Passivating protein coatings for implantable glucose sensors: evaluation of protein retention

The long-term function of implantable biosensors is limited by the foreign-body reaction (FBR). Since the acute phase of the FBR involves macrophage attachment mediated by adsorbed fibrinogen, preadsorption, and retention of other proteins might reduce the FBR. The retention of preadsorbed albumin, hemoglobin, von Willebrand's factor, and high-molecular-weight kininogen was therefore measured after exposure to plasma. The retention of preadsorbed proteins after incubation with monocyte cultures and implantation in rats was also measured. Fibrinogen adsorption from plasma to the preadsorbed surfaces was also measured. Hemoglobin adsorption was higher than that for other proteins, and it also had the greatest retention after exposure to blood plasma. When surfaces preadsorbed with hemoglobin were incubated with monocytes, more of the hemoglobin was displaced than that after incubation in plasma, while still more hemoglobin was displaced when the surfaces were implanted in vivo. Protein preadsorption on polystyrene greatly reduced fibrinogen adsorption. However, polyurethane surfaces used for glucose sensors had low fibrinogen adsorption compared with polystyrene, and this low level was not further reduced by preadsorption with other proteins. Preadsorbed proteins on polymers appear to be removed by passive exchange and/or displacement by plasma proteins and by proteases released by monocytes.     

The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin

The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to ±80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin.     

The effects of surface chemistry and adsorbed proteins on monocyte/macrophage adhesion to chemically modified polystyrene surfaces

Monocytes and macrophages play critical roles in inflammatory responses to implanted biomaterials. Monocyte adhesion may lead to macrophage activation and the foreign body response. We report that surface chemistry, preadsorbed proteins, and adhesion time all play important roles during monocyte adhesion in vitro. The surface chemistry of tissue culture polystyrene (TCPS), polystyrene, Primaria, and ultra low attachment (ULA) used for adhesion studies was characterized by electron spectroscopy for chemical analysis. Fibrinogen adsorption measured by (125)I-labeled fibrinogen was the lowest on ULA, higher on TCPS, and the highest on polystyrene or Primaria. Monocyte adhesion on protein preadsorbed surfaces for 2 h or 1 day was measured with a lactate-dehydrogenase method. Monocyte adhesion decreased over time. The ability of preadsorbed proteins to modulate monocyte adhesion was surface dependent. Adhesion was the lowest on ULA, higher and similar on TCPS or polystyrene, and the highest on Primaria. Monocyte adhesion on plasma or fibrinogen adsorbed surfaces correlated positively and linearly to the amount of adsorbed fibrinogen. Preadsorbed fibronectin, immunoglobulin G, plasma, or serum also promoted adhesion compared with albumin preadsorbed or uncoated surfaces. Overall, biomaterial surface chemistry, the type and amount of adsorbed proteins, and adhesion time all affected monocyte adhesion in vitro.     

Effect of adsorbed von Willebrand factor and fibrinogen on platelet interactions with synthetic materials under flow conditions

The effect of adsorbed fibrinogen (Fg) and von Willebrand factor (vWf) on platelet adhesion at low or high shear rate to several materials was studied. The materials studied were polyethylene terephthalate (PET), polystyrene (PS), glass, and tetraglyme-coated PET. The materials were preadsorbed with normal plasma, serum, and Fg-deficient plasma replenished with various amounts of Fg, and vWf-deficient plasma with or without added vWf. Platelet adhesion to PET preadsorbed with Fg-deficient plasma or serum was low at either low or high shear rate, but increased as Fg was added to the preadsorption media. However, the effect of added Fg on adhesion at the higher shear rate was much greater on surfaces preadsorbed with plasma than for serum, probably due to the much lower vWf concentration in serum in comparison to plasma. Platelet adhesion to either polystyrene or glass preadsorbed with normal plasma was much higher at high shear than low shear, but when vWf-deficient plasma was used to preadsorb these surfaces, adhesion was much less at the higher shear rate than at low shear rate. Platelet adhesion to polystyrene preadsorbed with vWf-deficient plasma to which vWf was added was higher at high shear rate than low shear rate. These results show that under high shear rate, both Fg and vWf are required for platelet adhesion on synthetic biomaterials. The results suggest that developing surfaces that adsorb low amounts of vWf is a good approach to improving the blood compatibility of biomaterials.     

