Plasma gas discharge deposited fluorocarbon polymers exhibit reduced elutability of adsorbed albumin and fibrinogen

The adsorption and subsequent detergent elutability of fibrinogen and albumin were measured on various treated and untreated polymer films in order to determine whether the relative adsorption of these proteins was responsible for the enhanced thromboresistance of Dacron vascular grafts treated with tetrafluoroethylene in a radio frequency glow discharge (RFGD) apparatus. Fluorocarbon-coated surfaces varying in the relative proportions of CF, CF₂' and CF groups and in the ratio of fluorine to carbon were prepared by RFGD treatment of poly(ethylene terephthalate) (PET) films with tetrafluoroethylene or perfluoropropane. The adsorption of fibrinogen and albumin to these fluorocarbon-coated surfaces was comparable to the adsorption of the proteins to polytetrafluoroethylene (PTFE) and PET. However, the elutability of fibrinogen and albumin from the RFGD fluorocarbon surfaces with sodium dodecyl sulfate was much lower than that from PTFE or PET. Other RFGD treatments of PET, such as ethylene deposition or argon etching, did not reduce the extent of albumin elutability as dramatically as did the RFGD fluorocarbon treatments. The strong albumin binding to RFGD fluorocarbon surfaces may be exploited clinically to enhance the retention of albumin preadsorbed to blood-contacting surfaces fo render them thromboresistant.     

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 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.     

Postadsorptive transitions in fibrinogen adsorbed to polyurethanes: changes in antibody binding and sodium dodecyl sulfate elutability.

Residence time-dependent changes in fibrinogen after adsorption to six different polyurethanes were examined by measuring polyclonal antifibrinogen binding to the adsorbed protein. The amount of adsorbed fibrinogen that could be eluted by sodium dodecyl sulfate (SDS) was also measured. Baboon fibrinogen was first adsorbed from dilute plasma to the polymers, which were then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the amount of antifibrinogen bound by the adsorbed fibrinogen was measured using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed fibrinogen was soaked in a 3% SDS solution, and the amount of retained 125I-radiolabeled fibrinogen was measured. With increasing residence time, decreases in both antibody binding and the SDS elutability of the adsorbed fibrinogen occurred, but the rate of change was dependent on the polyurethane to which the fibrinogen was adsorbed. In addition, the antibody binding per unit of adsorbed fibrinogen, when measured immediately after the adsorption step, varied by approximately a factor of 3 among the various polyurethanes. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen with residence time did not occur on some of the surfaces. This study shows that the chemical properties of the adsorbing surface influence the rate at which adsorbed fibrinogen undergoes change. The significance of the polymer-dependent changes in adsorbed fibrinogen with respect to blood reactions with polymers is discussed.     

Platelet adhesion and procoagulant activity induced by contact with radiofrequency glow discharge polymers: roles of adsorbed fibrinogen and vWF

The potential hemocompatibility of radiofrequency glow discharge (RFGD) polymers made by copolymerization of mixtures of hexafluoropropene and ethylene (C(3)F(6)/C(2)H(4)) or acrylic acid and 1,7-octadiene was investigated using in vitro assays for platelet adhesion and platelet catalyzed thrombin generation. Thrombin generation rate normalized to platelet number was used as a measurement of platelet activation (procoagulant activity). RFGD polymers produced by copolymerization of acrylic acid and 1, 7-octadiene contained varying amounts of carboxylic acid species as determined by electron spectroscopy for chemical analysis (ESCA). These polymers induced little variation in platelet adhesion, thrombin generation, or platelet activation. RFGD polymerization of C(3)F(6) and C(2)H(4) resulted in polymers with varying proportions of fluorinated species, as determined by ESCA. Fibrinogen adsorption from plasma was maximal on a polymer made with 25% C(3)F(6) (75% C(2)H(4)) in the feed. However von Willebrand factor (vWF) adsorption was greater on polymers made with increased %C(3)F(6) in the feed. Platelet adhesion decreased with increasing %C(3)F(6) in the feed. Thrombin generation was lowest for platelets adherent to polymers made from both C(3)F(6) and C(2)H(4). Therefore, procoagulant activity of platelets increased for polymers made with increased %C(3)F(6) in the feed, similar to the trend in vWF adsorption. These findings suggest that increased incorporation of fluorinated species into RFGD polymers leads to decreased platelet adhesion and increased platelet activation (which is possibly due to increased vWF adsorption).     

