Surface-attached PEO in the form of activated pluronicwith immobilized factor H reduces both coagulation andcomplement activation in a whole-blood model

Jonas Andersson,1,* Fredrik Bexborn,2,* Jeanna Klinth,2 Bo Nilsson,1 Kristina Nilsson Ekdahl1,2

1Division of Clinical Immunology, Uppsala University, Uppsala, Sweden2Department of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden

Received 4 January 2005; revised 15 February 2005; accepted 15 February 2005Published online 25 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30377

Abstract: In the present work we have bound Pluronic™, aclass of triblock copolymers consisting of a block of polypro-pylene oxide (PPO) surrounded on each side by polyethyl-ene oxide (PEO) blocks, to polystyrene surfaces and inves-tigated the thrombogenicity and complement activation ofthis construct upon exposure to whole blood. The surfacewas highly inert towards coagulation, unfortunately at theexpense of increased complement activation. We, therefore,as an alternative approach, used End-Group Activated Plu-ronic to conjugate factor H, a regulator of complement acti-vation (RCA), to the surface. The bound factor H did notdetach from the surface upon incubation with human se-rum. Furthermore, factor H bound in a physiological con-formation could to a significant degree attenuate comple-

ment activation at the Pluronic™ surface. Thus, we havecreated a hybrid surface in which the coagulation-inertproperties of the original Pluronic™ are supplemented witha specific complement-inhibitory effect. Medical device tech-nology includes numerous potential applications forcrosslinkers that are capable of specifically binding biomol-ecules to surfaces with retained activity. These applicationsinclude coupling of functional biomolecules to biomedicaldevices such as stents and grafts. The biomolecule may be anRCA, antibody, or other beneficial ligand. © 2005 WileyPeriodicals, Inc. J Biomed Mater Res 76A: 25–34, 2006

Key words: blood compatibility; coagulation; complement;factor H; regulator of complement activation (RCA)

INTRODUCTION

Surface-attached polyethylene oxide (PEO) isknown to reduce unspecific protein adsorption.1 Thepossibility to utilize this fact in blood contact applica-tions to reduce adverse reactions is a convenient wayto achieve a more biocompatible surface. When artifi-cial materials, such as biomaterials used in medicaldevices, come in direct contact with blood, there is aninstantaneous surface adsorption of proteins.2 Thisadsorption triggers activation of the coagulation andcomplement cascade systems. Unless properly con-trolled, these events lead to thrombotic and/or inflam-matory reactions, which may be detrimental to both

patient and biomaterial.3 Strategies for biomaterial de-sign have thus so far been mainly focused on under-standing and controlling the thrombogenicity of arti-ficial surfaces (e.g., by conjugating heparin or PEO tothe material surface); to date, technologies for attenu-ating complement activation at a biomaterial surfacehave not been as successful.4 There are two mainapproaches to achieve the nonthrombogenic surface:to minimize protein adsorption to the surface or tocoat the surface with biomolecules that actively down-regulates the cascade system. Studies indicate thatthese strategies need to be combined.5

In a huge number of studies different PEO surfaceshave been shown to be resistant to protein adsorptionand cell attachment. In almost all cases this has beenshown in systems using protein solutions, cell cul-tures, or blood fractions, such as plasma.6,7 Recently,in a review by Gorbet and Sefton, the relevance ofsuch studies was questioned. The different cascadesystems and cell types of blood are known to interact,and the true effect of a biomaterial surface on bloodcan only be known using whole blood.5 The use ofisolated systems are, of course, justified for identifyingspecific mechanisms, but the results should, if possi-

*These authors contributed equally to this work.Correspondence to: K. Nilsson Ekdahl; e-mail: Kristina.

Nilsson_Ekdahl@klinimm.uu.seContract grant sponsor: Faculty grantsContract grant sponsor: the Knowledge Foundation

(granted to the University of Kalmar)Contact grant sponsor: the Swedish Research Council;

contract grant number: 5647

© 2005 Wiley Periodicals, Inc.

ble, be confirmed in the whole blood situation. Somestudies of PEO surfaces using whole blood are avail-able.4,8 In vivo studies have indirectly confirmed thelow interaction with blood, because polystyrene beadswith surface-adsorbed PEO were protected from rapidelimination in the blood circulation of rats when com-pared to noncoated beads.9 Hansson et al. have shownthe benefit of PEO coatings in whole blood, where thethrombogenic characteristic of titanium cups is im-proved.10