Effects of adsorbed proteins and surface chemistry on foreign body giant cell formation, tumor necrosis factor alpha release and procoagulant activity of monocytes

The adhesion and activation of monocytes and macrophages are thought to affect the foreign body response to implanted medical devices. However, these cells interact with devices indirectly, because of the prior adsorption of proteins. Therefore, we preadsorbed several "model" biomaterial surfaces with proteins and then measured foreign body giant cell (FBGC) formation, tumor necrosis factor alpha (TNFalpha) release, and procoagulant activity. The model surfaces were tissue culture polystyrene (TCPS), untreated polystyrene (PS), and Primaria, whereas the proteins used were albumin, fibronectin, fibrinogen, and immunoglobulin. FBGC formation, TNFalpha release, and procoagulant activity of monocytes were the highest for surfaces preadsorbed with IgG. FBGC formation was lower on surfaces with adsorbed fibrinogen and fibronectin than on uncoated surfaces. TNFalpha release and procoagulant activity of monocytes were similar on surface adsorbed with fibrinogen, fibronectin, or albumin. Monocyte activation was also affected by the surface chemistry of the substrates, because FBGC formation was the highest on PS and the lowest on TCPS. Monocyte procoagulant activity was the highest on Primaria. Adsorbed proteins and surface chemistry were found to have strong effects on FBGC formation, monocyte TNFalpha release, and procoagulant activity in vitro, providing support for the idea that these same variables could affect macrophage-mediated foreign body response to biomaterials in vivo.     

Interactions of fibrinolytic system proteins with lysine-containing surfaces

Studies on the interactions of tissue plasminogen activator (tPA) and plasminogen with polyurethane surfaces containing epsilon-lysine moieties (epsilon-amino group free) are reported. These surfaces are considered to have the potential to dissolve nascent clots that may be formed on them. For adsorption from both single protein solutions and plasma, the surfaces were found to have a high capacity for tPA as well as plasminogen. A significant fraction of preadsorbed tPA was displaced from the epsilon-lysine surfaces upon contact with plasma. These surfaces, when preadsorbed with tPA and then incubated with plasma, were able to dissolve incipient clots formed around them. However, the clot-dissolving capacity diminished as the time of plasma incubation increased, presumably due to loss of tPA. It was also shown that in plasma, preadsorbed tPA is displaced from these surfaces largely by plasminogen, which thus appears to have a greater binding affinity than tPA for the epsilon-lysine moieties. Finally, it was found that in plasma, the epsilon-lysine surfaces interact with plasminogen in a dynamic manner, and that about 70% of the bound plasminogen is exchanging continuously with plasminogen in the plasma.     

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

The purpose of this study was to further investigate the role of fibrinogen adsorbed from plasma in mediating platelet adhesion to polymeric biomaterials. Polystyrene was used as a model hydrophobic polymer; i.e., we expected that the role of fibrinogen in platelet adhesion to polystyrene would be representative of other hydrophobic polymers. Platelet adhesion was compared to both the amount and conformation of adsorbed fibrinogen. The strategy was to compare platelet adhesion to surfaces preadsorbed with normal, afibrinogenemic, and fibrinogen-replenished afibrinogenemic plasmas. Platelet adhesion was determined by the lactate dehydrogenase (LDH) method, which was found to be closely correlated with adhesion of 111In-labeled platelets. Fibrinogen adsorption from afibrinogenemic plasma to polystyrene (Immulon I(R)) was low and <10 ng/cm2. Platelet adhesion was absent on surfaces preadsorbed with afibrinogenemic plasma when the residual fibrinogen was low enough (<60 microg/mL). Platelet adhesion was restored on polystyrene preadsorbed with fibrinogen-replenished afibrinogenemic plasma. Addition of even small, subnormal concentrations of fibrinogen to afibrinogenemic plasma greatly increased platelet adhesion. In addition, surface-bound fibrinogen's ability to mediate platelet adhesion was different, depending on the plasma concentration from which fibrinogen was adsorbed. These differences correlated with changes in the binding of a monoclonal antibody that binds to the Aalpha chain RGDS (572-575), suggesting alteration in the conformation or orientation of the adsorbed fibrinogen. Platelet adhesion to polystyrene preadsorbed with blood plasma thus appears to be a strongly bivariate function of adsorbed fibrinogen, responsive to both low amounts and altered states of the adsorbed molecule.     

