Tetraglyme coatings reduce fibrinogen and von Willebrand factor adsorption and platelet adhesion under both static and flow conditions

Previous studies have showed that radio-frequency plasma deposited tetraglyme coatings greatly reduced fibrinogen adsorption (Gamma(Fg)) from highly diluted plasmas (0.1 and 1%) and subsequent platelet adhesion under static conditions. In this study, the protein resistant properties of tetraglyme were re-examined with high-concentration plasma, and subsequent platelet adhesion was measured under both static and flow conditions. The resistance of tetraglyme to vWf adsorption (Gamma(vWf)) and the role of vWf in platelet adhesion under flow were also investigated. Gamma(Fg) and Gamma(vWf) were measured with (125)I radiolabeled proteins. Flow studies were done at shear rates of 50 or 500 s(-1) by passing a platelet/red cell suspension through a GlycoTech flow chamber. When adsorbed from a series of increasing plasma concentrations, the adsorption of both proteins to tetraglyme increased steadily, and did not show a peak at intermediate dilutions, i.e., there was no Vroman effect. When plasma concentration was less than 10%, the tetraglyme surface was highly nonfouling, exhibiting ultralow Gamma(Fg) (less than 5 ng/cm(2)) and extremely low platelet adhesion under both static and flow conditions. However, when the adsorption was done from 100% plasma, Gamma(Fg) was much higher ( approximately 85 ng/cm(2)), indicating that tetraglyme surface may not be sufficiently protein-resistant in the physiological environment. To correlate platelet adhesion under flow with Gamma(Fg) and Gamma(vWf), a series of tetraglyme surfaces varying in ether content and protein adsorption was created by varying deposition power. On these surfaces, platelet adhesion at low shear rate depended only on the amount of Gamma(Fg), but under high shear, both Gamma(Fg) and Gamma(vWf) affected platelet adhesion. In particular, it was found that Gamma(vWf) must be reduced to less than 0.4 ng/cm(2) to achieve ultra low platelet adhesion under high shear.     

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.     

Glow discharge plasma treatment of polyethylene tubing with tetraglyme results in ultralow fibrinogen adsorption and greatly reduced platelet adhesion

Previous studies from our lab have shown that fibrinogen adsorption (Gamma(Fg)) must be reduced below 10 ng/cm(2) to significantly reduce platelet adhesion, and that radio frequency glow discharge (RFGD) treatment of polymeric films in the presence of tetraethylene glycol dimethyl ether (tetraglyme) can reduce Gamma(Fg) to the desired ultralow value. In this report, the effects of RFGD coatings of tetraglyme on the lumenal surface of PE tubing on Gamma(Fg) and on blood interactions both in vitro and ex vivo are described. Gamma(Fg) on the tetraglyme-coated PE tubing was reduced to the desired ultralow level (<10 ng/cm(2)), and we also observed a significant decrease in adsorption of von Willebrand's factor. In vitro platelet adhesion from washed platelet suspensions, platelet rich plasma, or whole blood to tetraglyme-coated PE tubing was decreased compared to PE, polyurethane, or silicone rubber tubes. In addition, thrombin generation by platelets adherent to tetraglyme-coated PE was also much less than by platelets adherent to PE. When inserted in an ex vivo carotid artery-carotid artery shunt in sheep, the RFGD tetraglyme-coated PE exhibited a very low number of adherent platelets compared to heparin-coated, chromic acid-etched, or plain PE. The RFGD tetraglyme-coated PE tubes exhibited high protein and platelet resistance in vitro, and high platelet resistance ex vivo. The improved hemocompatibility is attributed to the unique chemical structure of RFGD tetraglyme that makes it highly protein resistant.     

The role of complement C3 and fibrinogen in monocyte adhesion to PEO-like plasma deposited tetraglyme

The role of complement C3 in mediating adhesion of monocytes to plasma deposited tetraglyme surfaces was studied. Although fibrinogen (Fg) is usually considered the main factor in mediating phagocyte attachment, plasma deposited PEO-like tetraethylene glycol dimethyl ether (tetraglyme) coatings that have ultra-low Fg adsorption (<10 ng cm(-2)) from low concentration solutions and low monocyte adhesion in vitro still show high phagocyte adhesion after short implantations and later become encapsulated when tested in vivo. To test whether higher Fg adsorption under in vivo conditions could explain the higher in vivo reactivity, we again measured the resistance of tetraglyme films to Fg adsorption. We found a surprising and previously unreported increased amount of adsorbed Fg on tetraglyme surfaces from higher concentration protein solutions. However, monocyte adhesion to tetraglyme did not markedly increase despite the increased Fg adsorption. We thus suspected proteins other than Fg must be responsible for the increased in vivo reactivity. We found that on tetraglyme preadsorbed with C3-depleted serum, monocyte adhesion was greatly reduced as compared to samples adsorbed with normal serum. Addition of exogenous pure C3 to the serum used to preadsorb the surfaces restored monocyte adhesion to tetraglyme coatings. While Fg clearly plays an important role in mediating monocyte adhesion to tetraglyme surfaces, the results show an additional role for adsorbed C3 in monocyte adhesion.     

