Postadsorptive transitions in fibrinogen adsorbed to biomer: changes in baboon platelet adhesion, antibody binding, and sodium dodecyl sulfate elutability

Residence-time-dependent changes in fibrinogen after its adsorption to Biomer were examined by measuring platelet adhesion and antibody binding to the adsorbed protein, and the amount of adsorbed fibrinogen which could be eluted by sodium dodecyl sulfate (SDS). Baboon fibrinogen was first adsorbed (from either pure solution or dilute plasma) to Biomer, which was then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the adherent protein layer was either probed for fibrinogen capable of mediating platelet adhesion using 111In radiolabeled, washed platelet suspensions under both static and shearing conditions, or for fibrinogen capable of binding antibody using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed protein layer was soaked in a 3% SDS solution, and the amount of 125I radiolabeled fibrinogen retained was measured. Decreases in platelet and antibody binding, and in the SDS elutability of the adsorbed fibrinogen after it was stored in buffer were detected, although different rates of decrease were observed for each method. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen was prevented. While each of the three assays measures a different property of adsorbed fibrinogen, this study suggests that the adherent protein undergoes time dependent conformational changes which render it less reactive toward platelets and antibodies, and more resistant to elution by SDS.     

Postadsorptive transitions in fibrinogen: influence of polymer properties

Changes in the fibrinogen molecule after its adsorption to biomaterials may be important in determining blood compatibility. Previously, postadsorptive transitions were detected by measuring the elutability of adsorbed proteins with surfactant solutions. The elutability decreased with increased residence time, suggesting that protein-surface interactions increased with residence time. In this study, we have examined the effects of polymer structure and composition, chain mobility, and hydrophobicity on the postadsorptive transitions of fibrinogen. Glassy, rigid polymers showed high fibrinogen adsorption, regardless of whether the polymer was hydrophilic or hydrophobic. However, the binding strength (as measured by elutability) was much lower on hydrophilic polymers and oxygen-containing hydrophobic polymers. Short-term transitions, requiring 2 h or less after adsorption, were observed only on hydrophobic polymers that contained no oxygen. More gradual transitions were observed on hydrophobic polymers containing oxygen, but only after a lag time of 1-4 h.     

Rapid postadsorptive changes in fibrinogen adsorbed from plasma to segmented polyurethanes

Fibrinogen adsorbed to biomaterials plays a key role in mediating platelet interactions that can lead to blood clotting so its behavior on surfaces is of fundamental interest. In previous work showing that fibrinogen adsorbed to surfaces quickly becomes non-displaceable upon exposure to blood plasma, the fibrinogen was adsorbed from buffer, so we performed new studies in which the displaceability of fibrinogen adsorbed from plasma was characterized. Fibrinogen was adsorbed from 1% plasma to seven different surfaces for 1-64 min and then transferred to 100% plasma lacking radiolabeled fibrinogen and the amount adsorbed before and after transfer measured. The surfaces were glass, Silicone rubber, and five different polyurethanes. As adsorption time increased, the fibrinogen became increasingly resistant to displacement during the 100% plasma step, but the rate of increase in resistance varied greatly with surface type. Fibrinogen adsorbed from 1% plasma evidently undergoes rapid, surface dependent transitions. This work shows that the transitions that occur when the fibrinogen is adsorbed from blood plasma are similar to what we have previously observed for fibrinogen adsorbed from buffer.     

Changes in the SDS elutability of fibrinogen adsorbed from plasma to polymers

Baboon fibrinogen was adsorbed from diluted plasma solutions to glass, polyethylene, polystyrene, polydimethylsiloxane, poly(ethylene)terephthalate, and Biomer. Following adsorption, half of the samples were immediately placed in sodium dodecyl sulfate (SDS) solutions to elute the protein, while the others were stored in buffer for up to 5 days and then eluted in SDS. The elution was typically incomplete, but depended on the plasma concentration and the residence time. The elutability was generally lower for fibrinogen adsorbed from more diluted plasma, and substantially lower for samples on which the fibrinogen had resided for 5 days. The non-elutable portion of the protein layer formed rapidly on polystyrene, while on polyethylene elutability was high initially, followed by a gradual decrease.     