In this study we aim at investigating the effect ofPEO on both coagulation and complement when at-tached to a model polymer surface such as polysty-rene. We adsorb PEO in the form of Pluronic™, a classof copolymers out of which the triblock copolymerF108 has been shown to be most efficient in reducingfibrinogen adsorption.1,11 This copolymer consists of ablock of polypropylene oxide (PPO) flanked on eachside by PEO blocks [Fig. 1(a)]. The PPO block mediatesthe adsorption to the surface [Fig. 1(b)]. This is asystem that also introduces the possibility of covalentimmobilization of biomolecules via chemically acti-vated forms of F108.12 The resulting End Group Acti-vated Pluronic (EGAP) is available in two different

forms, containing either a pyridyl disulfide (PDS)group or a nitrilotriacetic acid (NTA) group.13,14 ThePDS functionality used in this study allows binding ofproteins containing free thiol groups as shown in Fig-ure 1(c)–(g). Thiol groups can be introduced either bychemical means or by recombinant techniques. A pos-sible way to introduce thiol groups in proteins is touse the bifunctional crosslinker N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as shown in Figure1(d)–(e).15

We were able to confirm low thrombogenicity ofsurface-adsorbed PEO also in whole blood in accor-dance with the findings by Hansson et al.10 In contrast,this surfaced proved to be a potent complement acti-vator. This has been suggested before by Kidane etal.16 using a plasma system.

To overcome the complement activation at the Plu-ronic™ surface we studied the effect of immobilizingfactor H, a Regulator of Complement Activation(RCA), onto EGAP-PDS. Factor H functions both as acofactor for factor I-mediated inactivation of activatedC3 (C3b) and by competing with factor B and Bb forbinding to C3b in the formation of the alternativepathway convertase.17,18 This inhibition effectivelystops the amplification of complement activation, be-cause C3b is the central molecule in this system.19

Factor H is a soluble plasma protein, 49.5 nm long and3.4 nm wide, with a molecular weight of 155 kDa,which is often found folded back in a hairpin fash-ion.20 It is under normal conditions present in blood ata plasma concentration of 0.2–0.6 mg/mL. The singlepolypeptide chain of 1213 amino acid residues con-sists of 20 consecutive homologous short consensusrepeats (SCRs).21–23 Each repeat consists of about 60amino acid residues, and the structure is stabilized bytwo conserved disulfide bridges. The integrity of thedisulfide bridges is of critical importance for confor-mation and biological function of factor H.

Here, we successfully used the EGAP-PDS technol-ogy to specifically bind functionally intact, factor H topolystyrene surfaces. Using whole-blood models, wehave demonstrated the effectiveness of a hybrid sur-face in which the coagulation-inert properties of theoriginal Pluronic™ are supplemented with a specificcomplement-inhibitory effect.

MATERIALS AND METHODS

Reagents

Proteins

Human C3 and factor I were prepared according to Ham-mer et al. and Fearon et al., respectively.24,25 Factor H was

Figure 1. Schematic presentation of the Pluronic F108 sur-face modification system. Different aspects of Pluronic sur-face modification: (a) schematic representation of the Plu-ronic F108 blockcopolymer; (b) F108 adsorbed to a surface;(c) native F108 and EGAP-PDS; (d) protein and thecrosslinker SPDP mixed to achieve; (e) a SPDP activatedprotein; (f) surface adsorbed EGAP-PDS reduced to offer afree thiol; (g) the resulting immobilization of a SPDP acti-vated protein to reduced EGAP-PDS.

26 ANDERSSON ET AL.

prepared from human serum essentially according to Ham-mer et al. except that the first step consisted of a euglobulinprecipitation as described by Nilsson et al.26 C3 was di-gested with 1% (w/w) trypsin (Sigma Chemical Co, St.Louis, MO) for 5 min at 22°C, and the resulting C3a and C3bfragments were separated by gel filtration on SephadexG-100 (Amersham Pharmacia Biotech, Uppsala, Sweden)equilibrated in PBS.

