Adsorption of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: II. Competition with the other plasma proteins

In the preceding paper, we described results concerning the adsorption of purified thrombin and antithrombin III on two insoluble anticoagulant polystyrene derivatives. We now report similar results obtained in a plasma system. In each case, the purified protein was mixed with fresh platelet poor plasma in order to maintain the same concentrations of all the other plasma proteins. The thrombin molecule was modified by alkyl phosphorylation of the active serine site prior to mixing with plasma. The adsorption of antithrombin was found to be reduced 8 to 9 times when the protein solution was substituted by diluted plasma. In contrast the thrombin adsorption only depends on the substituents bound on the polymeric chain. These results are supported by those of the study of the competition between purified antithrombin and albumin.     

Adsorption of proteins out of plasma onto glass from a separated flow

Perturbations in the adsorption of plasma proteins caused by flow separation were studied quantitatively. An instrument was constructed that causes flow to separate over approximately half the width of a standard microscope slide and the pattern of protein deposition in and near the separated flow was observed by staining the slide with black iron oxide. The slide was mounted at the edge of a Couette flow field established between two concentric cylinders, the outer of which was rotating. The slide was located on the stationary, inner cylinder just downstream of a rectangular bar that causes the flow to separate. After exposure to dilute plasma injected upstream of the bar, the slide was removed and stained with oxide suspension. The resulting, visible pattern was scanned through a video camera and analyzed to yield relative values of stain density that could be quantified. The oxide patterns suggest that proteins were deposited onto the slide less rapidly in and just downstream of the separated flow region than farther downstream. At a shear rate of 6.61 s^-1, corresponding to a velocity of 1.32 cm s^-1 0.2 cm above the point of flow separation, overall amounts of adsorbed proteins increased with exposure time in the range 3-30 min with the exception of a period from 10 to 11 min when all data show a temporary decrease. In calibration experiments, oxide failed to adhere to slides exposed to purified albumin but adhered copiously to slides exposed to purified fibrinogen. These results suggest that the oxide patterns following plasma exposure are attributable primarily to fibrinogen and that the temporary decrease in the separated flow experiments is attributable to the displacement of fibrinogen by a less stainable protein, conjecturally high molecular weight kininogen and factor XII. This study yields quantitative information confirming earlier findings that were less controlled and non-quantitative. It confirms the hypothesis that the sequence of protein deposition from dilute plasma to glass surfaces is delayed in regions of separated flow.     

Adsorption of plasma proteins and adhesion of platelets onto novel polyetherurethaneureas--relationship between denaturation of adsorbed proteins and platelet adhesion

Novel polyetherurethaneureas which have been synthesized by the present authors were chosen for the substrate polymers, on which adhesion of platelets was investigated. The number of adhered platelets and the amount of serotonin released from platelets adhered on the polymers and the protein-coated polymers were determined by radioisotope method. Both of them were enhanced with increasing content of urea linkages in the polyetherurethaneureas. The platelet adhesion was discussed in terms of the denaturation of plasma proteins upon adsorption, which was determined by Fourier-transform infrared spectroscopy. With increasing degree of protein denaturation, the platelet adhesion and the serotonin release were enhanced. This relationship was particularly evident in the case of albumin adsorption. It was shown that the surface properties of substrate polymers affect the protein adsorption, which in turn influences the adhesion of platelets.     

Catalysis of the generation of thrombin-antithrombin complex by insoluble anticoagulant polystyrene derivatives

The inhibition of thrombin by antithrombin III is known to be accelerated by heparin through the formation of complexes between the muccopolysaccharide and both proteins. In the preceding papers, we reported that polystyrene derivatives absorb thrombin and its inhibitor with a higher affinity for the protease than for the antiprotease. These complexes are responsible for the catalysis of the generation of thrombin-antithrombin complex which was observed either with purified proteins or in plasma. The protease-antiprotease complex has an affinity for the polymer surface which is higher than that of antithrombin but lower than that of thrombin. Therefore, the thrombinantithrombin complex generated on the insoluble material is desorbed by thrombin and a catalytic anticoagulant effect can be observed with these polymers.     

Adsorption of plasma proteins on polyethylene oxide-modified lipid bilayers studied by total internal reflection fluorescence

Distearoylphophatidylcholine (DSPC) mixed with various mole percentages of polyethylene oxide (number average molecular weight 2000)-grafted distearoylphosphatidylethanolamine (PEO2000-DSPE) were deposited on DSPE-coated quartz surfaces by the Langmuir-Blodgett deposition. Structural transitions in PEO2000 from pancake to mushroom, and from mushroom to brush conformations were revealed from film balance experiments. Adsorption kinetics of proteins from 1% platelet-poor plasma (PPP) on the supported lipid bilayers were studied using intrinsic total internal reflection fluorescence. All the supported lipid bilayers exhibited over a magnitude reduction in adsorbed plasma proteins, compared with the quartz substrate. The increase of PEO2000-DSPE density in the mixed bilayers slightly increases the amount of adsorbed proteins on the bilayers.     

