulation of free radical processes in the cells. Thus, AGP produces anticoagulation action on blood theology.     

The measurement of conformational stability of proteins adsorbed on siloxanes

The paper investigates the conformational stability of bovine serum albumin (BSA) and fibrinogen during 24-h incubation in turn with a linear silicone polymer (polydimethylsiloxane (PDMS)), with linear silicone oligomers (hexamethyldisiloxane and octamethyltrisiloxane) and with cyclic silicone oligomers (octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5)). Ten-fold and 100-fold excesses of siloxanes with respect to the proteins were used. Using fluorescence spectroscopy of tryptophan located in the domain of proteins and fluorescence of 8-anilino-1-naphthalenesulfonic acid (1,8-ANS), which interacts with hydrophobic domains of proteins, changes in the tertiary structure of the protein were recorded. The results demonstrated that BSA does not change its native form during 24-h incubation with siloxanes. In contrast, the tertiary structure of fibrinogen was found to be altered by both short-chain linear siloxanes: (hexamethyldisiloxane and octamethyltrisiloxane) and long-chain PDMS. The changes can be observed only at a 100-fold excess of siloxanes with respect to the protein. No conformational changes in fibrinogen exposed to cyclic siloxanes were observed.     

The measurement of conformational stability of proteins adsorbed on siloxanes

The paper investigates the conformational stability of bovine serum albumin (BSA) and fibrinogen during 24-h incubation in turn with a linear silicone polymer (polydimethylsiloxane (PDMS)), with linear silicone oligomers (hexamethyldisiloxane and octamethyltrisiloxane) and with cyclic silicone oligomers (octamethylcyclotetrasiloxane (D₄) and decamethylcyclopentasiloxane (D₅)). Ten-fold and 100-fold excesses of siloxanes with respect to the proteins were used. Using fluorescence spectroscopy of tryptophan located in the domain of proteins and fluorescence of 8-anilino-1-naphthalenesulfonic acid (1,8-ANS), which interacts with hydrophobic domains of proteins, changes in the tertiary structure of the protein were recorded. The results demonstrated that BSA does not change its native form during 24-h incubation with siloxanes. In contrast, the tertiary structure of fibrinogen was found to be altered by both short-chain linear siloxanes: (hexamethyldisiloxane and octamethyltrisiloxane) and long-chain PDMS. The changes can be observed only at a 100-fold excess of siloxanes with respect to the protein. No conformational changes in fibrinogen exposed to cyclic siloxanes were observed.     

Comparative rheology of nucleated and non-nucleated red blood cells

Deformation and dynamic flow behaviour of nucleated duck erythrocytes were compared to those of non-nucleated human cells during steady flow (at wall shear stresses up to 3 N·m^–2) through glass capillaries (diameter between 5 m and 12 m). While the deformation process of both cell species is qualitatively similar, significant quantitative differences demonstrate a lower deformability of the nucleated erythrocytes. In comparison to human cells, the avian red cells exhibit significantly lower stability of orientation during capillary flow. Viscometric measurements show that, in contrast to human red cell suspensions, the relative viscosity of nucleated cell suspensions does not decrease with increasing suspending medium viscosity. The results indicate that the hydrodynamic disturbance caused by avian erythrocytes in the flow of the suspending fluid is more pronounced than that of the mammalian cells. This is due to their lower ability to adapt to local shear forces.Supported by the Deutsche Forschungsgemeinschaft     

Responses to multiple injections with alum alone compared to injections with alum adsorbed to proteins in mice

New effects and mechanisms of alum on innate immunity have emerged in recent years. A number of cellular and molecular mechanisms induced by aluminum adjuvant have been reported, while the role of NALP3 and inflammasome in the cellular pathway induced by alum is still controversial. The effect of injection of alum alone without vaccine antigen into human has not been reported so far. Recently, in a phase IIIa double-blinded placebo controlled clinical trial testing the therapeutic HBsAg-anti-HBs vaccine formulated with alum against chronic viral hepatitis B patients, the placebo group receiving alum only showed substantial therapeutic effects. To explore possible underlying therapeutic mechanisms, mice were treated either with multiple injections of alum alone or with alum adsorbed to proteins (HBsAg-anti-HBs). After 4 injections Gr1⁺/CD11b⁺ cells in the spleen were increased in both alum alone and alum adsorbed in proteins groups. Increased Gr1⁺/CD11b⁺ cells in spleens remained consistently high in the alum alone treated group, while Gr1⁺/CD11b⁺cells decreased in the alum adsorbed to proteins group after 6 injections. Both treatments triggered increased levels of TNF-alpha measured in the plasma, but only the alum alone treated mice showed increased levels of IL-10. Histology of the liver tissues revealed a higher number of spotty necrotic foci in the alum alone immunized group. Taken together, potent inflammatory responses were induced in the alum alone immunized mice, which suggests that the substantial therapeutic effects observed in chronic hepatitis B patients immunized with alum alone might be attributed to inflammatory responses.     