PEO-like plasma polymerized tetraglyme surface interactions with leukocytes and proteins: in vitro and in vivo studies

Polyethylene oxide (PEO) surfaces reduce non-specific protein and cell interactions with implanted biomaterials and may improve their biocompatibility. PEO-like polymerized tetraglyme surfaces were made by glow discharge plasma deposition onto fluorinated ethylene propylene copolymer (FEP) substrates and were shown to adsorb less than 10 ng/cm2 of fibrinogen in vitro. The ability of the polymerized tetraglyme surfaces to resist leukocyte adhesion was studied in vitro and in vivo. Polymerized tetraglyme and FEP were implanted subcutaneously in mice and removed after 1 day or 4 weeks. Histological analysis showed a similar degree of fibrous encapsulation around all of the 4-week implants. Darkly stained wells were present in the fibrous tissues at the tissue-material interface of both FEP and tetraglyme. Scanning electron micrographs showed that in vivo macrophage adhesion to polymerized tetraglyme was much higher than to FEP. After 2-hour contact with heparinized whole blood, polymorphonuclear leukocyte (PMN) adhesion to polymerized tetraglyme was much higher than to FEP, while platelet adhesion to polymerized tetraglyme was lower than to FEP. When PMNs isolated from blood were suspended in 10% autologous plasma, cell adhesion to polymerized tetraglyme was higher than to FEP; however when the cells were suspended in heat inactivated serum, cell adhesion to FEP was higher than to polymerized tetraglyme. The surface chemistry of polymerized tetraglyme did not change after 2-hour blood contact, but displayed nitrogen functional groups after 1-day implantation and became slightly degraded after 4-week implantation. The surface chemistry of FEP did not change significantly after blood contact or implantation. Loosely bound proteins such as fibrinogen on polymerized tetraglyme may contribute to the adhesion of PMNs and macrophages and ultimately to fibrous encapsulation (the foreign body response) around the implants.     

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.     

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.     

Fibrinogen adsorption and platelet lysis characterization of fluorinated surface-modified polyetherurethanes

A polyetherurethane (PU) was modified using fluorinated surface-modifying macromolecules (SMMs). A double radiolabel method was used simultaneously to measure the number of adhered platelets ((51)Cr) and the quantity of adsorbed Fg ((125)I), in a cone-and-plate instrument. The objectives were to determine if adsorbed Fg levels correlated to platelet adhesion on the surfaces, and to assess if any reductions in platelet adhesion for the SMM-treated surfaces resulted from surface-induced platelet lysis, rather than changes directly related to lower platelet activation and attachment on the novel surfaces. Platelet lysis was determined from lactate dehydrogenase (LDH) and unbound (51)Cr released into plasma isolated from whole blood exposed to test materials. The corresponding Fg adsorption, evaluated under the same platelet adhesion conditions, did not account for the reduced platelet adhesion on the treated surfaces. LDH and (51)Cr platelet release were very low and indicated no statistically significant differences between the materials. It was therefore concluded that platelet lysis did not contribute to the reduction in platelet adhesion characteristic observed on the SMM-treated surfaces. More importantly, the work emphasizes that the platelet activation cannot be inferred to by assessing the quantity of fibrinogen as is commonly done in the literature. The finding suggests a much more complex mechanism of action for the SMM surface modifiers. On-going work is investigating other Fg parameters such as protein binding affinity and protein conformational state in order to establish the mechanism by which the fluorinated surface modifiers may be reducing platelet adhesion via intermediary changes in initial protein adsorption.     