Chemicals

EGAP F108 (NF grade), with 141 mer PEO units and 44mer PPO chains, activated with pyridyl disulfide [EGAP-PDS; Fig. 1(c)] was supplied by allvivo, Inc. (Lake Forest,CA). The average molecular weight of the Pluronic F108 was14,600 (ranging from 12,700 to 17,400). Bovine serum albu-min (BSA) and the crosslinker N-succinimidyl 3-(2-pyri-dyldithio) propionate (SPDP) were purchased from Sigma.

Serum pool

Blood was collected from three to five donors and allowedto clot for 1 h in 7-mL Vacutainer™ glass tubes withoutadditives (Becton, Dickinson and Co., Plymouth, UK). Thesupernatants were collected and pooled after centrifugationat 3450 � g for 25 min at 4°C. The pool was stored at �70°Cprior to use.

Heparin coating

Whenever indicated, materials were heparin-coated usingthe Corline method (Corline Systems AB, Uppsala, Sweden)according to the manufacturer’s recommendations. The sur-face concentration of heparin was 0.5 �g/cm2, with an an-tithrombin binding capacity of 6 pmol/cm2.27

Modification and conjugation of factor H

SPDP activation of factor H

SPDP was dissolved in DMSO (5 mg/mL) and then addedto factor H in the desired mass ratios (1–17% w/w). Theconcentrations of the various batches of factor H rangedfrom 1 to 2 mg/mL. In a typical reaction mix, approximately10 �L SPDP was added to 1 mL factor H. Reagents wereincubated for 1 h at 22°C, and unbound SPDP was removedby gel filtration on NAP-5 Sephadex G-25 columns (Amer-sham Biosciences, Uppsala, Sweden).

Factor H activity assay

The activity of SPDP conjugated factor H was tested in acofactor assay for factor I-mediated cleavage of C3b withnative factor H as reference. Factor H in dilution series (0.5,

1 or 2 �g) was incubated with 10 �g C3b and 0.6 �g factorI in PBS for 60 min at 37°C. Samples were applied to a 10%SDS PAGE gel, along with samples of pure factor H anduncleaved C3b as a reference. Activity was measured bycomparing the C3b bands at 101 kDa (i.e., the alpha� chain);a weaker band meant that more C3b had been cleaved.

Surface preparation

EGAP-PDS was dissolved in PBS at a concentration of 1%(w/v) and incubated overnight at 22°C in the wells of 96-well Maxisorp™ microtiter plates (Nunc, Roskilde, Den-mark). After a rinse with PBS (pH 7.4) containing 1 mMEDTA (PBS-EDTA), the EGAP to be immobilized was re-duced with 25 mM dithioethriol (DTT; Merck, Darmstadt,Germany) in PBS-EDTA for 1 h. The DTT was washed awaywith PBS-EDTA, pH 6.0. SPDP-activated factor H, diluted inPBS-EDTA to 2-300 �g/mL, was incubated overnight at 4°C,and the surface was finally washed with wash buffer: PBScontaining 0.05%(v/v) Tween 20 and 0.02% (v/v) Anti-foam™ (Pharmacia Diagnostics, Uppsala, Sweden). The re-action scheme is outlined in Figure 1(d)–(g).

In separate experiments, SPDP-activated factor H waslabeled with the Alexa Fluor� 488 Protein Labeling Kit (Mo-lecular Probes, Leiden, The Netherlands). Labeled factor Hwas mixed with nonlabeled to 8% (w/w) and was incubatedin EGAP-coated polystyrene microtiter plates in total con-centrations of 100 �g/mL for 2 h at RT and for an additional22 h at 4°C. When wells were coated for subsequent expo-sure to human serum, 100% Alexa labeled factor H wasused.

Quantification of surface-bound factor H

Enzyme immunoassay (EIA) for immobilizedfactor H

Wash buffer containing 1% (w/v) BSA was used as aworking buffer. With the exception of the TAT analysis, theplates for all assays were stained with color solution con-taining the HRP color reagent DAB (Biorad, Hercules, CA)in 0.1 M citrate buffer, pH 5.0, for approximately 5 min andread at 492 nm.

Bound factor H was detected by incubation with a mixtureof biotinylated (1:500 dilution) and nonbiotinylated (1:300dilution) sheep antihuman factor H (The Binding Site, Bir-mingham, UK) in working buffer for 1 h, followed bystreptavidin-HRP (Amersham Biosciences, Little Chalfort,UK) diluted 1:500 in working buffer for 15 min.