Human serum albumin and fibrinogen interactions with an adsorbed RGD-containing peptide.

The modification of polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) with an arginine-glycine-aspartic acid cell adhesion peptide, RGD peptide (PepTite Adhesive Coating; Telios Pharmaceuticals, San Diego, CA) has been previously investigated. Initial animal studies showed this RGD peptide to accelerate healing and assist in the formation of an endothelial cell lining of the lumenal side of PET and PTFE fabrics in a cardiovascular application. It is of interest to determine how this RGD peptide is able to influence cellular events through intervening layers of plasma proteins that spontaneously adsorb upon implantation. This study examined the interaction of predeposited RGD-containing peptide with human serum albumin (HSA) or fibrinogen that was characterized using multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy, ellipsometry, and contact angle analysis. It was determined that fibrinogen-containing films consistently exhibited more mass than films of the RGD peptide, HSA, or HSA adsorbed onto RGD peptide-containing films. MAIR-IR spectra of RGD peptide films before and after HSA adsorption were similar in absorption and intensity; however, ellipsometry indicated HSA introduction had created thicker, less dense films. Fibrinogen, on the other hand, when adsorbed onto RGD peptide films provided increased relative mass in a more compact arrangement. Contact angle analyses of each of the dried films showed their surface energies to remain high, but the polar components of RGD peptide films were reduced after either serum protein adsorption. These phenomena may be related to the minimal thrombus accumulation that was noted during the initial animal studies, that promoted subsequent healing.     

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.     

Tight binding of albumin to glow discharge treated polymers.

Tetrafluoroethylene (TFE) glow discharge-treated Dacron vascular grafts resist thrombus deposition, embolization and thrombotic occlusion. In addition, albumin adsorbed on TFE-treated surfaces resists elution by sodium dodecyl sulfate (SDS). Since the tight binding of albumin to TFE-treated surfaces may contribute to their thromboresistant character, we decided to examine the mechanism responsible for this tenacious adsorption. We have investigated albumin adsorption and retention (after SDS elution) on a number of untreated and glow discharge-treated surfaces. Fluorocarbon glow discharge-treated polymers retain a larger fraction of the adsorbed albumin than ethylene and hexamethyldisiloxane glow discharge-treated surfaces. Albumin retention by surfaces appears to be closely related to their surface free energy (in air). Low energy surfaces (in air), whether untreated or glow discharge-treated, retain a larger fraction of the albumin adsorbed than higher energy surfaces. The lowest energy surfaces should have the highest interfacial energies in water, with correspondingly high driving forces for adsorption of proteins. This can lead to the formation of multiple binding sites upon adsorption, permitting strong hydrophobic interactions, which leads to the observed strong binding.     

Platelet adhesion to radiofrequency glow-discharge-deposited fluorocarbon polymers preadsorbed with selectively depleted plasmas show the primary role of fibrinogen

