Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer

A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 microg/cm2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.     

Platelet adhesion to heparin coated oxygenator fibers under in vitro static conditions: impact of temperature

Heparin coating of cardiopulmonary bypass (CPB) circuitry may attenuate the platelet consumption associated with CPB. We investigated the effect of temperature on the interaction between platelet and heparin coated surfaces under in vitro static conditions. Heparin coated and non coated oxygenator fibers were incubated with heparinized whole blood at 37 degrees C and 22 degrees C. The incubation time was set at 30, 60, 180, and 300 minutes. The number of platelets adhering to each fiber was assessed with enzyme immunoassay using monoclonal antibody against platelet receptor protein CD 61(GPIIbIIIa). As an index of platelet activation, plasma soluble(s) P-selectin levels were measured by enzyme-linked immunosorbent assay. Under normothermia, the number of adherent platelets on the non coated surface increased significantly after 300 min of incubation. Platelet adhesion was reduced significantly by heparin coating of the surface and was kept constant after 300 min. Under hypothermia, heparin coating was also associated with significant reduction of platelet adhesion. The levels of sP-selectin did not correlate with the extent of platelet adhesion. Our results suggest that heparin coating is effective in decreasing platelet adhesion to the synthetic surface tested regardless of the temperature under static conditions. Inhibition of platelet activation on the heparin coated surface may be masked by standard dose heparinization.     

The interactions between antithrombin III,thrombin and surface immobilized heparin

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

Hemocompatible albumin-heparin coatings prepared by the layer-by-layer technique. The effect of layer ordering on thrombin inhibition and platelet adhesion

Using the layer-by-layer technique, ELISA polystyrene plates were coated with multilayer assemblies of albumin with various heparins or with multilayer assemblies of albumin. The coatings containing heparin were tested for their ability to potentiate thrombin inhibition by antithrombin and its dependence on the layer arrangement. The order of activities of surface bound heparins matched their order in solution; however their activity was reduced to less than 10% due to binding. The increasing number of layers increased the activity of the coatings suggesting that heparin inside the assemblies is available for the interaction. The albumin-heparin assemblies overcoated with albumin layers preserved about half of heparin activity. Platelets adhered in similar amounts to albumin-heparin and albumin coatings; however, in both cases platelets adhered more to single layer than to multilayer coatings. The adhesion of platelets to single layer coatings was also affected by the crosslinking of the coatings; more platelets adhered to less crosslinked single layer coatings while multilayer coatings remained essentially unaffected by crosslinking. If the coatings were dried and reswollen, a substantial number of platelets adhered to the reconditioned single layer coatings but the two layer coatings were affected much less and the adhesion of platelets to the coatings with three layers was close to normal. A minimum of three albumin-heparin or albumin layers is apparently required to shield the underlying surface and to achieve proper functioning of the coatings.     

The measurement of respiratory burst induced in polymorphonuclear neutrophils by IgA and IgG anti-gliadin antibodies isolated from coeliac serum

The properties of IgA and IgG anti-gliadin antibodies from the serum of patients with coeliac disease have been compared. The antibodies were quantified by ELISA using microtitre plates coated with crude gliadin fractions. Their specificity was confirmed by immunoblotting. Heat-treated sera containing IgA antibodies stimulated chemiluminescence when added together with neutrophils to microtitre plates coated with crude gliadin. Sera containing only IgG antibodies were less efficient. When IgA and IgG were purified from a serum containing both classes of anti-gliadin antibodies, each of the preparations was able to stimulate neutrophil chemiluminescence in plates coated with gliadin. Although the yield of anti-gliadin antibody determined by ELISA was high, the ability of the purified immunoglobulins to stimulate neutrophil chemiluminescence was much less than that of the unfractionated serum. This loss of activity was shown to be due to the ability of each class of antibody to potentiate the activity of the other in the whole serum.     

Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion.

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO(2)), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO(2) with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO(2) coated with alb-hep conjugate was twice as high as on PS-CO(2) coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO(2) had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating.     