QCM-D

Quartz crystal microbalance with dissipation monitoring(QCM-D) is a technology that measures the mass of a boundsubstance and can provide information about the structuraland conformational properties of the bound substance (Q-sense AB, Gothenburg, Sweden). The QCM-D system uti-

SURFACE-ATTACHED PEO IN THE FORM OF ACTIVATED PLURONIC 27

lizes the fact that an applied AC voltage will cause a quartzcrystal to oscillate due to its piezoelectric properties. Ad-sorbed mass causes a proportional change in the resonancefrequency of the oscillating crystal, which can be measured.The relationship between frequency (f) and adsorbed mass(m) is described by the Sauerbrey equation.28 In addition tothe frequency shift, it is also possible to measure the dissi-pation of the adsorbed layer, which gives some informationon the viscoelasticity of the layer. The QCM-D system hasbeen shown to work both in gaseous and liquid environ-ments.29 Unfortunately, the Sauerbrey equation is only validwhen the adsorbed mass is a rigid layer.

For analysis by QCM-D, factor H preparations conjugatedwith 0, 1, 3.5, 7, and 17% (w/w) SPDP were prepared asdescribed above. The sensor crystal of the QCM-D wasspin-coated with polystyrene (Q-sense AB) and then incu-bated with EGAP overnight or longer. After a rinse withPBS-EDTA, pH 7.5, the crystal was mounted in the chamberand equilibrated in PBS-EDTA. All subsequent steps werecarried out at 25°C. The surface was reduced with PBS-EDTA containing 25 mM DTT for 1 h and then rinsed withPBS-EDTA, pH 6.0, six times for 5 min. SPDP conjugatedfactor H (100 �g/mL) was incubated on the crystal for 2 h(overnight for native factor H), then with PBS-EDTA, pH 7.5,for 2 � 10 min.

Fluorescence measurements

The fluorescence of Alexa 488-labeled factor H (expressedas cps) was measured in a Fluorolog 3-22 spectrofluorometer(Jobin Yvon Inc., Edison, NJ) at 518 nm. The factor H thatremained in solution after incubation in EGAP-coated poly-styrene microtiter wells was analyzed using a microcell(Hitachi, Tokyo, Japan). The factor H that was bound to theplate was analyzed using a F3000 fiberoptic interface (JobinYvon Inc.) integrated with Fluorolog 3-22.

Exposure of surface-conjugated factor H to serumor whole blood

Serum incubation

Serum without additions or containing 10 mM EDTA (toinactivate complement) was incubated for up to 1 h inpolystyrene microtiter wells at 37°C. Different surfaces weretested, including surfaces with immobilized factor H (withor without treatment with SPDP); EGAP, either DTT-treatedor nontreated; and PBS-incubated only. Prior to addition ofserum, the surfaces were incubated with working buffercontaining 1% BSA to obtain a complement-activating BSAsurface. After incubation, the serum was transferred to tubescontaining 10 mM EDTA (final concentration) and stored at�70°C until analyzed. The surfaces of the microtiter wellswere washed with wash buffer prior to detection of boundC3 fragments by EIA. Alternatively, the amount of plate-bound factor H after incubation with serum or PBS wasmeasured by fluorescence spectroscopy or by EIA.

Slide chamber model

Fresh whole-blood samples, collected from healthy volun-teers who had received no medication for 10 days, with theaddition of 50 �g/mL r-hirudin, leprirudin (Refludan™,Aventis Pharma) or 1 IU/mL heparin (Løvens Kemiske Fab-rik, Ballerup, Denmark), were incubated in the slide cham-ber model previously described.30 In this model 1.3-mLblood was added to a surface-heparinized well, a slidecoated with the surface of interest was placed on top of thewell, and the chamber was allowed to rotate at 37°C for 1 h.After incubation, the chamber was opened, and the bloodwas transferred to tubes containing EDTA (10 mM finalconcentration). Cell counts were collected with a Coulter�

AC � T diff™ Analyzer from Coulter Corporation (Miami,FL). Plasma was collected after centrifugation at 3450 � g for25 min at 4°C and stored at �70°C.

EIAs for activation products

Surface-bound C3 fragments

The total amount of C3 fragments was measured usingmixed rabbit antihuman C3c (Dako, Glostrup, Denmark)diluted 1:200 and antihuman C3c-HRP (Dako) diluted 1:400.