Fluorinated surface-modifying macromolecules: modulating adhesive protein and platelet interactions on a polyether-urethane

Polyether-urethanes (PEUs) have been the materials of choice for the manufacture of conventional blood-contacting devices. Nevertheless, biostability and blood compatibility are still among the principal limitations in their long-term application. Studies investigating the development of protective coatings for PEUs have shown that degradation can be reduced with the use of fluorinated surface-modifying macromolecules (SMMs). It has also been hypothesized that SMM-modified PEU surfaces may exhibit improved blood compatibility because other studies have shown a modulation in fibrinogen adsorption onto these surfaces. To determine the blood compatibility of a PEU-containing fluorinated SMMs, a series of in vitro experiments were designed to study the pattern of protein adsorption from plasma and then to assess the nature of platelet adhesion and activation on each substrate. Western blot analysis as well as single protein studies revealed that the dominant "adhesive proteins" [fibrinogen (Fg), fibronectin (Fnc), and vitronectin (Vnc)] were adsorbed on two of the SMM-containing PEUs in lower amounts relative to unmodified base. Platelet adhesion and activation data further highlighted the differences among the various substrates. It was shown that the unmodified base had a higher number of adhered platelets relative to the SMM-modified surfaces, and that of the SMM-containing substrates, which showed the lowest levels of adhesive proteins also, exhibited significantly lower platelet densities. Close morphological examination further revealed that platelets residing on these latter substrates were not appreciably activated. Based on the current evidence, it is believed that the fluorinated SMMs demonstrate good potential for the development of surfaces with minimal thrombogenic character in in vivo applications.     

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.     

Fluorine doping into diamond-like carbon coatings inhibits protein adsorption and platelet activation

The first major event when a medical device comes in contact with blood is the adsorption of plasma proteins. Protein adsorption on the material surface leads to the activation of the blood coagulation cascade and the inflammatory process, which impair the lifetime of the material. Various efforts have been made to minimize protein adsorption and platelet adhesion. Recently, diamond-like carbon (DLC) has received much attention because of their antithrombogenicity. We recently reported that coating silicon substrates with fluorine-doped diamond-like carbon (F-DLC) drastically suppresses platelet adhesion and activation. Here, we evaluated the protein adsorption on the material surfaces and clarified the relationship between protein adsorption and platelet behaviors, using polycarbonate and DLC- or F-DLC-coated polycarbonate. The adsorption of albumin and fibrinogen were assessed using a colorimetric protein assay, and platelet adhesion and activation were examined using a differential interference contrast microscope. A higher ratio of albumin to fibrinogen adsorption was observed on F-DLC than on DLC and polycarbonate films, indicating that the F-DLC film should prevent thrombus formation. Platelet adhesion and activation on the F-DLC films were more strongly suppressed as the amount of fluorine doping was increased. These results show that the F-DLC coating may be useful for blood-contacting devices.     

Hemocompatibility of heparin-coated surfaces and the role of selective plasma protein adsorption.

Although several studies have shown that heparin-coated surfaces reduce the activation of both the complement system and the coagulation system, there is still inadequate understanding of the factors initiating and controlling blood activation at these surfaces. We investigated the adsorption profile of 12 common plasma proteins (and the platelet receptor CD41) to a heparin coating (Carmeda BioActive surface (CBAS)) compared to uncoated controls (PVC) by using an in vitro whole blood Chandler-Loop model. Surface bound proteins were studied kinetically by a direct ELISA technique. Western blots were performed on the SDS eluates in order to detect adsorbed cleavage products and denatured proteins. Changes in plasma levels of neutrophil activation markers, platelet activation, coagulation activation, complement activation and the inflammatory response were measured by conventional ELISAs. This study showed significant differences in adsorption patterns among the heparin-coated and the uncoated surfaces, notably for fibronectin, fibrinogen, C3 and high molecular weight kininogen (HMWK). The kinetic studies confirmed the results obtained from Western blots and indicated specific adsorption profiles of plasma proteins. We assume that at least some of the improved blood compatibility of the heparin-coated surfaces may be ascribed to the selective uptake and cleavage of plasma proteins.     

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.     

Platelet inhibition and endothelial cell adhesion on elastin-like polypeptide surface modified materials

Platelet adhesion and activation are important early markers of biomaterial blood compatibility, while surfaces that promote enhanced endothelial cell adhesion and eNOS expression are strategic targets for long term vascular graft applications. Materials surface modified with fluorinated surface modifiers, containing peptides inspired from elastin cross-linking domains, have been used for the cross-linking of elastin-like polypeptide 4 (ELP4) macromolecules onto polyurethane surfaces. In the present study, ELP4 modified polyurethanes were evaluated in vitro to assess platelet adhesion, microparticle formation and bulk platelet activation following blood-material interactions. Reduced platelet adhesion and bulk platelet activation were observed following contact between reconstituted human blood and the ELP4 materials, relative to the uncoated base polyurethane controls. ELP4 modified materials also promoted endothelial cell adhesion and retention over a period of one week and showed that the endothelial cells exhibited an organized actin cytoskeleton and enhanced endothelial nitric oxide synthase (eNOS) expression relative to the control surfaces. These results indicate that polyurethane elastomers modified with ELP4 covalently bound to fluorinated surface modifiers provide a promising approach for endowing synthetic elastomers with both reduced blood platelet activation properties and enhanced endothelial cell adhesion for potential use in vascular graft applications.     

