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

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

Effect of adsorbed von Willebrand factor and fibrinogen on platelet interactions with synthetic materials under flow conditions

The effect of adsorbed fibrinogen (Fg) and von Willebrand factor (vWf) on platelet adhesion at low or high shear rate to several materials was studied. The materials studied were polyethylene terephthalate (PET), polystyrene (PS), glass, and tetraglyme-coated PET. The materials were preadsorbed with normal plasma, serum, and Fg-deficient plasma replenished with various amounts of Fg, and vWf-deficient plasma with or without added vWf. Platelet adhesion to PET preadsorbed with Fg-deficient plasma or serum was low at either low or high shear rate, but increased as Fg was added to the preadsorption media. However, the effect of added Fg on adhesion at the higher shear rate was much greater on surfaces preadsorbed with plasma than for serum, probably due to the much lower vWf concentration in serum in comparison to plasma. Platelet adhesion to either polystyrene or glass preadsorbed with normal plasma was much higher at high shear than low shear, but when vWf-deficient plasma was used to preadsorb these surfaces, adhesion was much less at the higher shear rate than at low shear rate. Platelet adhesion to polystyrene preadsorbed with vWf-deficient plasma to which vWf was added was higher at high shear rate than low shear rate. These results show that under high shear rate, both Fg and vWf are required for platelet adhesion on synthetic biomaterials. The results suggest that developing surfaces that adsorb low amounts of vWf is a good approach to improving the blood compatibility of biomaterials.     

Adsorption of baboon fibrinogen and the adhesion of platelets to a thin film polymer deposited by radio-frequency glow discharge of allylamine.

Platelet adhesion under static and flow conditions from a washed platelet suspension containing albumin to a polymer deposited by radio-frequency glow discharge of allylamine vapour on a poly(ethylene terephthalate) substrate was measured. Electron spectroscopy for chemical analysis was used to characterize the surface. Fibrinogen adsorption from a series of dilute plasma solutions to radio-frequency glow discharge/allylamine, measured using 125I radiolabelled baboon fibrinogen, increased with decreasing plasma dilution to a level much higher than that previously observed on polyurethanes. Elutability by sodium dodecyl sulphate of fibrinogen adsorbed from dilute plasma also increased with increasing plasma concentration, but fibrinogen preadsorbed from plasma became non-elutable when surfaces were stored in buffer for 5 d before contact with sodium dodecyl sulphate. Platelet adhesion to substrates which had been pre-adsorbed with dilute plasma was measured using baboon platelets radiolabelled with 111In. Adhesion greatly decreased as the plasma concentration used for preadsorption increased, suggesting that non-specific platelet binding to the bare surface occurs when protein coverage is incomplete. Non-specific platelet binding was inhibited to varying degrees by preadsorption of different proteins to the surface. Platelet adhesion to surfaces preadsorbed with dilute (1.0%) baboon and human plasmas lacking fibrinogen (i.e. serum, heat-defibrinogenated plasma and congenitally afibrinogenemic plasma) was diminished compared with normal plasma. Addition of exogenous fibrinogen to the deficient plasma partially restored platelet adhesion to normal levels. Adhesion to surfaces preadsorbed with human plasma deficient in von Willebrand factor was comparable to that observed with normal plasma. The plasma preadsorption studies with fibrinogen deficient media suggested that adsorbed fibrinogen is necessary for platelet adhesion to the radio-frequency glow discharge/allylamine substrate at high protein coverage. However, since adhesion was greatly reduced when the plasma preadsorbed substrate was stored in buffer before platelet contact, the conformation of adsorbed fibrinogen is also important in mediating platelet adhesion to radio-frequency glow discharge.     

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

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

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene.

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

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.     

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

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

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

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

Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin

Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat(?) or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies.     

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.     

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

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

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.     

Fibrinogen and von Willebrand factor mediated platelet adhesion to polystyrene under flow conditions