Soluble C3a

Plasma diluted 1:1000 was incubated in wells coated withmAb 4SD17.3, which served as capture antibody. As previ-ously described, C3a was detected using a biotinylated an-tihuman C3a, followed by HRP-conjugated streptavidin.Zymosan-activated serum calibrated against a solution ofpurified C3a served as a standard; values are given in ng/mL.31 It has previously been shown that iC3 (i.e., C3 with abroken thiol ester), which is generated at the interface be-tween air and blood in the slide chamber model, reacts in theC3a EIA. Therefore, control experiments with heparin fur-nished test slides were run in parallel, and the C3a valuesobtained from these slides were subtracted from the valuesobtained from the test surfaces.

Thrombin–antithrombin complex

Thrombin–antithrombin complexes (TAT) were measuredusing antibodies from Enzyme Research Laboratories (SouthBend, IN). TAT was captured in wells coated with antihu-man thrombin diluted 1:200. HRP-coupled antihuman anti-thrombin antibody diluted 1:200 was used for detection. Asa standard, purified thrombin mixed with an excess of an-tithrombin in the presence of heparin was used. The highestpoint in the standard curve was 200 �g/L.

Calculations and statistics

Because of pronounced individual variations in the re-sponse of coagulation and complement activation in whole

28 ANDERSSON ET AL.

blood, the differences were calculated as a percentage of thevalues obtained for whole-blood controls. The results areexpressed as mean � SD. Statistical significance was calcu-lated with Student’s t-test for unpaired samples, using Stat-view 4.01 (Abacus Concepts, Berkley, CA) for Macintosh(Apple Computer, Cupertino, CA). This study was per-formed with the consent of the Ethical Committees at theUniversity Hospitals of Uppsala and Linkoping.

RESULTS

Modification and conjugation of factor H

Optimizing factor H immobilization

Factor H was activated as previously described, andthe products from reactions with SPDP/factor H ra-tios ranging from 1 to 17% (w/w) were tested inactivity assays. The activity of factor H dropped whenthe ratio was above 3.5%, with no activity remainingat 17% (data not shown). In QCM-D, the binding offactor H conjugated with 1, 3.5, and 7% SPDP to theEGAP surface showed identical binding that was closeto four times that with native (0% SPDP) factor H (Fig.2). An SPDP/factor H ratio of 17% showed evenhigher binding than did the lower ratios, but thispreparation had already been shown to lack activity.Because activated factor H incubated with 3.5% SPDPshowed activity similar to native factor H and didbind to the surface, it was chosen for later experi-

ments. Using the Sauerbrey equation for the thirdharmonic a �180 Hz shift is equivalent to a surfaceconcentration of 1.1 �g/cm2. This result is, however,valid only under the assumption that the bound pro-teins form a rigid layer. When considering a viscoelas-tic layer this represents an overestimation.

Characterizing factor H immobilization

The binding of activated factor H to untreated poly-styrene, nonreduced EGAP, and reduced EGAP wasassessed using EIA (Fig. 3). Binding was similar onunmanipulated polystyrene and reduced EGAP, whileonly slight binding was seen to nonreduced EGAP.The incorporation of SPDP-activated factor H reacheda plateau at 50 to 100 �g/mL, and the higher coatingconcentration was used for subsequent experiments.In the wells incubated with 100 �g/mL of fluores-cently labeled factor H, 97 � 5% remained in solutionafter incubation in EGAP coated wells and 93 � 2% innoncoated wells, respectively after 24 h. Because thearea in contact with fluid in each well represents a 0.95cm2 surface this corresponds to surface concentrationsof 0.32 �g/cm2 and 0.74 �g/cm2, respectively, whichcorresponds to a molar factor H surface concentrationof 2 and 4 pmol/cm2, respectively.

Characterizing factor H stability

Wells coated with SPDP-activated factor H wereincubated with PBS, serum, or EDTA-serum for up to

Figure 3. Binding of factor H to noncoated or EGAP-coatedpolystyrene, as detected by EIA. The diagram shows bind-ing of SPDP-conjugated factor H to: polystyrene (circles);polystyrene coated by DTT-reduced EGAP-PDS (triangles);and polystyrene coated by nonreduced EGAP-PDS (squares).Data from three separate experiments performed in duplicateare presented as means � SD.