Scaled interfacial activity of proteins at the liquid-vapor interface

A principal conclusion drawn from observations of time- and concentration-dependent liquid-vapor (LV) interfacial tension gamma(lv) of a diverse selection of proteins ranging from albumin to ubiquitin spanning nearly three decades in molecular weight (MW) is that concentration scaling substantially alters perception of protein interfacial activity as measured by reduction in gamma(lv). Proteins appear more similar than dissimilar on a weight/volume basis, whereas molarity scaling reveals a "Traube-rule" ordering by MW, suggesting that adsorption is substantially driven by solution concentration rather than diversity in protein amphilicity. Scaling as a ratio-to-physiological-concentration demonstrates that certain proteins exhibit the full possible range of interfacial activity at and well-below physiological concentration, whereas others are only weakly surface active within this range, requiring substantially higher solution concentration to achieve reduction in gamma(lv). Important among this latter category of proteins are the blood factors XII and XIIa, assumed by the classical biochemical mechanism of plasma coagulation to adsorb to procoagulant surfaces, even in the presence of overwhelming concentrations of other blood constituents such as albumin and immunoglobulin that are shown by this work to be among the class of highly surface-active proteins at physiologic concentration. A comparison of pendant drop and Wilhelmy balance tensiometry as tools for assessing protein interfacial activity shows that measurement conditions employed in the typical Wilhelmy plate approach fails to achieve the steady-state adsorption condition that is accessible to pendant drop tensiometry.     

Mediation of biomaterial-cell interactions by adsorbed proteins: a review

An appropriate cellular response to implanted surfaces is essential for tissue regeneration and integration. It is well described that implanted materials are immediately coated with proteins from blood and interstitial fluids, and it is through this adsorbed layer that cells sense foreign surfaces. Hence, it is the adsorbed proteins, rather than the surface itself, to which cells initially respond. Diverse studies using a range of materials have demonstrated the pivotal role of extracellular adhesion proteins--fibronectin and vitronectin in particular--in cell adhesion, morphology, and migration. These events underlie the subsequent responses required for tissue repair, with the nature of cell surface interactions contributing to survival, growth, and differentiation. The pattern in which adhesion proteins and other bioactive molecules adsorb thus elicits cellular reactions specific to the underlying physicochemical properties of the material. Accordingly, in vitro studies generally demonstrate favorable cell responses to charged, hydrophilic surfaces, corresponding to superior adsorption and bioactivity of adhesion proteins. This review illustrates the mediation of cell responses to biomaterials by adsorbed proteins, in the context of osteoblasts and selected materials used in orthopedic implants and bone tissue engineering. It is recognized, however, that the periimplant environment in vivo will differ substantially from the cell-biomaterial interface in vitro. Hence, one of the key issues yet to be resolved is that of the interface composition actually encountered by osteoblasts within the sequence of inflammation and bone regeneration.     

Oxidation of proteins adsorbed on hemodialysis membranes and model materials

The cleaning of cellulosic hemodialysis membrane Cuprophan and model materials (glass; polystyrene and polypropylene, as such and surface-oxidized), conditioned by adsorption of blood plasma proteins (HSA, fibrinogen, IgG) was investigated in vitro. Sodium hypochlorite (NaClO) and Renalin, a product containing hydrogen peroxide and peroxyacetic acid, were used as cleaning reagents. X-ray photoelectron spectroscopy and the use of radiolabeled fibrinogen demonstrated the presence of varying amounts of a polypeptidic residue, with sulfur brought to a high oxidation stage (sulfonate-like). The trends were the same for the three proteins regarding the effectiveness of the oxidizer and the influence of the surface properties. NaClO was much more effective than Renalin to remove the adsorbed proteins. The proteins adsorbed on Cuprophan were more sensitive to the oxidizers, when compared with proteins adsorbed on other materials. This may be due to both the lower protein-surface affinity, as indicated by radiochemical measurements, and the sensitivity of the material itself to the oxidizer, as revealed by weight loss measurements. These results support the attribution of hemocompatibility improvement after regeneration of Cuprophan with Renalin to the masking of the activating surface by a residue from previously adsorbed proteins.     

Effects of oxidized fibrinogen on the functions of blood cells,blood clotting,and rheology

Oxidatively-modified fibrinogen induces platelet aggregation and potentiates ADP-induced platelet aggregation and production of active oxygen forms in zymosan-stimulated leukocytes. Fibrinogen induces IL-8 production in primary culture of endothelial cells from human umbilical vein; the oxidized form of fibrinogen is more active, similarly as during induction of the expression cell adhesion molecules (P-selectin and ICAM-1). Oxidized fibrinogen (10 and 20% oxidation degree) impairs microrheological properties of the blood, sharply reduces erythrocyte deformability, modifies blood viscosity, and reduces suspension stability of the blood. Oxidized fibrinogen modified blood clotting parameters and ADP-, ristocetin-, and collagen-induced platelet aggregation in whole blood. Oxidized fibrinogen disordered the formation of fibrin clot and blood clotting process. Platelet aggregation was activated in response to ADP, but not to ristocetin and collagen, the degree of activation increased in direct proportion to the degree of fibrinogen oxidation. This indicates the “dysregulatory” effect of oxidized fibrinogen on platelets. The formation of platelet complexes with polymorphonuclear leukocytes was intensified in the presence of oxidized fibrinogen; polymorphonuclear leukocyte luminol-dependent fluorescence intensity in the presence of platelets increased after incubation with oxidized fibrinogen in comparison with native fibrinogen. Hence, oxidized fibrinogen plays an important role in the development of atherosclerosis and its complications (thromboses). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Supplement 2, pp. 21–32, April, 2007