John L. Brash

Exploiting the current paradigm of blood–material interactions for the rational design of blood-compatible materials

The paradigm of tissue-material interactions, which holds that protein adsorption is the first event following contact and determines the later interactions of cells, is invoked to propose a design strategy for biocompatibility. Control of protein interactions is the key element, and it is suggested that nonspecific protein adsorption must be prevented while the adsorption of specific proteins that are expected to result in appropriate bioactivity must be promoted. Modification with polyethylene oxide has been investigated extensively as a means of preventing nonspecific adsorption. Examples of proteins that could be targeted for specific adsorption are antithrombin III to prevent coagulation and albumin to minimize platelet adhesion. Two examples of surfaces designed for specific adsorption from the authors laboratory are discussed: the incorporation of thrombin binding peptides to give a thrombin scavenging surface, and the incorporation of lysine to give a plasminogen specific surface with the potential to dissolve clots.

Dynamics of interactions between human albumin and polyethylene surface

Abstract The adsorption of human albumin to polyethylene was studied using radioiodinated (125I or 131I) protein. Adsorption at 23°C under laminar flow conditions over a range of shear rates up to 2500 sec−1 and from 0.1 to 3.8 mg ml−1 was investigated. A steady-state surface concentration (SSSC) was reached within 2–3 hr under all conditions. The value of SSSC was independent of flow conditions. Concentration dependence followed a Langmuir-like isotherm. The existence of a dynamic equilibrium with equal rates of adsorption and desorption was established. Adsorption and desorption were measured simultaneously by the use of double labeling (125I-labeled albumin on the surface, exchanging with 131I-labeled albumin in solution). Two characteristics of the dynamic steady state were investigated: the extent and the rate of turnover. Both extent and rate were found to increase with increasing shear rate and protein concentration, with essentially zero turnover at zero concentration or flowrate. Turnover data were fitted to a first-order model to obtain estimates of the fractional extent of turnover and the fractional turnover rate. These parameters varied from 0 to 0.8 and from 0 to 0.045 hr−1, respectively. The results are discussed in relation to the problem of thrombus formation at surfaces in contact with blood.

Patterns of adsorption of proteins from human plasma onto foreign surfaces.

The deposition of proteins on blood-contacting surfaces is known to be a determining factor in subsequent thromboembolic events. The composition of the protein layers and how they change with time are unknown. To generate information relevant to these questions, the quantities of albumin, fibrinogen and IgG adsorbed on seven surfaces from human plasma as a function of time were measured using a tracelabeling method. Materials studied include several segmented polyether-urethanes, glass, siliconized glass (SG), polystyrene (PS) and polyethylene (PE). Fibrinogen, surprisingly, was not adsorbed from plasma to any of the hydrophilic surfaces. On PE and SG adsorption passed through an early maximum (before 2 min) then declined to near zero. Only on PS was adsorption substantial and constant with time. Albumin was also not detected on the hydrophilic surfaces. IgG but was adsorbed substantially on the hydrophobic surfaces. IgG was detected on all surfaces, although in relatively low surface concentrations. These results suggest: 1. that the plasma itself interacts with initially adsorbed proteins, 2. that the role of fibrinogen adsorption in foreign-surface initiated thrombosis may need to be reevaluated and 3. that since the major plasma proteins are only minimally adsorbed, trace proteins may be important in blood-material interactions.

Protein-Resistant Poly(ethylene oxide)-Grafted Surfaces: Chain Density-Dependent Multiple Mechanisms of Action

A clear understanding of the mechanisms responsible for the protein-resistant nature of end-tethered poly(ethylene oxide) (PEO) surfaces remains elusive. A barrier to improved understanding is the fact that many of the factors involved (chain length, chain density, hydration, conformation, and distal chemistry) are inherently correlated. We hypothesize that, by comparing systems of variable but precisely known chain density, it should be possible to gain additional insight into the effects of the other factors. To evaluate this hypothesis, chain-end-thiolated PEOs were chemisorbed to gold-coated silicon wafers such that a range of chain densities was obtained. Three different PEOs were investigated: hydroxy-terminated chains of molecular weight 600 (600-OH), methoxy-terminated chains of molecular weight 750 (750-OCH3), and methoxy-terminated chains of molecular weight 2000 (2000-OCH3). In situ null ellipsometry was used to determine PEO chemisorption kinetics, ultimate PEO chain densities, protein adsorption kinetics, and ultimate protein adsorbed quantities. With this approach, it was possible to ascertain the effects of PEO distal chemistry (-OH, -OCH3), chain length, and layer hydration on protein adsorption. The data obtained suggested that properties related to chain density (conformational freedom, hydration) were the main determinants of protein resistance at chain densities up to a critical value of approximately 0.5 chain/nm2; at this value, protein adsorption was a minimum for the methoxy-terminated PEOs. For the hydroxyl-terminated PEO, adsorption leveled off at the critical value. Thus distal chemistry appears to be a major determinant of protein resistance at chain densities greater than the critical value.