Complement activation on poly(ethylene oxide)-like radiofrequency glow discharge-deposited surfaces

Nonspecific protein adsorption, particularly fibrinogen (Fg), is thought to be an initiating step in the foreign body response (FBR) to biomaterials by promoting phagocyte attachment. In previous studies, we therefore prepared radiofrequency glow discharge (ethylene oxide)-like tetraglyme (CH(3)O(CH(2)CH(2)O)(4)CH(3)) coatings adsorbing <10 ng/cm(2) Fg and showed that they had the expected low monocyte adhesion in vitro. However, when these were implanted in vivo, many adherent inflammatory cells and a fibrous capsule were found, suggesting the role of alternative proteins, such as activated complement proteins, in the FBR to these materials. We therefore investigated complement interactions with the tetraglyme surfaces. First, because of its well-known role in complement C3 activation, we measured the hydroxyl group (-OH) content of tetraglyme, but found it to be low. Second, we measured C3 adsorption to tetraglyme from plasma. Low amounts of C3 adsorbed on tetraglyme, although it displayed higher binding strength than the control surfaces. Finally, complement activation was determined by measuring C3a and SC5b-9 levels in serum after incubating with tetraglyme, as well as other surfaces that served as positive and negative controls, namely poly(vinyl alcohol) (PVA) hydrogels, Silastic sheeting, and poly(ethylene glycol) self-assembled monolayers with different end groups. Despite displaying low hydroxyl group concentration, relatively high C3a and SC5b-9 levels were found in serum exposed to tetraglyme, similar to the values in our positive control, PVA. Our results support the conclusion that complement activation by tetraglyme is a possible mechanism involved in the FBR to these biomaterials.     

Rapid postadsorptive changes in fibrinogen adsorbed from plasma to segmented polyurethanes

Fibrinogen adsorbed to biomaterials plays a key role in mediating platelet interactions that can lead to blood clotting so its behavior on surfaces is of fundamental interest. In previous work showing that fibrinogen adsorbed to surfaces quickly becomes non-displaceable upon exposure to blood plasma, the fibrinogen was adsorbed from buffer, so we performed new studies in which the displaceability of fibrinogen adsorbed from plasma was characterized. Fibrinogen was adsorbed from 1% plasma to seven different surfaces for 1-64 min and then transferred to 100% plasma lacking radiolabeled fibrinogen and the amount adsorbed before and after transfer measured. The surfaces were glass, Silicone rubber, and five different polyurethanes. As adsorption time increased, the fibrinogen became increasingly resistant to displacement during the 100% plasma step, but the rate of increase in resistance varied greatly with surface type. Fibrinogen adsorbed from 1% plasma evidently undergoes rapid, surface dependent transitions. This work shows that the transitions that occur when the fibrinogen is adsorbed from blood plasma are similar to what we have previously observed for fibrinogen adsorbed from buffer.     

The kinetics of baboon fibrinogen adsorption to polymers: in vitro and in vivo studies