Changes in fibrinogen adsorbed to segmented polyurethanes and hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Fibrinogen adsorption from blood to biomaterials may regulate platelet adhesion and thrombus formation because of fibrinogen's central role in the coagulation cascade and its ability to bind specifically to the platelet membrane glycoprotein (GP) IIb-IIIa. Adsorption of fibrinogen from blood plasma to many materials exhibits a maximum with respect to plasma dilution and exposure time (the Vroman effect). In this study fibrinogen adsorption to several polymers was examined to ascertain the influence of controlled changes in surface chemistry on the Vroman effect. The materials included hydroxyethylmethacrylate-ethylmethacrylate (HEMA/EMA) copolymers, Biomer, and a series of segmented polyurethanes (PEUs), two of which contained fluorinated chain extenders. Each material exhibited maximal adsorption of fibrinogen at intermediate plasma concentrations. Little effect of soft-segment type or molecular weight was observed and no significant differences in fibrinogen adsorption to the fluorinated PEUs were seen. Changes in the strength of fibrinogen attachment to these materials with time after adsorption were also assessed. Fibrinogen adsorbed for 1 min was displaced more readily by blood plasma than that adsorbed for 1 h, regardless of the material. The more hydrophobic polymers exhibited greater retention of adsorbed fibrinogen. In addition, the fraction of fibrinogen retained by polyethylene depended on the amount of fibrinogen adsorbed to the surface, being greatest when the surface loading was the least. These studies indicate that spreading or transition of adsorbed fibrinogen molecules from a weakly to tightly bound state is a general consequence of protein adsorption to solid surfaces.     

Changes in the SDS elutability of fibrinogen adsorbed from plasma to polymers

Baboon fibrinogen was adsorbed from diluted plasma solutions to glass, polyethylene, polystyrene, polydimethylsiloxane, poly(ethylene)terephthalate, and Biomer®. Following adsorption, half of the samples were immediately placed in sodium dodecyl sulfate (SDS) solutions to elute the protein, while the others were stored in buffer for up to 5 days and then eluted in SDS. The elution was typically incomplete, but depended on the plasma concentration and the residence time. The elutability was generally lower for fibrinogen adsorbed from more diluted plasma, and substantially lower for samples on which the fibrinogen had resided for 5 days. The non-elutable portion of the protein layer formed rapidly on polystyrene, while on polyethylene elutability was high initially, followed by a gradual decrease.     

Residence-time dependent changes in fibrinogen adsorbed to polymeric biomaterials.

It has generally been accepted that biomaterials adsorbing the least amount of the plasma protein fibrinogen following exposure to blood will support less platelet adhesion and therefore exhibit less thrombogenicity. Several studies suggest, however, that the conformation or orientation of immobilized fibrinogen rather than the total amount adsorbed plays an important role in determining the blood compatibility of biomaterials. The purpose of this study was to investigate time-dependent functional changes in fibrinogen adsorbed to polytetrafluoroethylene (PTFE), polyethylene (PE), and silicone rubber (SR). Fibrinogen was adsorbed to these materials for 1 min and then allowed to 'reside" on the surfaces for up to 2 h prior to assessing its biological activity. Changes in fibrinogen reactivity were determined by measuring the adhesion of 51Cr-labeled platelets, the binding of a monoclonal antibody (mAb) directed against an important functional region of the fibrinogen molecule (the gamma-chain dodecapeptide sequence 400-411), and the ability of blood plasma to displace previously adsorbed fibrinogen. Platelet adhesion differed among the polymeric materials studied, and PTFE and PE samples exhibited a small decrease in adhesion with increasing fibrinogen residence time. Platelet adhesion to SR was the least among all materials studied and showed no variation with residence time. When using PTFE and SR as substrates, mAb recognition of adsorbed fibrinogen did not change with residence time whereas that on PE decreased slightly. The mAb binding was least to fibrinogen adsorbed to SR, which is in agreement with the platelet adhesion results. Finally, the ability of plasma to displace previously adsorbed fibrinogen decreased dramatically with increasing residence time on all materials. These in vitro studies support the hypothesis that fibrinogen undergoes biologically significant conformational changes upon adsorption to polymeric biomaterials, a phenomenon that may contribute to the hemocompatibility of the materials following implantation in the body.     