Principles, problems, and strategies in the use of antigenic mixtures for the enzyme-linked immunosorbent assay

Competition between proteins and other macromolecules for adsorption sites on plastic was studied with the enzyme-linked immunosorbent assay (ELISA) to determine effects of the use of antigenic mixtures or extracts of organisms on assays of antibodies and antigens by ELISA. A comparison of a number of different polystyrene microplates with bovine albumin and human immunoglobulin G (IgG) as antigens showed two major classes of plates; those which adsorbed albumin poorly and those which adsorbed albumin well. IgG adsorbed well on all plates, but plates which adsorbed albumin best also gave significant background levels of nonspecific binding of conjugate. When mixtures of IgG and bovine serum albumin were used as coating antigens, significant competition was observed; the component present at 1% or less in the mixture was essentially undetectable unless excessive amounts of conjugate were used. The important factor was the ratio of competitor to antigen, not the absolute amount. Other proteins (ovalbumin, rabbit albumin, human albumin, and gelatin) were equally effective competitors for adsorption sites on plastic. Nonionic detergents (Tween 20, and Triton X-100) were strong competitors even at 10:1 competitor-to-antigen ratios. In antigen capture assays, normal serum components blocked attachment of antigen-specific IgG, but this competition could be lessened to a degree by the use of strongly binding polystyrene plates. In indirect ELISA for measurement of serum antibody, the use of antigenic mixtures gave significantly lower antibody titers when the desired antigen was less than 1% of the total protein coated. Therefore, the use of mixed or crude antigens in ELISA presents significant problems concerning the sensitivity and specificity of tests.     

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

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

An in vitro study of the adhesion of blood platelets onto vascular catheters. Part I

The adhesion of human blood platelets onto vascular catheters was studied using a specially designed perfusion chamber. Polyurethane catheters were exposed to citrated human blood for different periods (up to 20 min) and at different wall shear rates (190, 260, 330 sec-1). The rate of platelet adhesion was determined using 111In-labeled platelets, while the morphology of adhering platelets was investigated using scanning electron microscopy. A linear increase in platelet adhesion was found within the first 10 min of perfusion, after which a plateau value was reached. The number of adhering platelets did not vary significantly with the shear rates applied, which may indicate that within the range of shear rates studied, the adhesion of platelets onto the catheter surface is mainly determined by the rate of the reaction between the platelets and the material surface. Catheters coated with a conjugate of heparin and albumin showed a four- to five-fold reduction in platelet adhesion as compared to uncoated catheters. This reduction in platelet adhesion was not only due to the presence of albumin moieties at the surface but also to the presence of heparin residues in the adsorbed albumin-heparin conjugate.     

Interaction of plasma proteins with heparinized gel particles studied by high-resolution two-dimensional gel electrophoresis.

In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.     

Titanium-protein interaction: changes with oxide layer thickness

Since titanium is being used for various biomedical applications requiring enhanced blood compatibility, which may be partly due to its extremely stable oxide layer, an attempt is made here to understand the effect of oxide layer thickness on protein adsorption. Different thickness of oxide layers have been coated on titanium foil using anodizing method and thickness of oxide layers deposited have been measured by an ellipsometer. Studies of competitive adsorption of proteins, using I125 labelled protein from a mixture of 25 mg% albumin, 15 mg% gamma-globulin and 7.5 mg% fibrinogen indicate an increased adsorption of proteins onto the oxide layer coated surfaces compared to the bare surface.     