Figure 2. Binding of SPDP-conjugated factor H (w/w) toEGAP-coated polystyrene surfaces, as measured by QCM-D.Changes in frequency were measured on the third harmonic(n � 3, f � 15 MHz). EGAP was incubated on polystyrene-coated quartz crystals overnight. After rinsing, factor Hconjugated with SPDP in various ratios (w/w) was incu-bated on the crystal for 2 h and then rinsed with PBS-EDTA.

SURFACE-ATTACHED PEO IN THE FORM OF ACTIVATED PLURONIC 29

60 min. Thereafter, the amount of bound factor H wasdetected by EIA or fluorescence spectroscopy. WhenPBS or EDTA-serum (in which the complement sys-tem is inactivated) was incubated in wells with SPDP-activated EGAP-bound factor H, there was no loss offactor H during the first 60 min, as detected by EIA.When active serum without additives was incubatedon immobilized factor H, there was a continuous de-cline in the factor H available on the surface [Fig.4(A)].

When serum without EDTA was added to Alexa-labeled SPDP-bound factor H, an initial increase influorescence was seen, most likely as a result of lightscattering by deposited plasma proteins [Fig. 4(B)].After incubation for 60 min, the fluorescence had re-turned to the initial levels (p � 0.78). Taken together,these data suggest that the apparent loss of factor Hseen by EIA in the wells incubated with complement-sufficient serum was a result of antigen maskingrather than detachment of the immobilized protein.

Attenuation of complement activation by surface-conjugated factor H

Complement activation in serum

To achieve a moderate complement challenge, thesurfaces were coated with 1% BSA for 1 h prior toserum incubation. Immobilized factor H induced adelay of approximately 10 min in the binding of C3fragments from serum to the surface, when comparedto nonreduced EGAP (Fig. 5).

Coagulation and complement activation in whole-blood models

The effect of EGAP on the coagulation system wasvisualized by incubating whole blood containing 1IU/mL heparin in the slide chamber model on poly-styrene with (1) no coat; (2) nonreduced EGAP; or (3)immobilized factor H for 1 h. The level of consump-tion of platelets was 55% on polystyrene but only 14and 12% on EGAP and immobilized factor H, respec-tively, when the initial value of platelets found inwhole blood was set to 100% [p � 0.0001; Fig. 6(A)].This profile mirrored that of TAT generation, forwhich the values obtained for nonmodified controlpolystyrene were set to 100% [Fig. 6(B)].

Because heparin, even in low doses, inhibits thecomplement system,32 we instead used 50 �g/mL r-hirudin to obtain total inhibition of coagulation whencomplement parameters were being studied.33 In thissetting, complement activation, as indicated by thegeneration of C3a, was significantly higher on EGAP:100% compared to 64% for the noncoated polystyrenereference (p � 0.0019). The increased complement ac-tivation was almost completely reversed, to 76%, byimmobilization of factor H on EGAP (p � 0.11 versuspolystyrene control; Fig. 7).

DISCUSSION

The present study was undertaken to test the effectof surface-adsorbed PEO in a whole-blood model,with regard to its interaction with the coagulation andcomplement systems. Using whole-blood models ispreferred, because many interactions between differ-ent systems in blood will not be revealed using iso-

Figure 5. Effect of EGAP, with or without immobilizedfactor H, on complement activation in serum. Complementactivation in serum, as assessed by binding of C3 fragmentsto polystyrene plates coated with EGAP with (squares) orwithout (diamonds) SPDP-conjugated factor H. Data aregiven as means � SD (n � 5).

Figure 4. Stability of EGAP-bound factor H in serum andPBS. (A) SPDP-conjugated factor H was bound to EGAP-coated microtiter wells, which were then incubated withserum (triangles), EDTA-serum (circles) or PBS (squares).Thereafter, factor H was detected by EIA. Data are given asmeans � SD (n � 2). (B) PBS or serum was incubated inEGAP-coated microtiter wells with (gray) or without (open)immobilized Alexa-labeled SPDP-conjugated factor H.Thereafter, remaining surface immobilized factor H wasdetected by fluorescence spectroscopy. Data are given asmeans � SD (n � 3).