Changes in binding affinity of a monoclonal antibody to a platelet binding domain of fibrinogen adsorbed to biomaterials

Previously, we found that when fibrinogen-coated polyurethanes resided in a buffer for a period of time (the 'residence time') platelet adhesion to these materials decreased. Other changes in adsorbed fibrinogen such as decreases in polyclonal antibody binding and SDS elutability supported the conclusion that fibrinogen undergoes postadsorptive conformational changes. Subsequently we measured the binding of monoclonal antibodies to the three putative platelet binding sites on fibrinogen, using a single mid-range concentration of antibody. We found that binding of a monoclonal antibody to the platelet binding site at the C-terminus of the gamma chain of fibrinogen changed little with residence time, while binding of monoclonal antibodies to the other two putative binding sites on fibrinogen either increased with residence time (RGDF at Aα 95-98), or first increased and then decreased with residence time (RGDS at Aα 572-575). In the current study, we measured antibody binding affinity, K_a, by measuring antibody binding at a series of antibody concentrations. This is a more sensitive method for detecting changes in adsorbed fibrinogen than measuring antibody binding from a single antibody concentration. The K_a was determined for two antibodies, M 1 (4A5), which binds to a platelet binding domain of fibrinogen (y 402-411) and R 1 (155 B1616), which binds to residues 87-100 of the Aα chain (containing an RGDF site). A summary of the results for the M1 antibody are as follows. The Ka was higher for M1 binding to fibrinogen adsorbed to Immulon I® than to Biomer®, Biospan® or poly(ethylene terephthalate), suggesting that fibrinogen adsorbed to Immulon I® is more platelet adhesive than fibrinogen adsorbed to the other polymers. On Biospan®, the K_a decreased from 2.8 x 10⁹ to 1.0 x 10⁹ M^-1 after a 24 h 37°C residence time, which correlated with the decrease in platelet adhesiveness of adsorbed fibrinogen observed previously under these conditions. The change in K_a was greater when adsorbed fibrinogen was kept under denaturing conditions. For example, the K_a decreased from 2.8 x 10⁹ to 0.8 x 10⁹ M^-1 after a 1 h 70°C residence time whereas it remained approximately the same, 2.9 x 10⁹ M^-1, after a 24 h 0°C residence time.     

Variations in the ability of adsorbed fibrinogen to mediate platelet adhesion to polystyrene-based materials: a multivariate statistical analysis of antibody binding to the platelet binding sites of fibrinogen

Platelet adhesion to the surfaces of biomaterials preadsorbed with plasma previously has been shown to be mediated exclusively by surface-bound fibrinogen and does not seem to involve the other adhesion proteins in plasma (Tsai et al., J Biomed Mater Res 2002;60:348-359). In this study, the influence of surface-bound fibrinogen on platelet adhesion to five different types of polystyrene-based microtiter plates preadsorbed with plasma was analyzed relative to the amount of adsorbed fibrinogen and monoclonal antibody binding to the adsorbed fibrinogen. There was no significant correlation between platelet adhesion and the absolute amount of adsorbed fibrinogen. However, platelet adhesion was positively correlated to the ability of the adsorbed fibrinogen to bind three types of monoclonal antibodies. The antibodies used bound to the sites on fibrinogen thought to be involved in platelet binding (the two gamma chain C-terminal dodecapeptides and the RGDF and RGDS sequences in each of the Aalpha chains). A partial least-squares calibration model was used to analyze the relative importance of these binding sites in fibrinogen to platelet adhesion. The gamma chain C-terminal dodecapeptide was shown to be the most important site in adsorbed fibrinogen in mediating platelet adhesion.     

Changes in the state of ionization of carboxyl groups in elastin in response to the binding of sodium dodecyl sulfate.

The interaction of sodium dodecyl sulfate with elastin has been studied by complexometric titration. Approximately 1.2 mumoles of protons with a pKapp of 5.45 are taken up by 10 milligrams of insoluble elastin upon the binding of detergent, apparently due to the protonation of normally ionized carboxylate functions in this protein. Since ionized carboxylate functions of elastin are essential for its interaction with elastase and, possibly, metallic cations, these results may have physiological significance in view of the affinity of elastin for lipid-like ligands.     

Polarity of stripping of tobacco mosaic virus by alkali and sodium dodecyl sulfate.

Polar stripping of coat protein of TMV by both alkaline and sodium dodecyl sulfate treatment occurs from the 5′-end of the RNA, contrary to previous conclusions.     

Quantitative immunoelectrophoretic assay for murine oncornavirus p30: noncovalent facilitation by sodium dodecyl sulfate.