Adsorption of plasma proteins to the derivatives of polyaminoetherurethaneurea: the effect of hydrogen-bonding property of the material surface

Polyaminoetherurethaneureas bearing tertiary amino groups in the main chain (M-PAEUU) were synthesized, quaternized (Q-M-PAEUU) and heparinized (H-M-PAEUU). With increasing portions of diisocyanate and with decreasing portions of polyaminoether in the feed, M-PAEUU containing more hydrogen-bonded urea carbonyl groups was prepared. With increasing hydrogen-bonding character of M-PAEUU, the adsorbed bovine serum albumin (BSA) was more denatured. By quaternization of M-PAEUU, the protein adsorption increased, but the denaturation of adsorbed proteins was suppressed. With increasing ratio of hydrogen-bonded urea carbonyl groups in Q-M-PAEUU, the adsorptions of BSA, bovine serum gamma-globulin (B gamma G), and bovine plasma fibrinogen (BPF) were decreased, but the degree of denaturation of adsorbed proteins was increased. In the adsorption to H-M-PAEUU, both the amount and the degree of denaturation of adsorbed proteins were strongly decreased. The dynamic adsorption experiments of plasma proteins showed the behaviors which are similar to the equilibrium adsorption experiments. The decrease of hydrogen-bonded urea linkages and the increase of hydrophilicity by quaternization and heparinization of the polymer surface may be favorable for building up a hydration layer on the surface, thus suppressing the denaturation of plasma proteins which may trigger blood clotting and thrombus formation.     

Adsorption of fibronectin derived from serum and from human endothelial cells onto tissue culture polystyrene

Human endothelial cells (HEC) suspended in a culture medium containing 20% human serum (CMS) adhere and spread on(to) moderately wettable polymers, such as tissue culture polystyrene (TCPS). We have previously shown that serum derived-fibronectin, which is a cell adhesion promoting protein, has a high affinity for TCPS, but that the amount of fibronectin which adsorbed from CMS was relatively small. In this study we investigated whether fibronectin derived from HEC contributes to the adhesion and spreading of the cells on(to) TCPS. Therefore, HEC were seeded in the presence of fibronectin-depleted CMS. The amount of fibronectin detected on TCPS increased with both cell seeding density and incubation time. Although initial HEC adhesion is delayed on TCPS which has been precoated with albumin (Alb), high density lipoprotein (HDL) or immunoglobulin G (IgG), maximal numbers of adhering and spreading HEC were found on these surfaces 6 h after seeding of HEC. Fibronectin was detected on these surfaces, but an exchange of preadsorbed Alb, HDL, or IgG for fibronectin could not be demonstrated. We conclude that HEC deposit fibronectin onto TCPS, irrespective of the presence of a preadsorbed layer of proteins which delay cell adhesion.     

Interactions of proteins in human plasma with modified polystyrene resins

Investigations are reported on the composition of protein layers adsorbed from plasma to various modified polystyrene resins. As well as polystyrene itself, polystyrene bearing sulfonate groups in the benzene rings, and polystyrene sulfonate in which the sulfonate groups were converted to amino acid sulfamide, were investigated. Some of these resins were shown in previous work to have anticoagulant properties. To study the adsorption of proteins from plasma, the resins were exposed to citrate anticoagulated human plasma for 3 h. Adsorbed proteins were then eluted sequentially by 1M Tris buffer and 4% SDS solution, and examined by SDS-PAGE. The gel patterns were similar on all resins except polystyrene. From the MWs of the gel bands, the major protein component appeared to be fibrinogen. Smaller amounts of plasminogen, transferrin, albumin, and IgG were also present. In addition, Ouchterlony immunoassay of the eluates from one resin gave positive identification of complement C3, fibronectin, IgG, and IgM. Many other minor gel bands remain unidentified. A consistent finding for all resins was the presence of plasmin-type fibrinogen degradation products though the amounts varied with resin type. It is concluded from this (and from experiments showing FDP formation when fibrinogen was absorbed to the resins, from buffer containing a trace of plasminogen) that the functional groups in these materials promote the adsorption of plasminogen and its activation to a plasmin-like molecule. It appears from the substantial quantities of fibrinogen adsorbed to these materials after 3 h exposure to plasma that the Vroman effect (giving transient adsorption of fibrinogen) is not operative on these materials. It is hypothesized that specific interactions occur between fibrinogen and sulfonate groups.     