The roles of adsorbed fibrinogen (Fg) and von Willebrand factor (VWF) in mediating platelet adhesion to synthetic surfaces under flow were investigated using polystyrene (PS) as a model hydrophobic surface. We measured platelet adhesion to PS pre-adsorbed with Fg, VWF, normal plasma, afibrinogenemic plasma, VWF-deficient plasma and deficient plasmas with various concentrations of added Fg or VWF. Platelets in a red blood cell suspension were passed through a flow chamber at either low (50 or 100 s(-1)) or high (500 or 1000 s(-1)) shear. Adhesion to PS pre-adsorbed with afibrinogenemic plasma was very low under both low and high shear conditions, but was restored in a dose-dependent manner with addition of Fg. Less than 20 ng/cm(2)of adsorbed Fg was sufficient to support full-scale platelet adhesion under flow. At high shear rate, platelet adhesion on PS pre-adsorbed with VWF-deficient plasma was much less than on PS pre-adsorbed with normal plasma, but adhesion to PS pre-adsorbed with VWF-deficient plasma with added VWF was very similar to adhesion to PS pre-adsorbed with normal plasma. At low shear, adhesion to PS pre-adsorbed with VWF-deficient plasma was the same as on PS pre-adsorbed with normal plasma. As little as 1 ng/cm(2) of VWF adsorbed from plasma made platelet adhesion higher under high shear than under low shear. The effects of adsorbed Fg and VWF on the morphologies of platelets that adhered from suspensions flowing at high shear rates were also investigated. The lack of either Fg or VWF resulted in marked decreases in the extent of platelet spreading. Real-time observation of platelet adhesion under an epifluorescent microscope showed that platelets adhered to the surface in a linear pattern aligned in the direction of flow under high shear conditions.     

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.     

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.     

Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions.     

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.     

Osteoblast adhesion and morphology on TiO2 depends on the competitive preadsorption of albumin and fibronectin

This study aims at assessing the influence of the competitive preadsorption of human serum albumin (HSA) and human plasma fibronectin (FN) from binary solutions and 10% plasma on MC3T3-E1 osteoblast adhesion and morphology on two types of TiO2 substrates. One was commercially pure titanium with a titanium oxide layer formed in an H2O2 solution and the other TiO2 sputtered on Si (Sousa et al., Langmuir 2004; 20:9745-9754.). The strategy applied in the present investigation was to compare osteoblast adhesion to surfaces preadsorbed with HSA, FN, HSA/FN = 1, HSA/FN = 200, and 10% plasma. The adsorption of proteins was evaluated measuring the amount and the effectiveness of binding with radiolabeled proteins, 125 I-FN and 125 I-HSA. Our results indicated that MC3T3-E1 osteoblast adhesion correlates well with the amounts of FN and HSA adsorbed on TiO2 surfaces. Also, we found that fewer osteoblasts adhered to both substrates preadsorbed with HSA, HSA/FN = 200, and 10% plasma, after 4 and 24 h, than to the surfaces preadsorbed with FN and HSA/FN = 1. For the latter, FN was able to compensate the inhibitory effect of HSA on osteoblast adhesion. Therefore, the presence of lower amounts of coadsorbed albumin may improve presentation of FN in a more integrin-recognized conformation, suggesting that some degree of molecular packing prevents loss of integrin-binding activity. FN reversibility does not seem to be dependent on the HSA/FN adsorption mass ratio in solution, suggesting that FN competitively adsorbs to TiO2 in a favorable conformation and does not suffers subsequent conformational changes allowing exchange with other FN molecules in solution.     

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.     

PDMS-based polyurethanes with MPEG grafts: synthesis, characterization and platelet adhesion study.

Polyurethane (PU), based on poly(dimethyl siloxane) (PDMS) as a soft segment, with monomethoxy poly(ethylene glycol) (MPEG) grafted onto it, was synthesized as a new polymeric biomaterial for coating PDMS-based biomedical devices. Two different chain extenders, ethylene glycol (EG) and diethyl bis(hydroxymethyl) malonate (DBM), were used for the synthesis of PDMS-based PUs and then MPEG was grafted onto them by allophanate and esterification reactions, respectively. Their molecular structures were confirmed qualitatively and quantitatively using FT-IR and 1H-NMR measurements. PDMS-based PU was more hydrophobic than Pellethane, which is a commercial biomedical-grade poly(ether urethane), due to the PDMS-rich phase at the polymeric surface. However, the incorporation of MPEG in PDMS-based PU induced an increase in hydrophilicity. Analyses of their morphology using dynamic mechanical analysis and differential scanning calorimetry showed that the degree of phase separation increased with the content of hard segments. It also showed that MPEG is compatible with a hard segment consisting of 4,4'-diphenylmethane diisocyanate (MDI) and DBM, while being incompatible with one consisting of MDI and EG. Platelet adhesions with PDMS-based PUs were significantly reduced when compared with Pellethane. It was also observed from a platelet adhesion experiment that the incorportion of MPEG further reduced platelet adhesion. PDMS-based PUs with MPEG grafts, which have few hard segments and a distinct PEG phase, exhibited the least platelet adhesion among the polymer samples tested.