Treatment of Rauscher murine leukemia virus lysates with the anionic detergent sodium dodecyl sulfate (SDS) at concentrations between 0.2 to 2.0% SDS per mg of viral protein greatly increased the anodal electrophoretic mobility of p30, the major internal polypeptide. SDS treatment did not reduce p30 antigenicity or cause nonspecific precipitation of normal serum proteins during subsequent immunoanalysis. The increased anodal electrophoretic mobility allowed assay of Rauscher murine leukemia virus p30 by Laurell rocket immunoelectrophoresis. An SDS-facilitated rocket immunoelectrophoresis assay is described that was highly reproducible (coefficient of variability, less than 3.0%) and capable of detecting 125 ng of viral protein. To our knowledge, this is the first report of a quantitative immunoelectrophoretic assay for an oncornavirus antigen. Since SDS binding is a general property of proteins, this method of noncovalently altering electrophoretic mobility appears to be applicable to other antigen-antibody systems.     

Heterogeneity of glycoproteins of the human erythrocyte membrane. Fractionation in sodium dodecyl sulfate containing solutions.

Human erythrocyte membranes and the glycoprotein isolated by phenol-water extraction of membranes were fractionated by gel filtration on Bio-Gel P-300 in 1% SDS. It was shown by this procedure that lipids (and possibly other membrane components) promote the dissociation of glycoprotein aggregates in SDS-containing solutions. The isolated glycoprotein gives several fractions on gel filtration in 1% SDS. The main fraction contains the major sialogylcoprotein (MN), which can be obtained in an electrophoretically homogeneous form (PAS-1) when only a part of the major peak is pooled and re-chromatographed. The purified glycoprotein has elevated MN blood group activity, does not show I activity, and contains components of alkalibabile oligosaccharide chains in slightly higher proportion than crude glycoprotein. The other fractions have distinctly different carbohydrate composition and contain glycoproteins of different electorphoretic mobility in SDS-PAGE.     

The role of adsorbed fibrinogen in platelet adhesion to polyurethane surfaces: a comparison of surface hydrophobicity, protein adsorption, monoclonal antibody binding, and platelet adhesion

Ten specially synthesized polyurethanes (PUs) were used to investigate the effects of surface properties on platelet adhesion. Surface composition and hydrophilicity, fibrinogen (Fg) and von Willebrand's factor (vWf) adsorption, monoclonal anti-Fg binding, and platelet adhesion were measured. PUs preadsorbed with afibrinogenemic plasma or serum exhibited very low platelet adhesion, while adhesion after preadsorption with vWf deficient plasma was not reduced, showing that Fg is the key plasma protein mediating platelet adhesion under static conditions. Platelet adhesion to the ten PUs after plasma preadsorption varied greatly, but was only partially consistent with Fg adsorption. Thus, while very hydrophilic PU copolymers containing PEG that had ultralow Fg adsorption also had very low platelet adhesion, some of the more hydrophobic PUs had relatively high Fg adsorption but still exhibited lower platelet adhesion. To examine why some PUs with high Fg adsorption had lower platelet adhesion, three monoclonal antibodies (mAbs) that bind to sites in Fg thought to mediate platelet adhesion were used. The antibodies were: M1, specific to gamma-chain C-terminal; and R1 and R2, specific to RGD containing regions in the alpha-chain N- and C-terminal, respectively. Platelet adhesion was well correlated with M1 binding, but not with R1 or R2 binding. When these mAbs were incubated with plasma preadsorbed surfaces, they blocked adhesion to variable degrees. The ability of the R1 and R2 mAbs to partially block adhesion to adsorbed Fg suggests that RGD sites in the alpha chain may also be involved in mediating platelet adhesion and act synergistically with the C-terminal of the gamma-chain.     

Inhibitory effect of sodium dodecyl sulfate in detection of Mycobacterium tuberculosis by amplification of rRNA.

The concentrations of sodium dodecyl (lauryl) sulfate (SDS) found in the sediments of the respiratory samples pretreated with SDS-NaOH varied between 3.36 and 12.42 mg/ml. These concentrations of SDS were higher than the level considered critical (> or = 0.16 mg/ml) to obtain negative results with the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. Extensive washing, after neutralization of sediment, is sufficient to remove all traces of detergent which interfere with the enzymes used by the Mycobacterium Tuberculosis Direct Test assay.     