Adsorption of plasma proteins to the derivatives of polyetherurethaneurea carrying tertiary amino groups in the side chains

Polyetherurethaneureas carrying tertiary amino groups in the side chains were synthesized, quaternized, and heparinized. The adsorption of plasma proteins to the polyurethane derivatives was investigated using Fourier-transform infrared, surface fluorescence, ultraviolet, and circular dichroism spectroscopy. Bovine gamma-globulin and bovine plasma fibrinogen were easily adsorbed to hydrophobic polyurethanes and denatured. It was found that the beta structure was generated during the irreversible conformational change of the proteins on adsorption to hydrophobic polyurethane materials. On the other hand, bovine serum albumin was easily adsorbed to hydrophilic quaternized and heparinized polyurethanes without a serious conformational change. The conclusion was drawn that a polymer material that selectively adsorbs bovine serum albumin in a native state could be antithrombogenic.     

Platelet adhesion onto polyetherurethane urea derivatives: effect of cytoskeleton proteins of the platelet

Adhesion and activation of platelets upon adhesion onto synthetic polymers were investigated with reference to participation of the cytoskeleton proteins. Platelets were treated with cytoskeleton breakers, and then the adhesion of platelets onto polyetherurethane urea derivatives and serotonin release from adhered platelets were investigated. In the adhesion onto glass, platelets were strongly stimulated and accompanied rearrangement of the cytoskeleton system, and also serotonin release involved the action of the cytoskeleton system. On the other hand, platelets were not strongly stimulated upon adhering onto polyetherurethane urea derivatives. The platelet adhesion onto cationic polymers exceptionally accompanied the rearrangement of the cytoskeleton system. The participation of the cytoskeleton in platelet adhesion onto polyetherurethane urea derivatives was influenced by the presence of plasma proteins. It was found that protein layers deposited on the material surface play an important role in platelet adhesion.     

Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures

Enhancement of the speed and sensitivity of an ELISA technique was achieved by doing it on a polystyrene microtiter plate preactivated by a simple photochemical reaction. Immobilization of Epicoccum nigrum antigen (allergenic antigen) or goat anti-rabbit IgG onto the photoactivated plates was found to occur in only 45 min with higher binding than that obtained through adsorption during the same period onto the untreated surface. Nearly 1.5-2-folds higher readings were obtained when the ELISA was carried out with the solid phase prepared on the photoactivated surface rather than on the untreated surface. Moreover, solid phases prepared on the activated surface could detect IgE (E. nigrum antibody) even at 1/50 (v/v) dilutions, whereas a solid phase prepared on the untreated surface failed to do so. Around three times higher ELISA values were obtained in the activated plate than the untreated plate when IgE was diluted to 1/5 (v/v). Such photoactivated surface could be of great importance in diagnostic tests involving the ELISA technique particularly to confirm false negative cases and for other immunoassays such as radioimmunoassay procedures.     

Adherence of coagulase-negative staphylococci onto polyethylene catheters in vitro and in vivo: a study on the influence of various plasma proteins

Bacterial adherence on PE catheters inserted into the carotid artery of rabbits was assessed at 5, 30, and 240 min after injection with bacteria of five coagulase-negative staphylococci (CN Staph). These studies revealed that CN Staph adhere onto PE catheters 5 min after injection with bacteria. At 240 min after injection with bacteria almost all catheters were sterile, indicating that initially adherent bacteria on PE catheters disappear with time. EM studies revealed high numbers of platelets and leukocytes and many fibrin deposits on the surface of the PE catheter. In addition, the adherence of the five test strains on PE catheters was determined in vitro. In these in vitro studies the bacteria and catheters were pretreated with either fibrinogen, fibronectin, albumin or citrated whole plasma or the liquid adherence medium was supplemented with these proteins or the adherence assay was done in citrated whole plasma. The presence of proteins in the adherence medium or pretreatment of the catheter or bacteria with either fibronectin, albumin or whole citrated plasma markedly inhibited bacterial adherence. In contrast, pretreatment of bacteria or both bacteria and catheters with fibrinogen enhanced bacterial adherence suggesting the presence of ligands for fibrinogen on the cell surface of CN Staph.     

Adsorption of proteins from plasma at polyester non-wovens.