Phenomenology and mechanism of the transient adsorption of fibrinogen from plasma (Vroman effect)

Abstract Additional data are presented on the transient adsorption of fibrinogen from blood plasma (the Vroman effect) reported previously (Brash, J. L., and ten Hove, P., Thromb. Haemostasis 51, 326, 1984; Horbett, T. A., Thromb. Haemostasis 51, 174, 1984). This effect is believed to result from rapid initial adsorption followed by displacement by other proteins. Adsorption was measured using radioiodine-labeled fibrinogen added to plasma as a tracer and surfaces were in the form of tubing. Plasma dilution and time were the main experimental variables. Initial adsorption was found to be diffusion-limited and diffusivities of about 1.5 × 10−7 cm2 s−1 were obtained independent of the surface. Various surfaces were compared on the basis of fibrinogen adsorption versus plasma concentration curves, all of which showed peaks at about 1% plasma concentration in accord with the Vroman effect. For 5-min adsorptions it was found that the height of the peaks was inversely related to the displacement “rate.” At 24 h the peak heights were in a different relative order and indeed one surface (a polyurethane) showed no peak. At these long times, the fibrinogen adsorption appears to be determined by a surface-solution equilibrium. None of the characteristics of the adsorption-concentration curves (peak height, displacement phase, “equilibrium” adsorption) were correlated with water contact angle of the different surfaces. Preadsorbed fibrinogen showed rates of displacement after exposure to plasma considerably slower than the rates observed “in situ.” This effect is tentatively attributed to orientational and/or conformational changes occurring as a function of residence time on the surface. Experiments on glass in which the plasma fibrinogen concentration was changed over a wide range (about one-eighth normal to twice normal) gave 5-min adsorption-concentration curves which differed in peak height and initial slope. The latter was proportional to fibrinogen concentration as expected. The peak heights did not show any such simple relationship but were used to demonstrate that about 50% of the glass surface may be covered with fibrinogen under normal conditions, again demonstrating the high surface activity of this protein.

Adsorption of fibrinogen on glass: reversibility aspects

Abstract The interactions of human fibrinogen with borosilicate glass have been studied using a radio-labeling technique. Adsorption in this system is not significantly affected by shear rate. Concentration dependence follows a Langmuir-like isotherm with plateau surface concentration of about 0.7 μg cm −2 in 0.05 M Tris, pH 7.35. With increasing NaCl or increasing Tris concentration, the level of adsorption decreases. Fibrinogen turnover between surface and solution is demonstrated by a double labeling technique. Only a fraction of the adsorbed layer is exchangeable under given conditions. The ratio of exchangeable to nonexchangeable fractions is found to increase with increasing concentrations of fibrinogen, NaCl, and Tris at pH 7.35. However this ratio is not significantly affected by shear. The rate of exchange is considerably faster than that observed previously (Brash and Samak, J. Colloid Interface Sci. 65 , 495, 1978) for albumin on polyethylene. Although there is no desorption into the starting buffer, desorption (up to 80% of the adsorbed layer at the isotherm plateau) does take place into solutions of increased Tris concentration, even in the presence of fibrinogen. These data provide evidence of inherent reversibility in this system.

Protein resistant surfaces: Comparison of acrylate graft polymers bearing oligo-ethylene oxide and phosphorylcholine side chains

