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.     

Hydrophilic hybrid IPNs of segmented polyurethanes and copolymers of vinylpyrrolidone for applications in medicine.

The preparation and biocompatibility properties of thermoplastic apparent interpenetrating polymer networks (T-IPNs) of a segmented polyurethaneurea, Biospan (BS), and vinylpyrrolidone-dimethylacrylamide (VP-DMAm) copolymers, are described. The biological interaction between the obtained materials and blood was studied by in vitro methods. The addition of the VP-DMAm copolymers to form T-IPNs with BS substantially increased the equilibrium water uptake and water diffusion coefficients. Investigation of the proteins adsorption, platelet adhesion, thrombus formation and factor XII activation is presented. Investigations of the proteins adsorption of the BS/VP-DMAm T-IPNs surfaces show that the segmented polyurethane (BS) containing VP-DMAm copolymers with higher VP content adsorb more albumin than fibrinogen and gamma-globulin. The platelets adhesion, thrombus formation and factor XII activation are effectively suppressed with respect to the segmented polyurethane when VP-DMAm copolymers with high VP contents are incorporated into BS as T-IPNs.     

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.     

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.     

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.     

Haemocompatibility of segmented polyurethanes investigated in vivo

Samples of segmented polyurethanes differing in composition and in surface morphology were introduced into the left ventricles of hearts of goats for 72 h. After removal of the samples, their surface and the surface of the heart endothelium were evaluated visually with respect to the formation of thrombi. Differences in the interaction of the individual polyurethanes with blood were examined by XPS photoelectron spectroscopy, SEM, and by infrared (i.r.) reflexion spectroscopy. The results suggest ways for improving the haemocompatibility of the surfaces of polyurethanes used as parts of the total artificial heart.     

ESCA studies of surface chemical composition of segmented polyurethanes.

Surface chemical analysis of two commercially available polyurethanes, i.e., Avcothane and Biomer was carried out by electron spectroscopy for chemical analysis (ESCA). The depth which is subject to analysis is in the range of 50-100 A. The variables studied in this study are the difference in exposure to air or to the mold substrate during the solvent casting process. Model compounds such as a pure polydimethylsiloxane, polyether soft segment and hard segment copolymer were used to identify and assign various ESCA peaks. The air facing surface of Avcothane which is the blood contacting surface is found to be covered mostly with polydimethylsiloxane polymer, with a small amount of polyether soft segment mixed with silicone. Therefore, the hard segment of the polyurethanes is hidden beneath the blood contact surface in Avcothane. In Biomer films, the air facing surface contains a greater concentration of polyether soft segment than the substrate surface. These results are consistent with our previous results obtained by Fourier transform IR internal reflection and Auger electron spectroscopy.     

Effect of sulfonation of segmented polyurethanes on the transient adsorption of fibrinogen from plasma: possible correlation with anticoagulant behavior.

The influence of polyurethane sulfonation on fibrinogen adsorption from plasma and on plasma coagulation has been investigated. Sulfonated polyurethanes were synthesized using a two-step solution polymerization in which a diamino disulfonic acid was used as chain extender, thus incorporating sulfonate groups into the hard segments. Polymer molecular weights were determined by size exclusion chromatography and weight average values were in the range of 50,000 to 200,000. Equilibrium water uptake of solid polymer specimens was substantial and was found to increase with increasing sulfonate content. Titration of sulfonate groups allowed an estimate of the retention of free sulfonate in the polymers which ranged from 50 to 85%. Loss of free sulfonate is attributed to reaction of isocyanate with sulfonate groups during chain extension. Both surface chemistry and hydrophilicity were assessed using a combination of ESCA and water contact angle measurements. The ESCA data indicate enrichment of soft segment in the surface. Contact angles show increasing hydrophilicity with increasing sulfur content. Fibrinogen adsorption from plasma to the sulfonated polyurethane surfaces was studied using radioiodine labeling. Fibrinogen surface concentration was found to increase strongly as sulfonate content increased. Fibrinogen adsorption behavior is quite different from that of conventional unsulfonated polyurethanes in the sense that the adsorption levels are much higher and there is little displacement of initially adsorbed fibrinogen (Vroman effect). The data are interpreted in terms of two mechanisms: fibrinogen uptake (i.e., absorption) into a polymer-plasma "gel" hypothesized to exist at the surface of these materials, and adsorption in the usual sense. Thrombin times of human plasma in which polymer particles were suspended were prolonged and were found to increase with increasing sulfonate content of the polymers, suggesting that sulfonate groups confer a measure of anticoagulant activity on these materials.     

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.     

Interactions between segmented polyurethane surfaces and the plasma protein fibrinogen.

Surfaces of a segmented polyurethane were varied by casting on poly(ethylene terephthalate) (PET) and glass substrates, and were characterized through infrared-attenuated total-reflection spectroscopy (ATR). Surfaces cast on glass substrates showed a higher content of polyether segments, whereas those cast on PET contained a higher relative concentration of aromatic segments. Adsorption, and possible conformational changes of fibrinogen, were found to be more substantial on polymer surfaces having a higher content of polyether segments. It is concluded that the relatively good blood compatibility of segmented polyurethanes is partly due to the presence of peptide-like bonds on aromatic segments.     