In vitro hemocompatibility of albumin-heparin multilayer coatings on polyethersulfone prepared by the layer-by-layer technique

Polyethersulfone foils (PES)--a unique material for blood purification membranes--were coated with a multilayer assembly of heparin (unfractionated or high anticoagulant activity fraction heparin) and albumin (albumin-heparin coatings), or with a multilayer of albumin (albumin coating), using the layer-by-layer technique. The coatings combine advantages of albumin (reduction of nonspecific interactions) and heparin (specific interactions with blood coagulation proteins). The differences between the two heparins, while significant for their biological activity, had only a minor effect on the multilayer assembly with albumin monitored in situ by reflection infrared spectroscopy (FTIR MIRS). Uncoated as well as modified PES surfaces were evaluated using an in vitro assay with freshly drawn, slightly heparinized (1.5 IU heparin/mL) human whole blood. The blood was circulated with a roller pump over the sample surfaces in shear flow across rectangular slit channels ( app. 6 mL/min and 120 s(-1)) for 1.5 h at 37 degrees C. All coatings effectively reduced platelet adhesion and activation according to the PF4 release. The activation of coagulation evaluated as TAT generation was significantly lowered for the coating composed of albumin and high activity heparin. A further beneficial effect of the heparin containing coatings was reduced complement activation as determined by different complement fragments.     

In vivo protein adsorption on polymers: visualization of adsorbed proteins on vascular implants in dogs

The absorption of plasma proteins is an important event at the blood-material interface, and strongly affects subsequent cellular interaction and thrombus formation. Although considerable efforts have been expended to elucidate the mechanism of protein adsorption and the role of absorbed protein layer at the blood-material interface, there has been little knowledge of how the initial adsorbed proteins are maintained or changed in a time-variant process in in vivo long-term implantation. In this study, we described detailed analyses concerning the characterization of adsorbed proteins on HEMA-styrene block copolymer surfaces (HEMA-st) and poly(ethylene oxide)(PEO) grafted Biomer® (B-PE04K) for in vivo long-term canine vascular graft implants as well as in vitro short-term experiments. Biomer vascular grafts (6 mm I.D., 7 cm in length) were fabricated by a dip coating and the luminal surface was modified with PEO grafting, HEMA-st coating, or Biomer coating (control). These surface modified grafts were recirculated for different time intervals (5, 15, 30, 60 and 120 min) using citrated canine whole blood. The grafts were then implanted in the abdominal aortas of dogs and evaluated for graft patency and protein adsorption. The adsorbed proteins (albumin, IgG and fibrinogen) were quantified using an in situ radioimmunoassay. Surface protein layer thickness was measured by transmission electron microscopy (TEM). Visualization of absorbed plasma proteins (albumin, IgG and fibrinogen) was performed with TEM using an immunoperoxidase double antibody technique. In in vitro recirculation systems, albumin and IgG showed similar Langmuir type pattern onto all test surfaces. On B-PEO4K surfaces, fibrinogen adsorption kinetics demonstrated 'Vroman effect'. The Biomer and B-PE04K grafts occluded within 1 month, while HEMA-st grafts were patent for over 3 months. Biomer and B-PE04K showed thick multilayers of adsorbed proteins, and the thickness increased with implantation periods and the composition altered with time. In contrast, HEMA-st showed a monolayer-like adsorbed protein pattern, and the composition and thickness were consistent regardless of implantation time including in vitro short-time experiments, which may attribute to less conformational change of adsorbed proteins on HEMA-st surfaces. In terms of nonthrombogenicity, the stable monolayer-like adsorbed protein layer on HEMA-st surfaces exhibited improved blood compatibility over thick multilayered adsorbed proteins on Biomer and B-PE04K surfaces.     

In vivo protein adsorption on polymers: visualization of adsorbed proteins on vascular implants in dogs.