Protein adsorption on titanium surfaces and their effect on osteoblast attachment

The objective of this study was to investigate the adsorption of albumin and fibronectin on titanium (Ti) surfaces and the effect of preadsorbed albumin and fibronectin on osteoblast attachment in vitro. Bovine serum albumin and bovine fibronectin were used in this study. Maximum adsorption of bovine serum albumin and fibronectin on Ti surfaces was observed to occur after 180-min incubation. In the presence of preadsorbed proteins, osteoblast attachment on Ti surfaces was observed to be enhanced compared to control Ti surfaces. However, cell attachment was affected by the types of protein adsorbed. Preadsorbed albumin was observed to have no significant effect on the amount of osteoblast cells attached. In comparison to control Ti surface and Ti surfaces preadsorbed with albumin, Ti surfaces preadsorbed with fibronectin for 15 min was observed to significantly increase osteoblast cell attachment, whereas Ti surfaces preadsorbed with fibronectin for 180 min did not affect cell attachment. In addition, cell morphology of the attached cells on protein preadsorbed Ti surfaces was not affected by the type of protein used in this study. It was concluded from this study that the concentration of fibronectin adsorbed on Ti surfaces was higher compared to albumin. In addition, it was also concluded that the concentration of fibronectin on Ti surfaces plays a role in governing cell attachment.     

Adsorption of thrombin from buffer and modified plasma to polystyrene resins containing sulphonate and sulphamide arginyl methyl ester groups

The adsorption of albumin and thrombin to insoluble modified polystyrene resins bearing sulphonate (PSSO3) and L-arginyl methyl ester groups (PAOM) was investigated in both purified and plasma systems. Radioiodinated proteins were used to follow adsorption in a 'minicolumn' experiment. Albumin adsorption was found to follow the Langmuir model and specific surface areas of the various resins were evaluated from plateau albumin adsorption data. The adsorption isotherms of thrombin both in buffer and in antithrombin III/fibrinogen-free plasma were also found to be Langmuir-like, and the quantities adsorbed at the isotherm plateaux are in the monolayer range. Analysis of the isotherms at 4 degrees and 37 degrees C for the purified system shows that adsorption is endothermic. Adsorption capacities in plasma remain high (30-50% of those in the purified system) despite competition from the other plasma proteins. These data confirm the strong affinity and selectivity of these resins for thrombin.     

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.     

The role of fibronectin in platelet adhesion to plasma preadsorbed polystyrene

Platelet adhesion to synthetic surfaces that come in contact with blood is mediated by the adsorption of adhesive plasma proteins, especially fibrinogen. However, the roles of other adhesive proteins, such as fibronectin, vitronectin, and von Willebrand factor in platelet adhesion are not yet clear. In this study, the role of fibronectin in platelet adhesion to surfaces was assessed using three approaches. First, platelet adhesion was measured on Immulon I preadsorbed with fibronectin-depleted plasma or fibronectin-depleted plasma replenished with increasing amount of fibronectin. Under these conditions, fibronectin adsorbed from plasma did not have any effect on platelet adhesion, while fibrinogen played a major role in mediating platelet adhesion. Since fibronectin might play a role in platelet adhesion to surfaces which adsorb little or no fibrinogen, we also used two other strategies to assess the potential role of fibronectin. One was to use platelets treated with a platelet activation inhibitor, prostaglandin E1, which prevents the activation of platelet fibrinogen receptor GP IIb/IIIa. The adhesion of prostaglandin E1-treated platelets to Immulon I preadsorbed with plasma was greatly decreased compared to that of untreated platelets, but was increased by the addition of supernormal concentrations of fibronectin to the plasma. This suggests that GP Ic/IIa, rather than GP IIb/IIIa, might be the platelet receptor which is responsible for platelet adhesion to surface-bound fibronectin. Finally, we studied the effect of fibronectin on platelet adhesion to surfaces preadsorbed with fibronectin-depleted afibrinogenemic plasma. We found that fibronectin re-addition to fibronectin-depleted afibrinogenemic plasma increased platelet adhesion. However, our most important finding was that fibronectin seems to play little or no role in mediating platelet adhesion to polystyrene surfaces preadsorbed with normal plasma.     

