optimal heparin surface concentration and antithrombin binding capacity as evaluated with human...

Optimal heparin surface concentration and antithrombinbinding capacity as evaluated with human non-anticoagulated blood in vitro

Jonas Andersson,1 Javier Sanchez,1 Kristina Nilsson Ekdahl,1,2 Graciela Elgue,1 Bo Nilsson,1

Rolf Larsson1,3

1Department of Oncology, Radiology and Clinical Immunology, Section of Clinical Immunology, Rudbeck LaboratoryC5, Uppsala University Hospital, SE-751 85 Uppsala, Sweden2Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden3Corline Systems AB, Box 956, SE-751 09 Uppsala, Sweden

Received 6 August 2002; revised 5 November 2002; accepted 6 March 2003

Abstract: Contact between blood and a biomaterial surfacetakes place in many applications and is known to activatethe coagulation and complement systems. Heparin surfacecoatings have been shown to reduce blood activation uponcontact with artificial surfaces. To establish the optimal hep-arin surface concentration, blood was incubated in a tubingloop model at 37°C. The tubing was coated with differentsurface concentrations of heparin and rotated at three dif-ferent velocities. We demonstrate that the blood compatibil-ity of a surface with regard to coagulation, complement, andplatelet activation can be improved by increasing the hepa-rin surface concentration in the 6–12 pmol antithrombin/

cm2 concentration interval. The binding of factor H is notinfluenced by the increased heparin surface concentration,suggesting that this factor is not the primary regulator ofcomplement on heparin surfaces. In addition, the heparincoating has no effect on the complement activation thatoccurs on gas surfaces in extracorporeal circuits. © 2003Wiley Periodicals, Inc. J Biomed Mater Res 67A: 458–466,2003

Key words: biocompatibility; blood; coagulation; comple-ment activation; immobilized heparin

INTRODUCTION

Contact between blood and biomaterials triggers adefense response mediated by activation of the cas-cade systems in blood.1,2 This includes both coagula-tion and complement activation.3,4 Activation of thesesystems in turn activates cellular defense mecha-nisms.5 Many cell types are activated; some of themost important ones are neutrophils and platelets.3,6

During cardiopulmonary bypass there is a systemicinflammatory response that in part is caused by thebiomaterial contact. Complications caused by this ac-tivation are anaphylactic reactions, pulmonary andrenal dysfunctions, cerebral problems, and coagulopa-thy.7–9 In addition, vascular implants such as stents

might cause activation of coagulation leading to theinitiation of thrombosis.10 Heparin immobilized onthe biomaterial has been shown to lower activation ofboth the coagulation and complement systems.11–13

Many studies based on different heparin surfaces havebeen published, but in general, it is difficult to assessfrom the literature what the biological functions of thevarious heparin surfaces are and in particular how thebiological performance may be related to the chemicalconstitution. Although at least 10 different heparinsurfaces can be distinguished in the literature,14–17

there are only three that combine the stability of im-mobilized heparin and a sufficient capacity to bindand interact with antithrombin (AT). These are theCarmeda Bioactive Surface, the Medtronic HepamedSurface, and the Corline™ Heparin Surface (CHS).18

Soluble heparin down-regulates coagulation by bind-ing to AT and turning it into a more active state. Theability to inhibit different coagulation factors is thusincreased 1000-fold.19 It has been suggested that thebiological performance of immobilized heparin wouldimprove along with increased capacity to bind AT, butthere are no studies to confirm this assumption.

Correspondence to: J. Andersson; e-mail: [email protected]

Contract grant sponsor: Swedish Foundation for StrategicResearch

Contract grant sponsor: Swedish Research Council; con-tract grant numbers: 5647, 11578

© 2003 Wiley Periodicals, Inc.

Sanchez et al.20 concluded that a binding capacity of 4pmol AT/cm2 was required to obtain optimal effect ofCarmeda Bioactive Surface with regard to inhibition ofcontact activation in plasma. However, no informationwas provided on possible effects obtained by increas-ing the AT binding capacity in contact with wholeblood. Complement activation is basically a surface-induced process, which is initiated on either cell mem-branes or artificial surfaces. Regulation is assumed tobe accomplished by localizing soluble complementinhibitors to the surface. The affinity between heparinand the complement regulator factor H is well estab-lished.21–24 However, evidence that factor H mediatesthe complement regulatory effect of immobilized hep-arin remains to be presented. There are speculationsconcerning the importance of heparin surface concen-tration on complement activation. The coagulationsystem is inhibited by soluble heparin at much lowerconcentrations than the complement system.25 It istherefore reasonable to assume that the concentrationof surface-bound heparin optimized to inhibit activa-tion of the coagulation system is insufficient to down-regulate complement. To establish the optimal heparinsurface concentration able to avoid blood activation,CHS was prepared to express various surface concen-trations of heparin and consequently AT binding sites.The test surfaces were evaluated in a modified Chan-dler loop model using fresh human non-anticoagu-lated blood at three different blood flow rates. Activa-tion of coagulation, reflected in the generation ofcomplexes between AT and thrombin, activation ofplatelets as indicated by the release of �-thromboglob-ulin (TG) from the alpha-granules, and complementactivation assessed as generation of C3a and the ter-minal sC5b-9 complex were measured. In addition,binding of factor H to the immobilized heparin wasmonitored.