30 ANDERSSON ET AL.

lated blood fractions, such as cell cultures, plasma, orproteins. Gorbet and Sefton have reviewed this issueextensively, and they also stress the fact that the focuson thrombosis in the biomaterial field takes away theattention from the other cascade systems in blood.5 Amajority of the studies on PEO-coated surfaces arefocused on thrombosis and are not undertaken inwhole blood.6,7 There are some rare examples wherewhole blood is used.10

In the slide chamber model, using whole blood withan addition of minute amounts of heparin to avoidcomplete clotting, we found that platelet consumptionwas reduced on EGAP coated surfaces compared topolystyrene.30 This surface modification was also ac-companied by a lowered formation of TAT, indicatingimproved coagulation compatibility induced by thePluronic™-mediated PEO coating. This finding con-firms that minimized protein adsorption from proteinsolutions and plasma and reduced platelet adhesionfrom platelet-rich plasma correlates with lowered ac-tivation of the coagulation system as indicated bysome other whole-blood model systems.10

When investigating the activation of the coagulationsystem there were indications that the activation of thecomplement system was increased. In further experi-

ments, focused on complement activation, we avoidedthe use of soluble heparin, which is known to inhibitcomplement and interact with several complementcomponents, including factor H. Instead, we used thespecific thrombin-inhibitor hirudin to achieve com-plete inhibition of coagulation without affecting com-plement during the remaining whole-blood experi-ments.33 In this system, the levels of the complementactivation product C3a were, however, also increasedupon contact with the EGAP-coated surface whencompared to bare polystyrene. This has previouslybeen suggested by Kidane et al. in a plasma systemthat unfortunately contained EDTA, a unspecific in-hibitor of complement.16 They suggest that it is theblock separating the two PEO blocks in Pluronic™that activates complement. Another study has shownthat complement activation by surfaces is increased assubstrate molecular mobility increases.34 This mightindicate that it is the mobility of the PEO itself thatcauses complement activation.

The second aim of the study was to investigate thepossibility to specifically bind a complement inhibitorto the Pluronic-mediated PEO surface to overcome theincreased complement activation. For this purpose weused the already available system of chemically acti-vated Pluronic F108, EGAP.13 This system has previ-ously been shown to bind protein ligands with a re-tained native conformation, as reflected by retainedbiological function.12,14,35 A part of this aim was alsoto investigate the stability of the bound RCA in aphysiologically relevant milieu.

To investigate whether it is possible to use EGAP toimmobilize the RCA factor H, we decided to conjugate

Figure 7. Effect of EGAP, with or without immobilizedfactor H, on complement activation in whole blood. Com-plement activation was monitored as generation of C3a afterincubation of whole blood containing 50 �g r-hirudin/mL inthe chamber blood model. Data are given as means � SD(n � 4 separate experiments, each performed in duplicate).

Figure 6. Effect of EGAP, with or without immobilizedfactor H, on coagulation activation in whole blood. (A)Platelet loss after incubation of whole blood containing 1IU/mL heparin in the chamber blood model. Data are givenas means � SD (n � 4 separate experiments, each performedin duplicate). (B) Generation of TAT after incubation ofwhole blood containing 1 IU/mL heparin in the chamberblood model. Data are given as means � SD (n � 4 separateexperiments, each performed in duplicate).

SURFACE-ATTACHED PEO IN THE FORM OF ACTIVATED PLURONIC 31

the protein using the free thiol groups of reducedEGAP-PDS. Factor H is a soluble RCA present inblood, and this fact demonstrates that the proteinalone is stable in blood and that any lowered stabilityon the surface is due to chemical modifications. Thepreferred method of immobilization is to tag the pro-tein with a reduced thiol group. In the case of factor H,however, this was difficult because of the need ofprotecting internal disulfide bridges.36,37 Therefore,SPDP conjugation of factor H was not followed byreduction, and instead, the surface-adsorbed EGAP-PDS was reduced to introduce free thiol groups. Re-sults from the QCM measurements indicate a 1.1-�g/cm2 surface concentration, which includes associatedwater molecules. The Sauerbrey equation is only validfor rigid layers, and we are considering a protein layeron top of another layer of PEO. This indicates that thesurface density is highly overestimated.28,38 The studyof fluorescently labeled factor H confirms this conclu-sion by showing a surface density of 0.21–0.32 �g/cm2

corresponding to a molar surface concentration ofabout 2 pmol/cm2. This is corresponding to a mono-layer, and is in the same range as a surface conjugatedwith BMP-2 and a commercially available heparinsurface.27,39 The heparin surface has an antithrombinbinding capacity of 6 pmol/cm2, and considering thatonly every third heparin molecule possess antithrom-bin binding sites, this corresponds to 18 pmol hepa-rin/cm2. The BMP surface intended for cell culturescontained 60 ng/cm2, and taking the molecular weightof 26 kDa the molar concentration is 2.3 pmol/cm2.