Poly(ethylene oxide)-modified carboxylated polystyrene latices - immobilization chemistry and protein adsorption

α,ω-Diamino poly(ethylene oxides) (PEOs) with different molecular weights (148, 1000, and 3400) were covalently immobilized onto carboxylated polystyrene latices. The immobilization of PEO was carried out with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) in aqueous media. The reaction conditions were optimized to obtain a maximal coupling of PEO. The degree of coupling was determined by the surface concentration of amino groups. The maximal surface concentrations of amino groups were close to what is expected for a complete coverage of the surface with PEO. Adsorption of albumin from a buffer solution onto PEO-containing surfaces was about 85% less than the albumin uptake by unmodified polystyrene latices. Protein adsorption from plasma dilutions was lower on surfaces containing PEO molecules with a higher molecular weight. The reduction of the protein uptake from plasma by surfaces containing PEO-3400 molecules was only 40% compared to the adsorption to unmodified surfaces. These results indicate that plasma proteins have a low affinity for surfaces modified with PEO. However the PEO modified surfaces are by no means 'protein resistant' when exposed to plasma.     

The interactions between antithrombin III,thrombin and surface immobilized heparin

The interactions between antithrombin III (ATIII), thrombin, and surface immobilized heparin were investigated. Carboxylated polystyrene modified with covalently immobilized albumin-heparin conjugate contain sites which can bind ATIII from buffer and plasma solutions. Approximately 65% of the ATIII molecules present at the heparinized surface either adsorbed from a buffer or plasma solution, exchanged with ATIII in buffer solution. The exchange between surface bound ATIII and ATIII in solution was repeated several times on the same heparinized surface. The number of binding sites that could bind and release ATIII was much higher when the heparinized surface was exposed to an ATIII containing buffer solution than to a plasma solution. The reduction in binding sites available for ATIII using plasma solutions as compared to buffer solutions could be explained by the competition of other plasma proteins with ATIII for the heparinized surface. It was observed that heparin binding proteins were able to compete with ATIII for binding to the immobilized heparin. Furthermore adsorption of proteins on the heparinized surface significantly reduced the availability of binding sites for ATIII. Exposure of thrombin to the heparinized surface resulted in thrombin activity at the surface. The thrombin activity on the heparinized surfaces was lower on surfaces with a higher ATIII concentration. The activity of surface bound thrombin was not affected by the presence of other plasma proteins. Enzymatically active thrombin molecules present at the heparinized surface were completely inactivated when the surface was exposed to a solution containing ATIII. The inactivation rate of surface bound thrombin by ATIII was higher than the rate of the uncatalyzed inactivation of thrombin in solution. Part of the Thrombin-Antithrombin III (TAT) complexes (10-20%) that were formed upon inactivation of thrombin remained bound to the heparinized surface. In general it was concluded that only the surface immobilized heparin molecules that can bind ATIII in a reversible way determine the anticoagulant properties of the surface. The mechanism of inactivation of a protease on a heparinized surface depends either on the catalytic effect of heparin on the inactivation rate of proteases by ATIII or on an increased uncatalytic inactivation due to increased concentrations of ATIII near the surface as compared to the concentration of ATIII in the bulk phase.     

Co-adsorbed fibrinogen and von Willebrand factor augment platelet procoagulant activity and spreading

Previously we observed that platelets adherent to surfaces preadsorbed with blood plasma exhibited 1.3 to 2.4 times greater procoagulant activity than platelets on surfaces adsorbed with fibrinogen (Fg) only. These observations suggested that the adhesion proteins adsorbed from plasma may activate platelets in a cooperative, or synergistic manner. In the present study, polystyrene surfaces adsorbed with both Fg and vWF induced up to three times greater procoagulant activity than surfaces adsorbed with Fg or vWF only. The amounts of Fg and vWF adsorbed from binary mixtures that resulted in increased procoagulant activity were found to be similar to the amounts that adsorbed to PS from 100% plasma. The effect of adsorbed adhesion proteins on platelet spreading was also investigated. The proportion of fully spread platelets increased, depending on the adhesion protein preadsorbed to the surface, in the following order: vWF < Fg < Fn < (vWF + Fg) < Vn < plasma.     

