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.     

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.     

The role of adsorbed fibrinogen in platelet adhesion to polyurethane surfaces: a comparison of surface hydrophobicity, protein adsorption, monoclonal antibody binding, and platelet adhesion

Ten specially synthesized polyurethanes (PUs) were used to investigate the effects of surface properties on platelet adhesion. Surface composition and hydrophilicity, fibrinogen (Fg) and von Willebrand's factor (vWf) adsorption, monoclonal anti-Fg binding, and platelet adhesion were measured. PUs preadsorbed with afibrinogenemic plasma or serum exhibited very low platelet adhesion, while adhesion after preadsorption with vWf deficient plasma was not reduced, showing that Fg is the key plasma protein mediating platelet adhesion under static conditions. Platelet adhesion to the ten PUs after plasma preadsorption varied greatly, but was only partially consistent with Fg adsorption. Thus, while very hydrophilic PU copolymers containing PEG that had ultralow Fg adsorption also had very low platelet adhesion, some of the more hydrophobic PUs had relatively high Fg adsorption but still exhibited lower platelet adhesion. To examine why some PUs with high Fg adsorption had lower platelet adhesion, three monoclonal antibodies (mAbs) that bind to sites in Fg thought to mediate platelet adhesion were used. The antibodies were: M1, specific to gamma-chain C-terminal; and R1 and R2, specific to RGD containing regions in the alpha-chain N- and C-terminal, respectively. Platelet adhesion was well correlated with M1 binding, but not with R1 or R2 binding. When these mAbs were incubated with plasma preadsorbed surfaces, they blocked adhesion to variable degrees. The ability of the R1 and R2 mAbs to partially block adhesion to adsorbed Fg suggests that RGD sites in the alpha chain may also be involved in mediating platelet adhesion and act synergistically with the C-terminal of the gamma-chain.     

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.     

The effect of adsorbed fibrinogen, fibronectin, von Willebrand factor and vitronectin on the procoagulant state of adherent platelets

Procoagulant (activated) platelets provide a site for assembly of the prothrombinase complex which can rapidly convert prothrombin into thrombin (a potent inducer of clot formation). Previously, we reported that adhesion of platelets to surfaces preadsorbed with blood plasma caused them to become procoagulant. In the present study we investigated the effect of adsorbed adhesion proteins (fibrinogen (Fg), fibronectin (Fn), von Willebrand factor (vWF) and vitronectin (Vn)) on the procoagulant activity of adherent platelets. Adsorbed Fn, vWF and Fg promoted platelet adhesion in the following order: Fn < vWF = Fg. However, these proteins promoted platelet activation (thrombin generation per adherent platelet) in the following order: Fg < Fn < vWF. Adsorption with a series of dilutions of normal plasma, serum, and plasmas deficient in or depleted of von Willebrand factor (de-vWF), fibronectin (de-Fn), vitronectin (de-Vn), or both vitronectin and fibronectin (de-VnFn) resulted in varied platelet adhesion, but little difference in platelet activation. However, preadsorption with dilute de-vWF plasma induced lower procoagulant activity than normal plasma. Preadsorption with normal plasma resulted in higher levels of platelet activation than preadsorption with Fg, suggesting that adsorption of plasma proteins other than Fg caused the high levels of activation observed for plasma preadsorbed surfaces.     

Variations in the ability of adsorbed fibrinogen to mediate platelet adhesion to polystyrene-based materials: a multivariate statistical analysis of antibody binding to the platelet binding sites of fibrinogen

Platelet adhesion to the surfaces of biomaterials preadsorbed with plasma previously has been shown to be mediated exclusively by surface-bound fibrinogen and does not seem to involve the other adhesion proteins in plasma (Tsai et al., J Biomed Mater Res 2002;60:348-359). In this study, the influence of surface-bound fibrinogen on platelet adhesion to five different types of polystyrene-based microtiter plates preadsorbed with plasma was analyzed relative to the amount of adsorbed fibrinogen and monoclonal antibody binding to the adsorbed fibrinogen. There was no significant correlation between platelet adhesion and the absolute amount of adsorbed fibrinogen. However, platelet adhesion was positively correlated to the ability of the adsorbed fibrinogen to bind three types of monoclonal antibodies. The antibodies used bound to the sites on fibrinogen thought to be involved in platelet binding (the two gamma chain C-terminal dodecapeptides and the RGDF and RGDS sequences in each of the Aalpha chains). A partial least-squares calibration model was used to analyze the relative importance of these binding sites in fibrinogen to platelet adhesion. The gamma chain C-terminal dodecapeptide was shown to be the most important site in adsorbed fibrinogen in mediating platelet 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.     

Fibrinogen and von Willebrand's factor adsorption are both required for platelet adhesion from sheared suspensions to polyethylene preadsorbed with blood plasma

Previous studies showed that platelet adhesion to biomaterials from static suspensions was greatly increased by the adsorption of even very small amounts (<5 ng/cm2) of fibrinogen (Fg). In this study, the sensitivity of platelet adhesion to Fg was reexamined by measuring platelet adhesion under flow conditions. The role of adsorbed von Willebrand's factor (vWf) was also studied. Polyethylene (PE) tubing was preadsorbed with Fg, vWf, vWf-deficient plasma, and Fg-deficient plasma or serum with added Fg, and Fg adsorption measured with 125I Fg. Platelets in a red blood cell suspension were passed through the tubes at either low (50 s(-1)) or high (500 or 1000 s(-1)) shear rates and adhesion measured with an improved LDH assay. Adhesion from flowing suspensions measured after preadsorption with afibrinogenemic plasma or serum was very low, but increased greatly with addition of Fg. Less than 10 ng/cm2 of adsorbed Fg was enough to greatly enhance platelet adhesion. Adhesion at high shear was also strongly affected by vWf, as platelet adhesion at 500 s(-1) to PE preadsorbed with vWf-deficient plasma decreased by more than tenfold compared to adhesion at 50 s(-1), but platelet adhesion to PE preadsorbed with normal plasma increased about eightfold when shear rate was increased. The results show that very low amounts of adsorbed Fg are able to support platelet adhesion under shear flow. However, adsorbed vWf also appears to play an important cofactor role in platelet adhesion to biomaterials, as its presence greatly augments platelet adhesion under high shear.     