The absorption of plasma proteins is an important event at the blood-material interface, and strongly affects subsequent cellular interaction and thrombus formation. Although considerable efforts have been expended to elucidate the mechanism of protein adsorption and the role of absorbed protein layer at the blood-material interface, there has been little knowledge of how the initial adsorbed proteins are maintained or changed in a time-variant process in in vivo long-term implantation. In this study, we described detailed analyses concerning the characterization of adsorbed proteins on HEMA--styrene block copolymer surfaces (HEMA-st) and poly(ethylene oxide) (PEO) grafted Biomer (B-PEO4K) for in vivo long-term canine vascular graft implants as well as in vitro short-term experiments. Biomer vascular grafts (6 mm I.D., 7 cm in length) were fabricated by a dip coating and the luminal surface was modified with PEO grafting, HEMA-st coating, or Biomer coating (control). These surface modified grafts were recirculated for different time intervals (5, 15, 30, 60 and 120 min) using citrated canine whole blood. The grafts were then implanted in the abdominal aortas of dogs and evaluated for graft patency and protein adsorption. The adsorbed proteins (albumin, IgG and fibrinogen) were quantified using an in situ radioimmunoassay. Surface protein layer thickness was measured by transmission electron microscopy (TEM). Visualization of absorbed plasma proteins (albumin, IgG and fibrinogen) was performed with TEM using an immunoperoxidase double antibody technique. In in vitro recirculation systems, albumin and IgG showed similar Langmuir type pattern onto all test surfaces. On B-PEO4K surfaces, fibrinogen adsorption kinetics demonstrated 'Vroman effect'. The Biomer and B-PEO4K grafts occluded within 1 month, while HEMA-st grafts were patent for over 3 months. Biomer and B-PEO4K showed thick multilayers of adsorbed proteins, and the thickness increased with implantation periods and the composition altered with time. In contrast, HEMA-st showed a monolayer-like adsorbed protein pattern, and the composition and thickness were consistent regardless of implantation time including in vitro short-time experiments, which may attribute to less conformational change of adsorbed proteins on HEMA-st surfaces. In terms of nonthrombogenicity, the stable monolayer-like adsorbed protein layer on HEMA-st surfaces exhibited improved blood compatibility over thick multilayered adsorbed proteins on Biomer and B-PEO4K surfaces.     

Protein adsorption and platelet attachment and activation, on TiN, TiC, and DLC coatings on titanium for cardiovascular applications

The hemocompatibility of a TiN/TiC/diamond-like carbon (DLC) multilayer structure, deposited on titanium substrates for use as coatings for a heart valve prosthesis, has been studied through the adsorption of blood proteins and the adhesion and attachment of blood platelets. All of the surfaces were characterized by stylus profilometry and water contact angles. The adsorption of albumin and fibrinogen to the surfaces was assessed using the Amido Black assay, whereas platelet attachment was studied by scanning electron microscopy and quantified using stereological techniques. The degree of platelet spreading on the surfaces was seen to correlate with differences in surface energy, indicated from contact angle measurements. The greatest spreading was seen on the more hydrophilic surfaces. When studying protein adsorption to the surfaces, no correlation could be determined between contact angle results and levels of adsorption, although the most hydrophilic surfaces did appear to promote greater amounts of fibrinogen adsorption. Thrombus formation was observed to some degree on all of the surfaces, with the exception of the DLC coating. This coating also promoted less spreading of platelets than the other surfaces. The good hemocompatibility of the DLC coating is attributed to its hydrophobicity and smooth surface, resulting in a higher ratio of albumin to fibrinogen than any of the other surfaces.     

Use of the enzyme-linked immunosorbent assay for measurement of allergen-specific antibodies

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for allergen-specific IgG antibodies is described. Various solid-phase supports (microtiter plates), coating procedures, binding kinetics, and presentation of allergen in the assay were investigated. Using optimal conditions the indirect ELISA, in which the allergen is coated onto the well, was capable of detecting 2.4 ng/ml specific IgG antibodies to bee venom phospholipase A2(PLA2). The sandwich ELISA, in which the allergen was immobilized via specific antibody precoated onto the well, detected 0.24 ng/ml IgG antibodies to PLA2.     

Hemocompatibility of polymeric nanostructured surfaces

Tissue integration is an important property when inducing transplant tolerance, however, the hemocompatibility of the biomaterial surface also plays an important role in the ultimate success of the implant. Therefore, in order to induce transplant tolerance, it is critical to understand the interaction of blood components with the material surfaces. In this study, we have investigated the adsorption of key blood serum proteins, in vitro adhesion and activation of platelets and clotting kinetics of whole blood on flat polycaprolactone (PCL) surfaces, nanowire (NW) surfaces and nanofiber (NF) surfaces. Previous studies have shown that polymeric nanostructured surfaces improve cell adhesion, proliferation and viability; however it is unclear how these polymeric nanostructured surfaces interact with the blood and its components. Protein adsorption results indicate that while there were no significant differences in total albumin (ALB) adsorption on PCL, NW and NF surfaces, NW surfaces had higher total fibrinogen (FIB) and immunoglobulin-G (IgG) adsorption compared to NF and PCL surfaces. In contrast, NF surfaces had higher surface FIB and IgG adsorption compared to PCL and NW surfaces. Platelet adhesion and viability studies show more adhesion and clustering of platelets on the NF surfaces as compared to PCL and NW surfaces. Platelet activation studies reveal that NW surfaces have the highest percentage of unactivated platelets, whereas NF surfaces have the highest percentage of fully activated platelets. Whole blood clotting results indicate that NW surfaces maintain an increased amount of free hemoglobin during the clotting process compared to PCL and NF surface, indicating less clotting and slower rate of clotting on their surfaces.     

The influence of plasma proteins and platelets on oxygen radical production and F-actin distribution in neutrophils adhering to polymer surfaces

It is well known that blood cell interactions with artificial surfaces might have deleterious effects on host tissue, however, the mechanisms involved are far from understood. In this study, neutrophil-platelet interaction on uncoated or protein-coated polymer surfaces was investigated. Cell spreading, reorganization of actin filaments and release of oxygen metabolites (measured as luminol-amplified chemiluminescence) were used as criteria for cell activation on positively charged, hydrophilic 1,2-diaminocyclohexane, and negatively charged, hydrophobic hexamethylene-disiloxane. The model surfaces were made by radio frequency plasma discharge polymerization. Neutrophil contact with the uncoated polymers induced a prolonged generation of oxygen radicals. Precoating of the polymer surfaces with human serum albumin (HSA) or fibrinogen, markedly reduced neutrophil activation, whereas coating with human immunoglobulin G (IgG), a well-known opsonin, resulted in significantly higher levels of cell activation. Consequently, protein coating overruled the activating effects of the polymer surfaces. The presence of unstimulated or thrombin-stimulated platelets markedly increased the reactivity of neutrophils against fibrinogen- and IgG-coated surfaces. However, neutrophils remained relatively unreactive in the presence of platelets on HSA-treated surfaces. Comparison of the different types of surfaces used, reveals a correlation between the degree of cell spreading, reorganization of the actin cytoskeleton and the amount of oxygen radicals produced. Our results suggest that the acute inflammatory reaction on a biomaterial surface is highly dependent on the nature and composition of the first adsorbed protein layer and the extent of platelet activation.     

Thrombogenicity of polysaccharide-coated surfaces

Heparinization of artificial surfaces has been proven to reduce the intrinsic thrombogenicity of such surfaces. The mechanism by which immobilized heparin reduces thrombogenicity is not completely understood. In the present study heparin-, alginic acid- and chondroitin-6-sulphate-coated surfaces were examined for protein adsorption, platelet adhesion and thrombin generation. The protein-binding capacity from solutions of purified proteins was significantly higher for heparin-coated surfaces when compared with alginic acid- and chondroitin sulphate-coated surfaces. Yet, when the surfaces were exposed to flowing plasma, only the heparinized surface adsorbed significant amounts of antithrombin. None of the surfaces adsorbed fibrinogen under these conditions, and as a result no platelets adhered from flowing whole blood. Our results indicate that protein adsorption and platelet adhesion from anticoagulated blood cannot be used to assess the thrombogenicity of (coated) artificial surfaces. Indeed, the thrombin generation potentials of the different surfaces varied remarkable: while non-coated surface readily produced thrombin, alginic acid- and chondroitin sulphate-coated surfaces showed a marked reduction and virtually no thrombin was generated in flowing whole blood passing by heparinized surfaces.