Interaction of sodium dodecyl sulfate with poly(L-lysine) in aqueous solution

The interactions between a homopolypeptide, poly(L-lysine) [(Lys)_n], and an anionic surfactant, sodium dodecyl sulfate (NaDodSO₄), were studied by ¹³C NMR spectroscopy in D₂O solution. By observing the chemical shift of the ε-amino carbon of lysine monomer in D₂O solution at pH 7,3 in presence and absence of NaDodSO₄, direct evidence was found for electrostatic interaction between the protonated lysine ε-amino group and the head group of the anionic surfactant. At this pH, the (Lys)_n chain in NaDodSO₄ solution adopts the β-form conformation and the solubility of NaDodSO₄/(Lys)_n complexes is too small to observe ¹³C-spectra. At more alkaline solutions, where the (Lys)_n chain forms α-helices, the surfactant's polar group interacts with the uncharged lysyl side chain through hydrogen bonding. In the initial binding of the surfactant to (Lys)_n, both the surfactant head group and alkyl chain contribute to the association with (Lys)_n. With an excess of surfactant bound to α-helical (Lys)_n, NaDodSO₄/(Lys)_n complexes show high solubility and all carbon atoms of the surfactant molecules show micelle shifts. These results suggest that the surfactant molecules form micellelike clusters around the (Lys)_n helices exposing the surfactant polar head group to the medium and enhancing the solubility of NaDodSO₄/(Lys)_n complexes.     

The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to detect renal damage in Sprague-Dawley rats treated with gentamicin sulfate.

Renal toxicity is a common manifestation to the exposure of laboratory animals and humans to a wide range of xenobiotics. Traditional methods for evaluating renal damage by clinical chemistry such as blood urea nitrogen (BUN) and serum creatinine are not sensitive to early, mild changes. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to measure the molecular weight spectrum of urinary proteins allows for an evaluation of the functional changes associated with renal damage. The ability of the kidney to filter and reabsorb proteins is related to the functional ability of glomeruli and the proximal tubules. Gentamicin sulfate produces injury to the S-1 and S-2 segments of the proximal tubule in laboratory animals and humans. While severe damage to the tubules is associated with increased BUN, serum creatinine, and N-acetyl-beta-glucosiminadase (NAG), mild injury is not detected by these means. The evaluation of urinary proteins by SDS-PAGE demonstrated renal toxicity at a dose of 6 mg/kg after 2 days of sc treatment. The NAG: creatinine ratio was shown to be elevated after 2 days of treatment at 63 mg/kg. The use of SDS-PAGE as described in this paper provides a sensitive method for detecting renal injury.     

Batch emulsion copolymerization of butyl acrylate and methyl methacrylate in the presence of sodium dodecyl sulfate

Kinetics of batch emulsion copolymerization of butyl acrylate (BuA) with methyl methacrylate (MMA) have been investigated using sodium dodecyl sulfate as emulsifier and the redox system potassium peroxodisulfate (K₂S₂O₈)/sodium disulfite (Na₂S₂O₅) as initiator. Latexes have been characterized with regard to their copolymer composition through ¹H NMR analysis and particle size distribution by a quasi-elastic light scattering (QELS) technique or transmission electron microscopy. The effect of the monomer feed composition on the average copolymer composition vs. conversion was first studied in order to derive emulsion reactivity ratios. However, simulation of experimental data based on monomer partitioning (due to the high water solubility of MMA) and particle swelling was better accounted for when considering bulk reactivity ratios together with polymerization in the aqueous phase (in addition to that in the main locus of the polymer particles). Then, the influence of several pertinent parameters, such as emulsifier concentration, amount of initiator, monomer-to-water ratio and addition of an organic solvent has been examined in relation to the actual polymerization mechanism.     

Humoral immune response in tuberculosis: initial characterization by immunoprecipitation of 125iodine labelled antigens and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

125Iodine-labelled Mycobacterium tuberculosis antigens were immunoprecipitated with tuberculosis patients' sera and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A group of four polypeptide antigens of 55, 38, 28 and 18 kD were thus identified. The 38 and 28 kD polypeptides were the major antigens. Antibody response differed from one patient to another, both with respect to the number and quantity of antigens precipitated. Untreated patients and those undergoing treatment with antimycobacterial drugs also showed marked differences in their antibody response. Generally, immunoprecipitates from treated patients showed a larger number of antigen bands and the relative intensities of the bands was also greater. No correlation was observed between the immunoprecipitation profile and antibody titres determined by enzyme linked immunosorbent assay.