In a previous study we have demonstrated thatimmobilizing factor H is feasible and also beneficial interms of controlling complement activation.40 The re-ported method of immobilization, which involved thegeneration of an amine-coated surface, had not beenoptimized, and seemed to cause an activation of com-plement by itself. In the present study, we have im-proved the concept from our previous work by suc-cessfully using EGAP as the agent of immobilization,consistent with its previously demonstrated effective-ness in immobilizing many proteins.12,14,35

Our data indicate that we were able to functionalizeour protein with SPDP while retaining activity, andwe further demonstrated that this protein was able tospecifically bind to reduced but not to nonreducedEGAP. When tested in the whole-blood models, sur-face-bound factor H, despite its low concentration,was indeed able to overcome the increased comple-ment activation elicited by the EGAP surface, to yieldlevels approximating those for the original comple-ment-inert polystyrene surface. This finding indicatesthat the thiol groups introduced by the SPDP treat-ment had not altered the integrity of this conforma-tion-sensitive molecule. The very low thrombogenicityseen for the nonconjugated EGAP was preserved bythis treatment. Beneficial effect of factor H in serum

was only seen after challenging the complement sys-tem with adsorbed BSA, showing the importance ofusing a proper test system as whole blood. The factthat the generation of C3a is not below the levelsgenerated by polystyrene can be explained by thepresence of free thiol groups at the surface and thelack of site-specificity of factor H binding. This is aproblem that could be overcome by the use of recom-binant factor H as suggested below.

Our results also indicated that the EGAP-conju-gated factor H remained bound to the surface whenexposed to serum containing active complement forthe entire test period (60 min). This finding is in agree-ment with previous results indicating a stability ofEGAP coatings of up to 2 weeks in blood.11 Thisstability, in combination with the ability to bind pro-teins while preserving their physiological conforma-tion (as exemplified by the retained activity of factor Hdemonstrated here), makes EGAP an attractive candi-date for binding ligands to devices such as stents.

In the case of devices that include large areas ofbiomaterial surface, the release of inflammatory me-diators can give rise to a systemic inflammatory re-sponse. In cardiopulmonary bypass this responsemight lead, in the worst case, to multiorgan failure.3

There are two major approaches to lowering this re-sponse: to make the surface as inert as possible toprotein adsorption, or to change it in a way that ac-tively downregulates activation of the various cascadesystems. In the present article we have used a combi-nation of these two strategies. The method used forconjugation in the present study (i.e., the reversedapproach of first targeting the protein and then reduc-ing the EGAP coated onto the surface) demonstratesthat immobilization is possible but rather cumber-some. An alternative approach based on the use of atagged recombinant protein would be preferred. Theuse of a recombinant protein having a protein-engi-neered functional group would eliminate the need toreduce the EGAP-coated substrate and would, in turn,eliminate potential problems with oxidation of thefunctional groups on the EGAP. Furthermore, allbound proteins would bind in the same specific man-ner, and there would be no need to manipulate theprotein, thereby reducing the risk of disrupting pro-tein activity. If the EGAP-based approach were usedin combination with a recombinant protein, it wouldalso be possible to vary the density of the protein, inthis case factor H, to find the most beneficial balancebetween active and passive downregulation.

In summary, the data presented here confirms, inwhole blood, that Pluronic™-mediated surface-at-tached PEO on a model biomaterial surface results inincreased compatibility with the coagulation system,as previously shown in other systems and to someextent also in whole blood. This increased coagulationcompatibility is achieved at the price of increased

32 ANDERSSON ET AL.

activation of the complement system. Immobilizationof factor H is, however, able to counteract this activa-tion. This finding points to the possibility of creatinghybrid surfaces for blood applications by utilizing thegeneral coagulation-inert properties of the Pluronic™in conjunction with specific inhibitors of the othercascade systems of the blood.

We thank professor Karin Caldwell and PhD Jennifer Nefffor valuable discussions, and Kerstin Sandholm and Lil-lemor Funke for preparation of complement components.

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