Platelet adhesion to polystyrene-based surfaces preadsorbed with plasmas selectively depleted in fibrinogen, fibronectin, vitronectin, or von Willebrand's factor

Four plasma proteins have been shown to be able to mediate platelet adhesion to synthetic materials when they are adsorbed as purified proteins: fibrinogen (Fg), fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWF). Among them, Fg is thought to play a leading role in mediating platelet adhesion to plasma-preadsorbed biomaterials, but this has been established for only three types of materials so far in our laboratory. Furthermore, the role of Fn, Vn, and vWF in mediating platelet adhesion to plasma-preadsorbed surfaces is still unclear. The aim of the current study was to assess the importance of Fg, Fn, Vn, and vWF in mediating platelet adhesion to a series of polystyrene-based surfaces. The strategy applied in the present investigation was to compare platelet adhesion to surfaces preadsorbed with normal plasma, plasma selectively depleted in Fn or Vn or both Fn and Vn, plasma from donors who were genetically deficient in vWF, and serum. Few platelets adhered to the surfaces preadsorbed with serum, whereas depletion of Fn, Vn, or vWF from plasma did not decrease platelet adhesion significantly. Replenishment of exogenous Fg to serum before protein adsorption restored platelet adhesion to the surfaces, suggesting that Fg was the major plasma protein that mediated platelet adhesion. Also, we found that a surface density of adsorbed Fg far below the amount that usually adsorbs to synthetic surfaces was sufficient to support full-scale platelet adhesion.     

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.     

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.     

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.     

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.     

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.     

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.     

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 α-amino group leaving the ε-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.     

Introduction of surface functional groups onto biomaterials by glow discharges

An attempt was made to graft the monomer HEMA to the polymer surface by "Glow discharge" technique. Experiments were carried out for different surfaces varying the exposure times of samples to HEMA and also as a function of glow discharge time. It was found that as the percentage of grafting increases the hydrophilicity also increases. Contact angle measurements were performed on these substrates, which confirmed the hydrophilic nature of the grafted samples compared to the controls. The role of protein adsorption and their effects to modulate the blood polymer interaction is briefly discussed. When a foreign material comes in contact with blood, the initial event is the adsorption of plasma proteins in parallel with the adhesion of platelets to the material. Albuminated surfaces discourage platelet adhesion while fibrinogen enhances the platelet attachment and thrombosis. Hence a decreased ratio of fibrinogen to albumin on a substrate can be correlated as an indication to its improved blood compatibility. Fibrinogen to albumin ratios of the grafted samples showed a reduction, indicating that albumin adsorption is high; which may make the modified surfaces non-thrombogenic.     

Adsorption of proteins from plasma at polyester non-wovens.

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

Glow discharge plasma deposition of tetraethylene glycol dimethyl ether for fouling-resistant biomaterial surfaces.

The glow discharge plasma deposition (GDPD) of tetraethylene glycol dimethyl ether is introduced as a novel method for obtaining surfaces that are resistant to protein adsorption and cellular attachment. Analysis of films by x-ray photoelectron spectroscopy and several biological assays indicate the formation of a fouling-resistant, PEO-like surface on several substrata (e.g., glass, polytetrafluoroethylene, polyethylene). Adsorption of 125I-radiolabelled proteins (fibrinogen, albumin and IgG) from buffer and plasma was very low (typically less than 20 ng/cm2) when compared to the untreated substrata, which exhibited much higher levels of protein adsorption. Not all coated substrata adsorbed equal amounts of protein (e.g., coated glass samples typically adsorbed more protein than coated polyethylene or coated polytetrafluoroethylene samples), suggesting that the substratum used may affect the amount of protein adsorbed. Measurement of dynamic platelet adhesion, using epifluorescent video microscopy, and endothelial cell attachment further demonstrates the short-term nonadhesiveness of these surfaces.     

Platelet Function Analyzer: Shear Activation of Platelets in Microchannels

Platelet function is suggestive of pathological conditions in cardiovascular diseases. With current insufficient prognostic devices, the need exists for a device to assess complete platelet function. This work presents a preliminary microfluidic device for such analysis based on platelet adhesion under shear flow conditions. In this novel device, polydimethylsiloxane (PDMS) microchannels were coated using a layer-by-layer self-assembly technique to provide controlled nanometer-thick layers of fibrinogen. Anticoagulated platelet rich plasma labeled with a fluorescein isothiocynate-tagged anti-glycoprotein IIb/IIIa-antibody and acridine orange was passed through these micro-channels at various time-averaged shear rate values. Fluorescence assays confirmed shear-dependent adhesion of platelets in the microchannels. Control experiments showed that the extent of adhesion on bare PDMS surfaces was less than on the surfaces coated with fibrinogen at similar shear rates. Fluorescent microscopy demonstrated that the extent of platelet adhesion to the fibrinogen substrate depended on shear rate. The extent of adhesion was modeled as a third order polynomial in shear rate.