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.     

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 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.     

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.     

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 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.     

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.     

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.     

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.     

Effects of base material,plasma proteins and FGF2 on endothelial cell adhesion and growth

Blood-contacting materials rapidly acquire a coating of plasma proteins which can lead to local platelet activation and thrombus formation. This phenomenon seriously limits the usefulness of small diameter synthetic vascular grafts. One solution to this problem is to pre-seed or encourage in situ colonisation of the material with endothelial cells to maintain a non-thrombogenic surface. We have investigated the effect of contact with plasma and serum on the subsequent ability of human endothelial cells to adhere to model hydrophobic and hydrophylic plastic surfaces, and the effect of surface bound fibroblast growth factor 2 (FGF2) on endothelial cell proliferation. Cell adhesion was mainly dependent on adsorbed fibrinogen or vitronectin, depending on the polymer surface, and correlated with antibody binding to these molecules rather than quantitative surface concentrations. Cell proliferation was directly correlated with surface bound FGF2. Surface binding of the latter was controlled both by the chemical nature of the polymer surface and by the presence of FGF-binding molecules adsorbed on the surface. FGF2 bound specifically to surface-adsorbed fibrinogen, fibronectin and vitronectin as well as to pre-coated heparan sulphate proteoglycan, perlecan. Binding was significantly inhibited by plasma and serum which contained high levels of FGF2 binding proteins. To be effective in supporting endothelialisation of vascular grafts in vivo, surface-bound FGF2 would need to be protected from surface dissociation into the circulating blood.     

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.     

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.     

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

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

Changes in 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.     

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.     

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

The adsorption and subsequent detergent elutability of fibrinogen and albumin were measured on various treated and untreated polymer films in order to determine whether the relative adsorption of these proteins was responsible for the enhanced thromboresistance of Dacron vascular grafts treated with tetrafluoroethylene in a radio frequency glow discharge (RFGD) apparatus. Fluorocarbon-coated surfaces varying in the relative proportions of CF, CF2, and CF3 groups and in the ratio of fluorine to carbon were prepared by RFGD treatment of poly(ethylene terephthalate) (PET) films with tetrafluoroethylene or perfluoropropane. The adsorption of fibrinogen and albumin to these fluorocarbon-coated surfaces was comparable to the adsorption of the proteins to polytetrafluoroethylene (PTFE) and PET. However, the elutability of fibrinogen and albumin from the RFGD fluorocarbon surfaces with sodium dodecyl sulfate was much lower than that from PTFE or PET. Other RFGD treatments of PET, such as ethylene deposition or argon etching, did not reduce the extent of albumin elutability as dramatically as did the RFGD fluorocarbon treatments. The strong albumin binding to RFGD fluorocarbon surfaces may be exploited clinically to enhance the retention of albumin preadsorbed to blood-contacting surfaces to render them thromboresistant.     

Adsorbed serum proteins responsible for surface dependent human macrophage behavior

Substrate specific cellular responses are the result of a complex biological system that includes protein adsorption, receptor-ligand binding, and signal transduction. This investigation attempted to identify specific proteins adsorbed from human serum that may be responsible for the previously reported in vitro surface dependent behavior of human macrophages and foreign body giant cells (FBGCs). The adsorption of human albumin, alpha(2)-macroglobulin, complement factor 3b, fibronectin, IgG, thrombospondin, vitronectin (VN), and von Willebrand factor (vWF) from a 25% serum solution was quantified with (125)I-labeled protein. Adsorption substrates included clean glass, alkyl-silane modified glass, amino-silane modified glass, poly(ethylene oxide) (PEO)-coupled glass, and the reference biomaterials poly(etherurethane urea), Silastic(R), and poly(tetrafluoroethylene) (PTFE). Following quantification of 2-h adsorption, surfaces were treated with sodium dodecyl sulfate (SDS) and the level of adsorbed proteins remaining was quantified. The pre- and post-SDS adsorption were both compared to previously reported surface dependent in vitro macrophage and FBGC behavior on the same surfaces; however, no correlations could be made. Adsorption strength, defined as the percentage of initially adsorbed protein that remained adsorbed after SDS treatment, correlated well with previously reported in vitro cellular behavior indicating that adsorbed vWF, IgG, and VN may be involved in the modulation of adherent macrophage and FBGC behavior. Those surfaces that strongly adsorbed vWF also inhibited long-term macrophage adhesion, while those surfaces that strongly adsorbed IgG promoted long-term macrophage adhesion. In addition, the highest levels of FBGC formation had been observed only on those surfaces that strongly adsorbed VN. Subsequent human monocyte cultures on protein preadsorbed substrates confirmed the inhibitory effect of adsorbed vWF and the promoting effect of IgG on longterm macrophage adhesion as predicted by adsorption strength correlations. However, preadsorbed VN was not observed to modulate FBGC formation, which is in contrast to the conclusions of the adsorption correlations.     

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.     

Effects of base material,plasma proteins and FGF2 on endothelial cell adhesion and growth

Blood-contacting materials rapidly acquire a coating of plasma proteins which can lead to local platelet activation and thrombus formation. This phenomenon seriously limits the usefulness of small diameter synthetic vascular grafts. One solution to this problem is to pre-seed or encourage in situ colonisation of the material with endothelial cells to maintain a non-thrombogenic surface. We have investigated the effect of contact with plasma and serum on the subsequent ability of human endothelial cells to adhere to model hydrophobic and hydrophylic plastic surfaces, and the effect of surface bound fibroblast growth factor 2 (FGF2) on endothelial cell proliferation. Cell adhesion was mainly dependent on adsorbed fibrinogen or vitronectin, depending on the polymer surface, and correlated with antibody binding to these molecules rather than quantitative surface concentrations. Cell proliferation was directly correlated with surface bound FGF2. Surface binding of the latter was controlled both by the chemical nature of the polymer surface and by the presence of FGFbinding molecules adsorbed on the surface. FGF2 bound specifically to surface-adsorbed fibrinogen, fibronectin and vitronectin as well as to pre-coated heparan sulphate proteoglycan, perlecan. Binding was significantly inhibited by plasma and serum which contained high levels of FGF2 binding proteins. To be effective in supporting endothelialisation of vascular grafts in vivo, surface-bound FGF2 would need to be protected from surface dissociation into the circulating blood.     

Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry

Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDP peptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen.