Platelet adhesion to radiofrequency glow-discharge-deposited fluorocarbon polymers preadsorbed with selectively depleted plasmas show the primary role of fibrinogen.

Fluorocarbon radio-frequency glow-discharge (RFGD) treatment has previously been shown to cause decreased platelet adhesion despite the presence of adsorbed fibrinogen on the surfaces. In this study platelet adhesion to fluorocarbon RFGD-treated surfaces preadsorbed with human plasma was further examined. A series of plasma deposited fluorocarbon thin films were made by varying the C3F6/CH4 ratio in the monomer feed. The surfaces were preadsorbed with plasma, serum, or plasma selectively depleted of fibronectin, vitronectin, or Von Willebrand factor, and platelet adhesion was measured. We also measured fibrinogen adsorption to the surfaces from plasma, monoclonal antibody binding to adsorbed fibrinogen and SDS elutability of the adsorbed fibrinogen. The antibodies used bind to the three putative platelet binding sites on fibrinogen, namely, M1 antibody binds to the dodecapeptide at the C-terminus of the gamma chain, gamma (402-411), R1 antibody binds to a sequence in the Aalpha chain (87-100) which includes RGDF at Aalpha (95-98) and R2 antibody binds a sequence in the Aalpha chain (566-580) which includes RGDS at Aalpha (572-575). Fibrinogen was found to play a decisive role in mediating platelet adhesion to the fluorocarbon surfaces contacting plasma. Few platelets adhered to the fluorocarbon surfaces preadsorbed with serum, while preadsorption with plasma selectively-depleted of either fibronectin, vitronectin, or von Willebrand factor did not decrease platelet adhesion significantly. Replenishment of exogenous fibrinogen to serum restored platelet adhesion, while replenishment of the other proteins had no effect. Platelet adhesion to the fluorocarbon surfaces was lower than to PET or the methane glow-discharge-treated PET. However, there was no apparent correlation between platelet adhesion and the amount of fibrinogen adsorption or monoclonal antibody binding to surface-bound fibrinogen.

Quantitative analysis of binary adsorbed protein films by time of flight secondary ion mass spectrometry.

Time of flight secondary ion mass spectrometry (ToF-SIMS) is an ideal technique for the analysis of adsorbed protein films because of its surface sensitivity and chemical specificity. In this study, we examined ToF-SIMS with the multivariate calibration method partial least squares regression (PLSR) for the determination of the relative abundance of the components in binary protein films adsorbed onto mica, PTFE, and heptyl amine plasma polymer substrates. These results have been compared with independently measured 125I-radiolabeled protein adsorption experiments. By applying PLSR to the ToF-SIMS data, the relative abundance of the components in the binary adsorbed protein films was quantified, and the agreement between the ToF-SIMS and 125I-radiolabeling data was measured by the root mean square prediction error (RMSPE). Differences in protein quantification by PLSR and 125I-radiolabeling ranged from 5 to 25 mass % RMSPE and were highly dependent on the structure of the adsorbed protein film, the substrate surface chemistry and morphology, and the number of latent variables retained in the PLSR model. The limit of detection for the minor component in the adsorbed protein film was found to be approximately 10 mass %. This study demonstrates that the combination of ToF-SIMS and multivariate calibration provide complementary information to 125I-radiolabeling about the composition and structure of binary adsorbed protein films.

Characterizing multicomponent adsorbed protein films using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectrometry, and radiolabeling: capabilities and limitations.

Characterization of complex adsorbed protein films is a critical aspect of biomaterials science, particularly in understanding the in vivo response to biomaterials. The surface analysis techniques electron spectroscopy for chemical analysis (ESCA) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) are particularly suited to the analysis of complex adsorbed protein films due to their wide applicability to a variety of materials. We have investigated the applicability of ESCA for studying the structure of adsorbed serum and plasma protein layers. ESCA was able to monitor the thickness of the adsorbed protein film. Due to its chemical specificity, ToF-SIMS was used to estimate the composition of the plasma and serum protein layers by comparison of their spectra with the spectra of single protein films. The limit of detection of ToF-SIMS for the plasma protein fibrinogen was determined by comparison with independent radiolabeled fibrinogen adsorption measurements. While ToF-SIMS was able to determine some qualitative trends in the composition of the plasma protein films as a function of adsorption time, the detection limit of the minor components in multicomponent adsorbed protein films ultimately limits the ability of ToF-SIMS to quantify the composition of these films. However, both ESCA and ToF-SIMS can provide useful information on adsorbed plasma protein films without further sample treatment. This study outlines the strengths and weaknesses of ESCA and ToF-SIMS for studying multicomponent adsorbed plasma protein films.

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.

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

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

Inhibition of monocyte adhesion and fibrinogen adsorption on glow discharge plasma deposited tetraethylene glycol dimethyl ether.

Monocytes and macrophages play important roles in host responses to implanted biomedical devices. Monocyte and macrophage interactions with biomaterial surfaces are thought to be mediated by adsorbed adhesive proteins such as fibrinogen and fibronectin. Non-fouling surfaces that minimize protein adsorption may therefore minimize monocyte adhesion, activation, and the foreign body response. Radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme) was used to produce polyethylene oxide (PEO)-like coatings on a fluorinated ethylene-propylene (FEP) surface. Electron spectroscopy for chemical analysis (ESCA) and static time of flight secondary ion mass spectrometry (ToF-SIMS) were used to characterize the surface chemistry of tetraglyme coating. Fibrinogen adsorption to the tetraglyme surface was measured with 125I-labeled fibrinogen and ToF-SIMS. Adsorption of fibrinogen to plasma deposited tetraglyme was less than 10 ng cm(-2), a 20-fold decrease compared to untreated FEP or tissue culture polystyrene (TCPS). Monocyte adhesion to plasma deposited tetraglyme was significantly lower than adhesion to FEP or TCPS. In addition, when the surfaces were preadsorbed with fibrinogen, fibronectin, or blood plasma, monocyte adhesion to plasma deposited tetraglyme after 2 h or 1 day was much lower than adhesion to FEP. RF-GDPD tetraglyme coating provides a promising approach to make non-fouling biomaterials that can inhibit non-specific material-host interactions and reduce the foreign body response.

The 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.

Platelet adhesion and procoagulant activity induced by contact with radiofrequency glow discharge polymers: roles of adsorbed fibrinogen and vWF.

The potential hemocompatibility of radiofrequency glow discharge (RFGD) polymers made by copolymerization of mixtures of hexafluoropropene and ethylene (C(3)F(6)/C(2)H(4)) or acrylic acid and 1,7-octadiene was investigated using in vitro assays for platelet adhesion and platelet catalyzed thrombin generation. Thrombin generation rate normalized to platelet number was used as a measurement of platelet activation (procoagulant activity). RFGD polymers produced by copolymerization of acrylic acid and 1, 7-octadiene contained varying amounts of carboxylic acid species as determined by electron spectroscopy for chemical analysis (ESCA). These polymers induced little variation in platelet adhesion, thrombin generation, or platelet activation. RFGD polymerization of C(3)F(6) and C(2)H(4) resulted in polymers with varying proportions of fluorinated species, as determined by ESCA. Fibrinogen adsorption from plasma was maximal on a polymer made with 25% C(3)F(6) (75% C(2)H(4)) in the feed. However von Willebrand factor (vWF) adsorption was greater on polymers made with increased %C(3)F(6) in the feed. Platelet adhesion decreased with increasing %C(3)F(6) in the feed. Thrombin generation was lowest for platelets adherent to polymers made from both C(3)F(6) and C(2)H(4). Therefore, procoagulant activity of platelets increased for polymers made with increased %C(3)F(6) in the feed, similar to the trend in vWF adsorption. These findings suggest that increased incorporation of fluorinated species into RFGD polymers leads to decreased platelet adhesion and increased platelet activation (which is possibly due to increased vWF adsorption).

Plasma-deposited membranes for controlled release of antibiotic to prevent bacterial adhesion and biofilm formation.

Bacterial infection on implanted medical devices is a significant clinical problem caused by the adhesion of bacteria to the biomaterial surface followed by biofilm formation and recruitment of other cells lines such as blood platelets, leading to potential thrombosis and thromboembolisms. To minimize biofilm formation and potential device-based infections, a polyurethane (Biospan) matrix was developed to release, in a controlled manner, an antibiotic (ciprofloxacin) locally at the implant interface. One material set consisted of the polyetherurethane (PEU) base matrix radiofrequency glow discharge plasma deposited with triethylene glycol dimethyl ether (triglyme); the other set had an additional coating of poly(butyl methyacrylate) (pBMA). Triglyme served as a nonfouling coating, whereas the pBMA served as a controlled porosity release membrane. The pBMA-coated PEU contained and released ciprofloxacin in a controlled manner. The efficacy of the modified PEU polymers against Pseudomonas aeruginosa suspensions was evaluated under flow conditions in a parallel plate flow cell. Bacterial adhesion and colonization, if any, to the test polymers were examined by direct microscopic image analysis and corroborated with destructive sampling, followed by direct cell counting. The rate of initial bacterial cell adhesion to triglyme-coated PEU was 0. 77%, and to the pBMA-coated PEU releasing ciprofloxacin was 6% of the observed adhesion rates for the control PEU. However, the rate of adherent cell accumulation due to cell growth and replication was approximately the same for the triglyme-coated PEU and the PEU controls, but was zero for the pBMA-coated PEU releasing ciprofloxacin.

Design of infection-resistant antibiotic-releasing polymers: I. Fabrication and formulation.

Biomaterials-related infections are often observed with prosthetic implants and in many cases result in the failure of the devices. To design a biomedically useful polymer that is intrinsically infection-resistant, we have developed a ciprofloxacin-loaded polyurethane (PU) matrix that releases antibiotic locally at the implant surface, thereby minimizing bacterial accumulation. We report here the methods of fabrication and formulation for making such antibiotic-loaded devices, as well as evidence of their bactericidal properties. Specifically, various pore-forming agents and drug loadings were examined. An optimum formulation consisting of BIOSPAN PU, poly(ethylene glycol) and ciprofloxacin offered the longest effective period of sustained release (5 days). The bactericidal efficacy of the released ciprofloxacin against Pseudomonas aeruginosa (PA) was four times that of the control PU without antibiotics. This bactericidal efficiency was due to an increase in the PA detachment from the surface. These observations suggested that the released ciprofloxacin was biologically active in preventing the bacteria from permanently adhering to the substratum, and thus decreasing the possibility of biofilm-related infection.

Design of infection-resistant antibiotic-releasing polymers. II. Controlled release of antibiotics through a plasma-deposited thin film barrier.

In the first paper in this series, we described the methods to synthesize an antibacterial polyurethane (PU) incorporating ciprofloxacin as the releasable antibiotic and poly(ethylene glycol) as the pore-forming agent. Here, we report that a thin, RF-plasma-deposited, n-butyl methacrylate (BMA) overlayer on this drug-loaded PU can act as a rate-limiting barrier to achieve a constant, sustained release of ciprofloxacin. Deposition power and deposition time during the coating process were optimized to give an appropriate crosslinked coating barrier that yielded desirable release rates, above the minimum required killing rate, N(kill). Electron spectroscopy for chemical analysis (ESCA), also known as X-ray photoelectron spectroscopy (XPS), was used to characterize the coating, and its crosslinking degree was indirectly related to the C/O ratio. Increasing either deposition power (10-60 W) or duration (5-25 min) resulted in increased C/O ratios and decreased ciprofloxacin release rates. The correlation between increased C/O ratios and reduced release rates is believed to be due to the increased crosslinking, increased hydrophobicity and increased thickness of the coating. The optimal plasma conditions to attain an appropriate crosslinked plasma-deposited film (PDF) required argon etching, pre-treatment of the matrices with an 80W-BMA plasma for 1 min, followed by immediate BMA plasma deposition at 40 W and 150 mT for 20 min. By using these plasma deposition protocols, we eliminated the initial burst effect, significantly reduced the release rates, and closely approached the zero order release kinetics for at least five days. In this study, we also showed that ESCA could be used as a powerful tool to explain the release behavior of molecules through the plasma-deposited films (PDFs).

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

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

The role of 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.

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.

Hemocompatibility of treated polystyrene substrates: contact activation, platelet adhesion, and procoagulant activity of adherent platelets.

Platelet adhesion to biomaterials is often used as an index of blood compatibility, but a more clinically relevant issue is whether the adherent platelets are able to promote clot formation (i.e., if they are in the procoagulant state). Platelets rapidly generate thrombin when they are in the procoagulant state and the VA/Xa complex is present. We found that adherent platelets are procoagulant by three different methods: binding of FITC-Annexin V, acceleration of thrombin generation in the presence of Xa, Va, and prothrombin; and clotting of recalcified plasma. In the clotting times studies, the effect of adherent platelets on TCPS was completely eliminated by the addition of Annexin V, which is known to bind tightly to procoagulant platelets. The degree of procoagulant activity of adherent platelets was determined by measuring thrombin generation rates in the presence of the clotting factors Va, Xa, and prothrombin and normalizing to the number of adherent platelets. Two key observations were made in these studies. First, the procoagulant activity of platelets adherent to untreated and to several types of treated polystyrenes, as well as to glass and PET, was much greater than the procoagulant activity of unstimulated bulk phase platelets. Little difference in the procoagulant activity of adherent platelets was observed among the materials tested, however. Second, the procoagulant activity of platelets prestimulated with ionophore and subsequently allowed to adhere to Plastek M was much greater than when adherent platelets were stimulated by the adhesion event only. Measured values for platelet adhesion, platelet activation, and contact activation of blood plasma are discussed in the context of their potential combined impact on blood clotting.

Glow discharge plasma deposition (GDPD) technique for the local controlled delivery of hirudin from biomaterials.

PURPOSE: Biomaterials which release locally high concentrations of antithrombotic agents should lessen the thrombogenicity of the materials. To evaluate this approach, we prepared novel polyurethane matrices loaded with hirudin and coated them with 2-hydroxyethyl methacrylate (HEMA) by glow discharge plasma deposition (GDPD) to reduce the release rate. METHODS: Polyurethane (BioSpan) matrices containing hirudin and pore former (d-mannitol or BSA) were prepared by the solvent casting method. HEMA plasma deposition was then applied using GDPD technique to create a diffusional barrier film on the surface of the matrices. The effect of pore former and HEMA plasma coating on the release of hirudin was systematically investigated. Surface properties of matrices was also studied using Scanning Electron Microscopy (SEM) and Electron Spectroscopy for Chemical Analysis (ESCA). RESULTS: The release of hirudin from BioSpan matrix could be controlled by changing the weight fraction and particle size of pore former. HEMA plasma treatment of matrices produced a thin, highly cross-linked film on the surface. The initial burst and subsequent release of hirudin was significantly reduced after HEMA plasma coating, which suggested that the plasma disposition acted as a diffusional barrier and limited the release of hirudin incorporated in the polyurethane matrix. CONCLUSIONS: The plasma coating served as a diffusional barrier, and could work to control the release kinetics of hirudin by changing the various plasma coating conditions. Local delivery of hirudin using these biomaterials at the site of cardiovascular diseases can have the advantage of regional high levels of hirudin, as well as lowering systemic hirudin exposure, thereby minimizing the possibility of side effects.

Fibrinogen adsorption to receptor-like biomaterials made by pre-adsorbing peptides to polystyrene substrates.

Two peptides from the ligand-binding site of the platelet receptor GPIIb/IIIa, residues 296-306 of GPIIb, designated B12 by D'Souza et al. (1991), and 300-311 of GPIIb, designated G13 by Taylor et al., (1992), as well as two control peptides, designated C14 and C20, were adsorbed to treated polystyrene substrates. Fibrinogen adsorption to the peptide-coated substrates was characterized. The specificity of I-125 labeled fibrinogen binding to the peptide-coated substrates was investigated by measuring the amount of fibrinogen adsorbed to each substrate and the inhibition of fibrinogen binding by RGDS peptide, bovine serum albumin, a divalent ion chelator (ethylene diamine tetra-acetic acid disodium salt), unlabeled fibrinogen and the B12 peptide. The results show that non-specific binding of fibrinogen to the B12-coated substrate is predominant under most conditions. Binding of monoclonal antibodies to fibrinogen adsorbed to the peptide coated substrates was characterized. The failure of several antibodies to bind fibrinogen adsorbed to the B12 substrate suggested that adsorption of fibrinogen to the B12-coated substrate alters its conformation relative to fibrinogen adsorbed to the bare substrate.

Pharmacology and controlled release of hirudin for cardiovascular disorders.

Hirudin is the most potent specific inhibitor of thrombin known. Hirudin was originally isolated from leeches, but it is now also available in synthetic form (recombinant hirudin). The inhibitor is currently undergoing clinical trials as a potential replacement for the extensively used thrombin inhibitor heparin. In this review, the biochemical and pharmacokinetic characteristics of hirudin (native and recombinant) and the efficacy of hirudin in treating and preventing cardiovascular disorders is discussed. The advantages of local controlled delivery of hirudin for treating cardiovascular disorders are then presented. Several implantable polymers applicable for controlled delivery system also are introduced. Finally, the feasibility of controlled delivery of r-hirudin for local therapy of cardiovascular disorders is addressed.

Contact activation during incubation of five different polyurethanes or glass in plasma.

During blood-material interaction, the enzymes factor XII fragment (factor XIIf) and kallikrein are generated (contact activation). In this study, the enzymatic activities of factor XIIf and kallikrein were examined with an assay based on the conversion of tripeptide-p-nitroanilide substrate. With the use of aprotinin to inhibit kallikrein, the proteolytic activities of factor XIIf and kallikrein could be separately determined. In this in vitro study, two commercially available polyurethanes, Pellethane and Biomer; three custom synthesized polyurethanes; a biomerlike 2000 MW polytetramethyleneoxide containing polyurethane (PU-2000); an octadecyl extended (ODCE) biomer-like 2000 MW polytetramethyleneoxide containing polyurethane (PU-2000-ODCE); a hard-segment polyurethane (HS-PU); and glass (reference material) were incubated in 25% diluted plasma. In both series of experiments, glass caused the highest amidolytic activities by factor XIIf and kallikrein compared with any of the polyurethanes. In contrast, within the polyurethane group of materials, lower amidolytic activities by factor XIIf and kallikrein were measured on the custom-made polyurethanes than on the commercially available polyurethanes, although the differences among the polyurethanes were small. In addition, the influence of different ratios of material surface to the plasma incubation volume was studied. An increased ratio of surface area over plasma volume resulted in reduced contact activation, suggesting that plasma components are the limiting factor.

Platelet and monoclonal antibody binding to fibrinogen adsorbed on glow-discharge-deposited polymers.

The state of fibrinogen adsorbed on untreated and glow-discharge-treated surfaces was examined by measuring platelet adhesion, monoclonal antibody (mAb) binding, the amount of fibrinogen adsorbed, and the amount of adsorbed fibrinogen which could be eluted with sodium dodecyl sulfate (SDS). Tetrafluoroethylene (TFE) glow-discharge-treated polymers have a lower surface free energy (in air) and retain a larger fraction of adsorbed fibrinogen than untreated surfaces after SDS elution. Platelet adhesion was lowest on the TFE-treated surfaces which retain the highest amounts of fibrinogen after SDS elution. Fibrinogen may undergo unfolding or spreading on the TFE-treated surfaces to minimize interfacial free energy (in water) and maximize protein-surface interactions. When it is adsorbed on the TFE-treated surfaces, fibrinogen evidently assumes a state which somehow prevents its recognition and binding by platelet receptors. Monoclonal antibodies that bind to the three regions in fibrinogen thought to be involved in platelet adhesion were therefore used to detect changes in adsorbed fibrinogen. These regions and the antibodies which bind to them are: the COOH-terminal of the gamma-chain, mAb M1; the RGD peptide sequence at A alpha 95-98, mAb R1; the RGD sequence at A alpha 572-575, mAb R2. For fibrinogen adsorbed on the untreated or TFE-treated surfaces, M1 and R2 binding was relatively high compared to background, while R1 binding was low. However, the amount of binding of each mAb to fibrinogen adsorbed on the TFE-treated surfaces was equal to or greater than fibrinogen adsorbed to the untreated surfaces. Therefore, antibody-detectable changes in the platelet binding regions of adsorbed fibrinogen that might have been caused by conformational or orientational rearrangements were not observed for the TFE-treated surfaces. The data suggest that the tight binding of fibrinogen on a surface may directly affect the ability of the fibrinogen to interact with the platelet receptors--i.e., that fibrinogen must be loosely held to facilitate maximal interaction with platelet receptors.