Selective protein adsorption modulates platelet adhesion and activation to oligo(ethylene glycol)-terminated self-assembled monolayers with C18 ligands

This study focuses on the selective binding of albumin to a nanostructured surfaces to inhibit other blood proteins from adsorbing thereby reducing platelet adhesion and activation. Tetra (ethylene-glycol)-terminated self-assembled monolayers (EG4 SAMs) with different percentages of C18 ligands on the surface were characterized by contact angle measurements, X-ray photoelectron microscopy, infrared reflection-absorption spectroscopy, and ellipsometry. A specific surface (2.5% C18 SAM) was found to be selective for human serum albumin (HSA) in the presence of both albumin and fibrinogen (HFG). The importance of this concentration of C18 ligands was stressed in reversibility studies since that surface exchanged almost all the preadsorbed HSA by HSA in solution, but not by HFG. The effect of protein adsorption in the subsequent adhesion and activation of platelets was studied by pre-immersing the surfaces in albumin and plasma before contact with platelets. Scanning electron microscopy and glutaraldehyde induced fluorescence technique images showed that as surfaces got more hydrophobic due to the immobilization of C18 ligands, the number of adherent platelets increased and their morphology changed from round to fully spread. Pre-immersion in HSA led to an 80% decrease in platelet adhesion and reduction of activation. Pre-immersion in 1% plasma was only relevant in 2.5% C18 SAMs since this was the only surface that demonstrated less adhesion of platelets comparing with buffer pre-immersion. However, they still adsorb more platelets then when HSA was preadsorbed. This was confirmed in competition studies between HSA and plasma that suggested that other plasma proteins were also adsorbing to this surface.     

Osteoblast adhesion and morphology on TiO2 depends on the competitive preadsorption of albumin and fibronectin

This study aims at assessing the influence of the competitive preadsorption of human serum albumin (HSA) and human plasma fibronectin (FN) from binary solutions and 10% plasma on MC3T3-E1 osteoblast adhesion and morphology on two types of TiO2 substrates. One was commercially pure titanium with a titanium oxide layer formed in an H2O2 solution and the other TiO2 sputtered on Si (Sousa et al., Langmuir 2004; 20:9745-9754.). The strategy applied in the present investigation was to compare osteoblast adhesion to surfaces preadsorbed with HSA, FN, HSA/FN = 1, HSA/FN = 200, and 10% plasma. The adsorption of proteins was evaluated measuring the amount and the effectiveness of binding with radiolabeled proteins, 125 I-FN and 125 I-HSA. Our results indicated that MC3T3-E1 osteoblast adhesion correlates well with the amounts of FN and HSA adsorbed on TiO2 surfaces. Also, we found that fewer osteoblasts adhered to both substrates preadsorbed with HSA, HSA/FN = 200, and 10% plasma, after 4 and 24 h, than to the surfaces preadsorbed with FN and HSA/FN = 1. For the latter, FN was able to compensate the inhibitory effect of HSA on osteoblast adhesion. Therefore, the presence of lower amounts of coadsorbed albumin may improve presentation of FN in a more integrin-recognized conformation, suggesting that some degree of molecular packing prevents loss of integrin-binding activity. FN reversibility does not seem to be dependent on the HSA/FN adsorption mass ratio in solution, suggesting that FN competitively adsorbs to TiO2 in a favorable conformation and does not suffers subsequent conformational changes allowing exchange with other FN molecules in solution.     

Protein adsorption on biomedical polymers with a phosphorylcholine moiety adsorbed with phospholipid.

The effects of phospholipid adsorption onto the polymer surface during adsorption of plasma proteins were investigated. When a polymer with the phosphorylcholine moiety, 2-methacryloyloxyethyl phosphorylcholine (MPC) co-polymer, was treated with dipalmitoylphosphatidylcholine (DPPC) liposome solution, an organized adsorption layer of DPPC was formed on the MPC co-polymer surface, which was confirmed by differential scanning calorimetric analysis and X-ray photoelectron spectroscopy. On the other hand, an organized layer of DPPC on poly(n-butyl methacrylate) and poly(2-hydroxyethyl methacrylate) could not be found. The amount of albumin adsorbed on the polymer surfaces was decreased by pretreatment of the surface with DPPC liposome solution in every polymer case. The smallest amount of adsorbed proteins was found on the MPC co-polymer. Protein adsorption on the surface of MPC co-polymers from the plasma was also small. The difference in protein adsorption on the polymers probably reflects the difference in the orientation of the phospholipid molecules which cover the polymer surface.     

Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.     

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.     

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.