Fluorocarbon radio-frequency glow-discharge (RFGD) treatment has previously been shown to cause decreased platelet adhesion despite the presence of adsorbed fibrinogen on the surfaces. In this study platelet adhesion to fluorocarbon RFGD-treated surfaces preadsorbed with human plasma was further examined. A series of plasma deposited fluorocarbon thin films were made by varying the C3F6/CH4 ratio in the monomer feed. The surfaces were preadsorbed with plasma, serum, or plasma selectively depleted of fibronectin, vitronectin, or Von Willebrand factor, and platelet adhesion was measured. We also measured fibrinogen adsorption to the surfaces from plasma, monoclonal antibody binding to adsorbed fibrinogen and SDS elutability of the adsorbed fibrinogen. The antibodies used bind to the three putative platelet binding sites on fibrinogen, namely, M1 antibody binds to the dodecapeptide at the C-terminus of the gamma chain, gamma (402-411), R1 antibody binds to a sequence in the Aalpha chain (87-100) which includes RGDF at Aalpha (95-98) and R2 antibody binds a sequence in the Aalpha chain (566-580) which includes RGDS at Aalpha (572-575). Fibrinogen was found to play a decisive role in mediating platelet adhesion to the fluorocarbon surfaces contacting plasma. Few platelets adhered to the fluorocarbon surfaces preadsorbed with serum, while preadsorption with plasma selectively-depleted of either fibronectin, vitronectin, or von Willebrand factor did not decrease platelet adhesion significantly. Replenishment of exogenous fibrinogen to serum restored platelet adhesion, while replenishment of the other proteins had no effect. Platelet adhesion to the fluorocarbon surfaces was lower than to PET or the methane glow-discharge-treated PET. However, there was no apparent correlation between platelet adhesion and the amount of fibrinogen adsorption or monoclonal antibody binding to surface-bound fibrinogen.     

Inhibition of monocyte adhesion and fibrinogen adsorption on glow discharge plasma deposited tetraethylene glycol dimethyl ether.

Monocytes and macrophages play important roles in host responses to implanted biomedical devices. Monocyte and macrophage interactions with biomaterial surfaces are thought to be mediated by adsorbed adhesive proteins such as fibrinogen and fibronectin. Non-fouling surfaces that minimize protein adsorption may therefore minimize monocyte adhesion, activation, and the foreign body response. Radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme) was used to produce polyethylene oxide (PEO)-like coatings on a fluorinated ethylene-propylene (FEP) surface. Electron spectroscopy for chemical analysis (ESCA) and static time of flight secondary ion mass spectrometry (ToF-SIMS) were used to characterize the surface chemistry of tetraglyme coating. Fibrinogen adsorption to the tetraglyme surface was measured with 125I-labeled fibrinogen and ToF-SIMS. Adsorption of fibrinogen to plasma deposited tetraglyme was less than 10 ng cm(-2), a 20-fold decrease compared to untreated FEP or tissue culture polystyrene (TCPS). Monocyte adhesion to plasma deposited tetraglyme was significantly lower than adhesion to FEP or TCPS. In addition, when the surfaces were preadsorbed with fibrinogen, fibronectin, or blood plasma, monocyte adhesion to plasma deposited tetraglyme after 2 h or 1 day was much lower than adhesion to FEP. RF-GDPD tetraglyme coating provides a promising approach to make non-fouling biomaterials that can inhibit non-specific material-host interactions and reduce the foreign body response.     

Molecular and supramolecular structure of adsorbed fibrinogen and adsorption isotherms of fibrinogen at quartz surfaces

The adsorption of fibrinogen to quartz surfaces was measured by ellipsometry, ELISA, and electron microscopy. The initial adsorption at low concentrations was diffusion rate limited as determined by the ELISA and by counting the number of adsorbed molecules at electron micrographs. From ellipsometry, ELISA, and electron microscopy measurements it was found that the surface concentration of adsorbed fibrinogen increased continuously over four decades in bulk concentration of fibrinogen. At a hydrophilic quartz surface a plateau level of the adsorption isotherm was found at a surface concentration of 0.1 pmol/cm2 where the adsorbed molecules had a mean intermolecular distance of 10 +/- 5 nm between neighbors. At higher surface concentrations the molecules were densely packed and formed a layer where single molecules could not be identified. Adsorbed fibrinogen showed different structure at hydrophobic and hydrophilic quartz surfaces. At a hydrophilic surface, the fibrinogen molecules appeared as a 46 nm nodose rod consisting of 6-7 nodes with a diameter of 4 nm. At a hydrophobic surface, the molecule appeared as a binodular or trinodular rod with a node diameter of 5-9 nm, connected with a thin filament to form a 40-nm rod. Adsorption from higher concentrations of fibrinogen in solution resulted in adsorbed spheric structures with a diameter of 18-24 nm at the hydrophobic surface and in end-to-end polymers at the hydrophilic quartz membrane.     

Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin

Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat(?) or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies.     

Glow discharge plasma treatment of polyethylene tubing with tetraglyme results in ultralow fibrinogen adsorption and greatly reduced platelet adhesion

Previous studies from our lab have shown that fibrinogen adsorption (Gamma(Fg)) must be reduced below 10 ng/cm(2) to significantly reduce platelet adhesion, and that radio frequency glow discharge (RFGD) treatment of polymeric films in the presence of tetraethylene glycol dimethyl ether (tetraglyme) can reduce Gamma(Fg) to the desired ultralow value. In this report, the effects of RFGD coatings of tetraglyme on the lumenal surface of PE tubing on Gamma(Fg) and on blood interactions both in vitro and ex vivo are described. Gamma(Fg) on the tetraglyme-coated PE tubing was reduced to the desired ultralow level (<10 ng/cm(2)), and we also observed a significant decrease in adsorption of von Willebrand's factor. In vitro platelet adhesion from washed platelet suspensions, platelet rich plasma, or whole blood to tetraglyme-coated PE tubing was decreased compared to PE, polyurethane, or silicone rubber tubes. In addition, thrombin generation by platelets adherent to tetraglyme-coated PE was also much less than by platelets adherent to PE. When inserted in an ex vivo carotid artery-carotid artery shunt in sheep, the RFGD tetraglyme-coated PE exhibited a very low number of adherent platelets compared to heparin-coated, chromic acid-etched, or plain PE. The RFGD tetraglyme-coated PE tubes exhibited high protein and platelet resistance in vitro, and high platelet resistance ex vivo. The improved hemocompatibility is attributed to the unique chemical structure of RFGD tetraglyme that makes it highly protein resistant.     

Adsorption of baboon fibrinogen and the adhesion of platelets to a thin film polymer deposited by radio-frequency glow discharge of allylamine.

Platelet adhesion under static and flow conditions from a washed platelet suspension containing albumin to a polymer deposited by radio-frequency glow discharge of allylamine vapour on a poly(ethylene terephthalate) substrate was measured. Electron spectroscopy for chemical analysis was used to characterize the surface. Fibrinogen adsorption from a series of dilute plasma solutions to radio-frequency glow discharge/allylamine, measured using 125I radiolabelled baboon fibrinogen, increased with decreasing plasma dilution to a level much higher than that previously observed on polyurethanes. Elutability by sodium dodecyl sulphate of fibrinogen adsorbed from dilute plasma also increased with increasing plasma concentration, but fibrinogen preadsorbed from plasma became non-elutable when surfaces were stored in buffer for 5 d before contact with sodium dodecyl sulphate. Platelet adhesion to substrates which had been pre-adsorbed with dilute plasma was measured using baboon platelets radiolabelled with 111In. Adhesion greatly decreased as the plasma concentration used for preadsorption increased, suggesting that non-specific platelet binding to the bare surface occurs when protein coverage is incomplete. Non-specific platelet binding was inhibited to varying degrees by preadsorption of different proteins to the surface. Platelet adhesion to surfaces preadsorbed with dilute (1.0%) baboon and human plasmas lacking fibrinogen (i.e. serum, heat-defibrinogenated plasma and congenitally afibrinogenemic plasma) was diminished compared with normal plasma. Addition of exogenous fibrinogen to the deficient plasma partially restored platelet adhesion to normal levels. Adhesion to surfaces preadsorbed with human plasma deficient in von Willebrand factor was comparable to that observed with normal plasma. The plasma preadsorption studies with fibrinogen deficient media suggested that adsorbed fibrinogen is necessary for platelet adhesion to the radio-frequency glow discharge/allylamine substrate at high protein coverage. However, since adhesion was greatly reduced when the plasma preadsorbed substrate was stored in buffer before platelet contact, the conformation of adsorbed fibrinogen is also important in mediating platelet adhesion to radio-frequency glow discharge.     