The objective of this work was to compare poly(ethylene glycol) (PEG) and phosphorylcholine (PC) moieties as surface modifiers with respect to their ability to inhibit protein adsorption. Surfaces were prepared by graft polymerization of the methacrylate monomers oligo(ethylene glycol) methyl ether methacrylate (OEGMA, MW 300, PEG side chains of length n=4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC, MW295). The grafted polymers thus contained short PEG chains and PC, respectively, as side groups. Grafting on silicon was carried out using surface-initiated atom transfer radical polymerization (ATRP). Graft density was controlled via the surface density of the ATRP initiator, and chain length of the grafts was controlled via the ratio of monomer to sacrificial initiator. The grafted surfaces were characterized by water contact angle, x-ray photoelectron spectroscopy, and atomic force microscopy. The effect of graft density and chain length on fibrinogen adsorption from buffer was investigated using radio labeling methods. Adsorption to both MPC- and OEGMA-grafted surfaces was found to decrease with increasing graft density and chain length. Adsorption on the MPC and OEGMA surfaces for a given chain length and density was essentially the same. Very low adsorption levels of the order of 7 ng/cm2 were seen on the most resistant surfaces. The effect of protein size on resistance to adsorption was studied using binary solutions of lysozyme (MW 14 600) and fibrinogen (MW 340 000). Adsorption levels in these experiments were also greatly reduced on the grafted surfaces compared to the control surfaces. It was concluded that at the lowest graft density, both proteins had unrestricted access to the substrate, and the relative affinities of the proteins for the substrate (higher affinity of fibrinogen) determined the composition of the layer. At the highest graft density also, where the adsorption of both proteins was very low, no preference for one or the other protein was evident, suggesting that adsorption did not involve penetration of the grafts and was occurring at the outer surface of the graft layer. It thus seems likely that preference among different proteins based on ability to penetrate the graft layer would occur, if at all, at a grafting density intermediate between 0.1 and 0.39 /cm2. Again the MPC and OEGMA surfaces behaved similarly. It is suggested that the main determinant of the protein resistance of these surfaces is the “water barrier layer” resulting from their hydrophilic character. In turn the efficacy of the water barrier depends on the monomer density in the graft layer.

Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma.

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.

Interaction of fibrinogen with solid surfaces of varying charge and hydrophobic—hydrophilic balance: I. Adsorption isotherms

Abstract Adsorption of fibrinogen to a series of polyelectrolyte complex surfaces of varying charge density (bulk ion-exchange capacity ranging from +1.21 to −1.33 meq g −1 ) and hydrophilicity (water content from 4.5 to 60 wt%) was studied using an iodine-labeling technique under static conditions. In the high concentration region of the isotherms ( c > 0.5 mg ml −1 ) pseudoplateaux were observed with adsorption values in the range expected for a close-packed monomolecular layer. The surface of a material of +0.8 meq g −1 ion-exchange capacity showed a particularly high value. At low concentrations ( c −2 mg ml −1 ), the data were found to fit a Freundlich-type relation with identical slopes for all surfaces. Free energies of adsorption estimated from these data were of the order of 7 kcal mole −1 . These values are indicative of nonspecific relatively weak bonding perhaps of the hydrophobic interaction type. It is concluded that fixed surface charge of the order of magnitude of that used in the present work has little effect on the equilibrium (isotherm) aspects of protein adsorption.

Lysine-PEG-modified polyurethane as a fibrinolytic surface: Effect of PEG chain length on protein interactions, platelet interactions and clot lysis.

Fibrinolytic polyurethane surfaces were prepared by conjugating lysine to the distal terminus of surface-grafted poly(ethylene glycol) (PEG). Conjugation was through the alpha-amino group leaving the epsilon-amino group free. Lysine in this form is expected to adsorb both plasminogen and t-PA specifically from blood. It was shown in previous work that the PEG spacer, while effectively resisting nonspecific protein adsorption, was a deterrent to the specific binding of plasminogen. In the present work, the effects of PEG spacer chain length on the balance of nonspecific and specific protein binding were investigated. PEG-lysine (PEG-Lys) surfaces were prepared using PEGs of different molecular weight (PEG300 and PEG1000). The lysine-derivatized surfaces with either PEG300 or PEG1000 as spacer showed good resistance to fibrinogen in buffer. The PEG300-Lys surface adsorbed plasminogen from plasma more rapidly than the PEG1000-Lys surface. The PEG300-Lys was also more effective in lysing fibrin formed on the surface. These results suggest that the optimum spacer length for protein resistance and plasminogen binding is relatively short. Immunoblots of proteins eluted after plasma contact confirmed that the PEG-lysine surface adsorbed plasminogen while resisting most of the other plasma proteins. The hemocompatibility of the optimized PEG-lysine surface was further assessed in whole blood experiments in which fibrinogen adsorption and platelet adhesion were measured simultaneously. Platelet adhesion was shown to be strongly correlated with fibrinogen adsorption. Platelet adhesion was very low on the PEG-containing surfaces and neither surface-bound lysine nor adsorbed plasminogen promoted platelet adhesion.