Platelet adherence and detachment with adsorbed fibrinogen: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy.

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) with preadsorbed fibrinogen. Observations of the interactions of acridine-orange-labeled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). In some cases preadsorbed materials were incubated for 24 h, during which little or no loss of protein occurred. Protein surface concentration, by itself, was a poor indicator of expected cell adhesion and morphology. Surface chemistry was a second important factor which must be considered. A third observation is that for the 100% EMA copolymer, 24 h of incubation led to a large reduction in platelet adhesion when compared to the 100% EMA material without incubation. For the 0% and 100% EMA polymers, the percentage of contacting platelets which adhere and detach is greater for the 24-h incubation cases than for those not incubated. These results led to the conclusion that our most hydrophilic surface favors adhesion with detachment, transient cell contact, over long-term adhesion, as does incubation of adsorbed protein. A brief discussion is presented of a possible connection between this behavior and platelet consumption in vivo for hydrogels.     

Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers

The interaction of cells with solid surfaces is important in many settings, including the response of tissue to implanted materials. Protein adsorption to the surface plays a critical role in controlling cell interactions with surfaces. However, few comprehensive studies of both cell behavior and protein adsorption in complex protein mixtures (e.g., serum) have been done so the connection between these events is not well understood. In particular, methods to systematically perturb both protein adsorption and cell behavior in order to understand their relationship have been lacking. To induce changes in cell and protein behavior, the effects of serum dilution and substrate surface chemistry were studied. Surface chemistry was varied by using a series of polymers and copolymers of hydroxyethyl methacrylate (HEMA) and ethylmethacrylate (EMA) varying in their hydrophobic/hydrophilic balance. Large changes in cell spreading and fibronectin adsorption were observed when either serum concentration or polymer type was varied. The spreading of 3T3 cells in serum was found to be well correlated with the amount of fibronectin adsorption to the substrates. Attachment was not correlated with fibronectin adsorption, especially on glass preadsorbed with diluted serum. For 3T3 cells and perhaps other cells that have a receptor for a protein which is present in the medium, the amount of adsorption of this protein to the substrate appears to be a critical factor controlling cell interactions with the substrate.     

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.     

Solid-phase immunoassay of serum antibodies to peptides. Covalent antigen binding to adsorbed phenylalanine-lysine copolymers

Two small peptide antigens, glucagon and enkephalin (5-L-leucine), were covalently immobilised using either glutaraldehyde or bis-(sulphosuccinimidyl) suberate to an adsorbed layer of phenylalanine-lysine copolymer (PL) or partially acetylated PL (APL) on polystyrene. Both copolymers formed stable layers, particularly APL after adsorption from solution in distilled water. Adsorption of the copolymers under these conditions and subsequent coupling of the antigens yielded solid phases with low non-specific immunoglobulin binding characteristics in an enzyme-linked immunosorbent assay (ELISA) to detect peptide-specific antibodies in rabbit serum. The signal-to-noise ratio in this ELISA was dependent on the combination of copolymer, antigen and coupling reagent employed. Removal from the solid-phase of weakly bound antigen by washing with solutions containing Tween 20 or sodium dodecyl sulphate (SDS) increased assay sensitivity, which was 2-4-fold greater than when simple antigen adsorption was utilised. In the ELISA, the coefficient of variation was lower when covalent antigen coupling was employed.     

Fluorinated polyurethanes, 1. Synthesis and characterization of fluorine-containing segmented poly(urethane-urea)s

To produce high performance antithrombus elastomers, a series of new fluorine-containing segmented poly(urethane-urea)s was synthesized from 2,2,3,3,4,4,5,5-octafluorohexamethylene diisocyanate ( 1 ) and α-hydro-ω-hydroxypoly(oxyalkylene)s ( 5a, b and 6 ) or fluorinated dioxa-1,10-decanediols ( 3 and 4 ) (as the soft segments) via the prepolymers ( 7 ) which subsequently were reacted with various diamines including a fluorinated aliphatic diamine ( 2 ) as chain-extenders. High-molecular-weight polymers were obtained in high yields with 5a and nonfluorinated diamines. Spectroscopic analysis of the prepolymers and the chain-extended polymers indicated their structures to be highly ordered. Stress-strain curves of films cast from a DMF solution indicated the polymers to be typical elastomers with tensile strength as much as 600–700 kg/cm².     

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.     

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.     

Effect of soft segment chemistry on the biostability of segmented polyurethanes. I. In vitro oxidation

A series of segmented polyurethanes (SPUs) containing various polyol soft segments was prepared and their resistance to oxidative degradation was investigated after aging in AgNO3 solution. The SPU with the polyether soft segment showed a large reduction in mechanical strength after exposure to the oxidative environment. Surface cracking was often observed for these specimens. XPS measurements revealed that scission of the ether linkage occurs upon oxidation. The oxidative resistance of SPUs containing aliphatic hydrocarbon soft segments was significantly improved over the poly(tetramethylene oxide) (PTMO) based polyurethane.