Fibrinogen adsorption on polymers from blood may mediate or potentiate thrombosis because of its involvement in both the intrinsic clotting system and the formation of platelet aggregates. While the kinetics of fibrinogen adsorption from plasma in vitro have previously been found to be very different on polar and nonpolar surfaces [T. A Horbett, "The kinetics of adsorption of plasma proteins to a series of hydrophilic-hydrophobic copolymers," ACS Org. Coat. Plas. Chem. 40, 642-646 (1979)] the significance of this difference with respect to thrombogenesis in vivo has not been clarified. In this study, the kinetics of deposition of baboon 125I fibrinogen from plasma in vitro or from blood in vivo on a series of polymers was measured. The polymers chosen for this study had previously been found to have a large range in surface polarity and reactivity in the in vivo baboon shunt model. The kinetics of fibrinogen adsorption in vitro were observed to be of three types, depending on the polymer: high initial adsorption decreasing to a lower steady state value; constant throughout the time course; low initial adsorption rising steadily to a plateau value. In vivo, fibrinogen deposition kinetics were of two types: low, constant deposition throughout the time course, independent of heparinization; low deposition initially followed by a second phase of greatly increased deposition (probably as fibrin) which was prevented or greatly decreased by heparinizing the animals. Polymers for which fibrinogen adsorption increased to a plateau in vitro were found to have a heparin inhibitable second phase of enhanced in vivo fibrinogen deposition. These polymers also have been found in previous studies to enhance the rate of platelet destruction when used as in vivo shunts on baboons. Conversely, most polymers with high initial in vitro fibrinogen adsorption followed by a decrease had low fibrinogen deposition behavior in vivo and were also minimally destructive of platelets. The adsorption kinetics of fibrinogen to polymers from blood in vivo and in vitro and the consumption of platelets in vivo induced by the polymers all vary with polymer polarity. More polar polymers had in vitro fibrinogen kinetics characterized by a rise to a plateau, in vivo fibrinogen deposition characterized by a second stage of great increase inhibitable by heparin, and enhanced platelet consumption. The correlation of three separate indicators of surface thrombogenicity with surface polarity suggests that more polar materials may be more thrombogenic because of an influence on the way in which fibrinogen interacts with these surfaces.     

The effect of fluid shear and co-adsorbed proteins on the stability of immobilized fibrinogen and subsequent platelet interactions

The conformation adopted by the plasma protein fibrinogen upon its adsorption onto synthetic surfaces has been implicated to play an important role in determining the blood compatibility of biomaterials. It has recently been shown that adsorbed fibrinogen undergoes biologically significant conformational changes with increasing residence time on the surface of selected biomaterials. The purpose of this study was to examine the effects of co-adsorbed proteins and shear forces on such time-dependent functional changes in fibrinogen adsorbed onto polyethylene (PE), polytetrafluoroethylene (PTFE), and silicone rubber (SR). Fibrinogen was adsorbed onto these materials for 1 min and then allowed to 'reside' on these 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 and the ability of blood plasma to displace previously adsorbed fibrinogen. The magnitude of platelet adhesion to substrates adsorbed with pure fibrinogen increased in the presence of shear, compared with static conditions; at the lowest shear rate of 200 s(-1), samples exhibited a 20-fold increase in adhered platelet levels. In contrast, at a higher shear rate of 1000 s(-1), the three polymers supported minimal levels of platelet attachment. Surfaces pre-adsorbed with 10% plasma did not promote a significant increase in the number of adherent platelets with increasing shear when compared with the pure fibrinogen-coated substrates. The presence of shear also significantly altered the materials' ability to retain fibrinogen. Under static conditions, the amount of fibrinogen retained following incubation in blood plasma increased on all materials with increasing fibrinogen residence time. However, the materials varied distinctly in their ability to retain adsorbed fibrinogen with increasing fibrinogen residence time, shear rate, and nature of the co-adsorbed proteins. Thus, the results from this study indicate that fluid shear, residence time of the adsorbed protein, nature of the co-adsorbed proteins, and surface chemistry of the material all play important roles in influencing platelet-surface interactions and that they act in a complex manner to influence the biocompatibility of a material.     

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.     

Prevention of protein adsorption and platelet adhesion on surfaces by PEO/PPO/PEO triblock copolymers.

Fibrinogen adsorption and platelet adhesion on to dimethyldichlorosilane-treated glass and low-density polyethylene were examined. The surfaces were treated with poly(ethylene glycol) and poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) triblock copolymers (Pluronics). Poly(ethylene glycol) could not prevent platelet adhesion and activation, even when the bulk concentration for adsorption was increased to 10 mg/ml. Pluronics containing 30 propylene oxide residues could not prevent platelet adhesion and activation, although the number of ethylene oxide residues varied up to 76. However, Pluronics containing 56 propylene oxide residues inhibited platelet adhesion and activation, even though the number of ethylene oxide residues was as small as 19. Fibrinogen adsorption on the Pluronic-coated surfaces was reduced by more than 95% compared to the adsorption on control surfaces. The ability of Pluronics to prevent platelet adhesion and activation was mainly dependent on the number of propylene oxide residues, rather than the number of ethylene oxide residues. The large number of propylene oxide residues was expected to result in tight interaction with hydrophobic dimethyldichlorosilane-treated glass and low-density polyethylene surfaces and thus the tight anchoring of Pluronics to the surfaces. The presence of 19 ethylene oxide residues in the hydrophilic poly(ethylene oxide) chains was sufficient to repel fibrinogen and platelets by the mechanism of steric repulsion.