Polyester non-wovens in filters for the removal of leukocytes from platelet concentrates (PCs) must be platelet compatible. In PC filtration, the adsorption of proteins at the plasma-non-woven interface can be of great importance with respect to the yield of platelets. Unmodified and radio frequency glow discharge (RFGD) treated poly(ethylene terephthalate) non-woven (NW-PET) and two commercial surface-modified non-wovens were contacted with human plasma. Protein desorption by sodium dodecyl sulphate (SDS) was evaluated by X-ray photoelectron spectroscopy (XPS). The desorbed proteins were characterized by gel electrophoresis and immunoblotting. Compared to the commercial surface-modified non-wovens, unmodified and RFGD-treated NW-PETs adsorbed a relatively high amount of protein. Significantly more protein was removed from the hydrophobic NW-PET by SDS than from the hydrophilic RFGD-treated non-wovens. RFGD treatment of NW-PET reduces the reversibility of protein adsorption. Less albumin and fibrinogen were removed from the RFGD-treated non-wovens than from NW-PET. In addition, a large amount of histidine-rich glycoprotein was removed from RFGD-treated non-wovens, but not from NW-PET. The different behaviour of RFGFD-treated non-wovens towards protein adsorption is probably caused by differences in the chemical reactivity of the non-woven surfaces.     

Characterization of plasma proteins adsorbed onto biomaterials. By MALDI-TOFMS

The analysis of plasma proteins adsorbed onto a polyurethane (PU) biomaterial was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This article marks the first study on MALDI-TOFMS analysis of multiple proteins adsorbed from plasma, in vitro, onto the surface of a biomaterial to easily enable their characterization. Plasma standards from three different hosts were placed in contact with non-porous PU, a model biomaterial. Following the use of washing protocols developed in our laboratory, the biomaterial was analyzed, directly, with MALDI-TOFMS. Proteins with molecular weights (Mr) ranging from ca. 6.5 to 150 kDa were observed in the mass spectra and characterized upon comparison with proteins of known Mr. The proteins observed were tentatively identified as those known to adsorb onto PU, both in vitro and in vivo. In an attempt to model in vivo sorption, the PU biomaterial was exposed to freshly collected canine plasma, in vitro, for different lengths of time. Corresponding MALDI-TOFMS spectra displayed increasing protein signal for a number of different proteins with increasing times of exposure to plasma. This method provided qualitative and semi-quantitative analysis of the proteins adsorbed onto the biomaterial surface.     

Passive adsorption of human antirrabic immunoglobulin onto a polystyrene surface

The latex agglutination immunoassay technique uses polymer colloids as carriers of adsorbed proteins to enhance the antigen–antibody reaction. The aim of the present work is to study the adsorption of Human Antirrabic Immunoglobulin (HA-IgG) on polystyrene latex (PS). The physical adsorption of HA-IgG on PS latex was investigated as a function of pH at 2 mM ionic strength. The amount of HA-IgG adsorbed onto PS latex greatly depends on pH; its value showed a maximum in the neighborhood of the IEP of HA-IgG. The electrophoretic method was applied to characterize latex particles. The influence of the amount of HA-IgG adsorbed (J _ads) on the electrophoretic mobility and ζ -potential values was also studied.     

Passive adsorption of human antirrabic immunoglobulin onto a polystyrene surface

The latex agglutination immunoassay technique uses polymer colloids as carriers of adsorbed proteins to enhance the antigen-antibody reaction. The aim of the present work is to study the adsorption of Human Antirrabic Immunoglobulin (HA-IgG) on polystyrene latex (PS). The physical adsorption of HA-IgG on PS latex was investigated as a function of pH at 2 mM ionic strength. The amount of HA-IgG adsorbed onto PS latex greatly depends on pH; its value showed a maximum in the neighborhood of the IEP of HA-IgG. The electrophoretic method was applied to characterize latex particles. The influence of the amount of HA-IgG adsorbed (J(ads)) on the electrophoretic mobility and zeta-potential values was also studied.     

Affinity of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: I. In vitro adsorption studies

In previous papers, we described insoluble polystyrene derivatives which exhibit a heparin-like antithrombic activity in plasma. In order to ascertain the heparin-like mechanism of this activity we have studied the interactions of thrombin and antithrombin III with two polymers of this series: sulphonated polystyrene and sulphonate-glutamic acid sulphonamide polystyrene. The adsorption was measured using purified enzyme and enzyme inhibitor and polymer beads whose average diameter was about 25 μm. The maxima of adsorption approximately correspond to a monolayer of protein. The results are discussed with respect to the most common isotherms used in chemisorption and the affinities of the enzyme and its inhibitor for both materials are evaluated: k_T- 10⁷(M/I)⁻¹, k_AT- 3.10⁵(M/I)⁻¹.