Adorption of human fibrinogen and human serum albumin onto polyethylene

The adsorption of human fibrinogen (HFb) onto polyethylene at pH 7.4 and 37°C is irreversible with the saturated value 0.51 μg/cm². The established dependence of the concentration of irreversibly adsorbed HFb on the concentration of HFb in solution does not reflect an equilibrium between adsorption and desorption and cannot be described by an adsorption isotherm. No reversible adsorption of HFb and HSA at 37°C has been observed. At lower temperatures, both reversibly and irreversibly adsorbed HFb have been detected.     

The surface stability of polymers with adsorbed fibronectin

Linear low-density polyethylene (LLDPE), polypropylene (PP), polystyrene (PS), and polyethylene-co-ethacrylic acid (PE-EAA, 17.5% acid content) films were treated with an aqueous solution of fibronectin. Advancing contact angles of water (straight theta(a)) were used to monitor the change in the surface wettability of these films. With the exception of PE-EAA, all of the samples showed an increase in their wettability by water, indicating that the protein had adsorbed to the polymer surface. The stability of these protein-modified films against a buffered aqueous solution and against air under ambient conditions was monitored over time. The surface wettability of these protein-modified polymers, with the exception of PS, remained unchanged after heating against the buffer solution. In air, however, straight theta(a) increased over a period of 2 months. In addition, the effect of organic solvent extraction on the surface stability of these protein-modified films was investigated. Unmodified samples of LLDPE, PS, and PP were subjected to soxhlet extraction to remove impurities and low-molecular-weight oligomers prior to film preparation and subsequent treatment with fibronectin. These samples were left in air under ambient conditions for 2 months. There was no difference in the magnitude of the change in straight theta(a) for the protein-modified, extracted LLDPE film compared to the protein-modified, nonextracted polymer film. A slight decrease in the rate of thermal reconstruction was observed for the protein-modified extracted PS film compared to the protein-modified nonextracted sample, and a slight increase in the rate of thermal reconstruction was observed for the protein-modified extracted PP film compared to the protein-modified nonextracted sample.Copyright 2000 John Wiley & Sons, Inc.     

Character of adsorbed bovine serum albumin from adsorption enthalpies

Heats of adsorption of BSA onto polystyrene and polycarbonate were determined microcalorimetrically. The polymers were coated onto 50 nm alumina particles which were immersed in buffer for the measurements. Experimental variations to isolate the various component reactions of the integral heats included adsorption of native and denatured BSA from solution, adsorption onto substrates precoated with BSA, varying the thicknesses of adsorbed BSA by varying concentrations of the adsorbing solutions, and determining the denaturation energy by solution of the solid native and denatured BSA into a denaturing solvent.     

Preadsorption of polymers on glass and silica to reduce fibrinogen adsorption

Glass and silica beads were precoated with various polymers to obtain steric exclusion chromatography (SEC) supports which are nonadsorbant for hydrophilic macromolecules. The efficiency of this treatment was estimated by subsequent radiolabeled fibrinogen adsorption. The result obtained with a block copolymer was better than with various hydrophilic homopolymers. This ABA type block copolymer, where A is a poly(N-acetylethyleneimine) (PAEI) sequence and B a polyethylene oxide (PEO) sequence was preadsorbed at pH 4.5 and 25 degrees C; the fibrinogen adsorption was reduced to less than 5% of the value observed on untreated solid surfaces. Thus the hemocompatibility of solid supports should be increased by precoating with this block copolymer. Results for nonporous glass beads and porous silica particles were in good correlation.