MATERIALS AND METHODS

Heparinization

Polyvinyl chloride tubing with different surface concen-trations of the CHS was supplied by Corline System AB,Uppsala, Sweden. This surface is prepared by a conditioninglayer of polymeric amine onto which a macromolecularconjugate of heparin is attached. The conjugate is preparedby covalent binding of approximately 70 moles heparin/mole of carrier chain.18 This procedure produces a surfacewith a heparin surface concentration of 0.5 �g/cm2 and anAT binding capacity of 2–4 pmol AT/cm2. Higher levels ofheparin surface concentration and AT binding capacity wereachieved by repeating the application of polymeric amineand heparin conjugate up to three times.

Analysis using quartz crystal microbalance withdissipation monitoring (QCM-D)

To monitor the heparinization of a surface, the QCM-Dtechnique was used.26 This technique relies on the fact thata mass adsorbed onto the sensor surface of a shear-modeoscillating quartz crystal causes a proportional change in itsresonance frequency, f. Changes in f reflect the amount ofmass deposited onto the surface of the crystal. For thin,evenly distributed and rigid films, an adsorption-inducedfrequency shift (�f) is related to mass uptake (�m) via theSauerbrey relation, � f � �n�mC�1, where C (equivalent to17.7 ng cm�2 Hz�1) is the mass sensitivity constant and n (�1,3…) is the overtone number.27 However, for macromole-cules adsorbed from the aqueous phase, one must also beaware that water hydrodynamically coupled to the adlayeris included in the measured mass uptake.28 The dissipationfactor (D) reflects frictional (viscous) losses induced by de-posited materials such as proteins adsorbed on the surface ofthe crystal. Hence, changes in the viscoelastic properties ofadlayers (e.g., those induced by conformational changes) aswell as differences between various protein–surface interac-tions can be monitored.29–31

Analysis of adsorption kinetics by simultaneous measure-ment of both the frequency, f, and the energy dissipation, D,was performed on a QCM-D instrument (Q-Sense AB, Goth-enburg, Sweden), which is described in detail elsewhere.32

Sensor crystals (5 MHz), sputtered with stainless steelSIS2343, were used. Changes in D and f were measured onboth the fundamental frequency (n �1, i.e., f � 5 MHz) andthe third (n � 3, i.e., f � 15 MHz) and fifth harmonic (n � 5,i.e., f � 25 MHz). The third harmonic is shown in Figure 1.All measurements were performed at 25°C.

The chandler loop model

Fresh whole blood was drawn from healthy volunteers,who had received no medication for 10 days, using surfaceheparinized equipment only, without soluble anticoagu-lants. Blood was drawn to achieve negligible activation,resulting in preloop values of 410 � 58 IU/mL for �-TG,5.75 � 0.85 �g/L for thrombin-AT complexes (TAT), 113 �25 ng/mL for C3a, and 19.4 � 2.3 arbitrary units (AU)/mLfor sC5b-9. Pieces of polyvinyl chloride tubing furnishedwith immobilized heparin were used in the loops. Tubingwith a total volume of 6.3 mL (inner diameter � 4 mm,length � 500 mm) was filled with 4.5 mL of fresh wholeblood leaving a gas volume of 1.8 mL. The tubing wasturned into closed circuits using surface heparinized con-nectors of thin walled steel (length � 20 mm). The ends ofthe connector were tightly pushed into the lumen of thetubing and secured using copper seals. The tubing wasrotated vertically at 15, 33, or 50 rpm in a 37°C water bath for1 h. After incubation, 2-mL samples were collected in 13 mMcitrate and in 4 mM ethylenediaminetetraacetic acid, respec-tively, and centrifuged at 3300g for 25 min. At each velocity,blood from four different donors was used, except for thelowest velocity in which one donor was excluded. Bloodfrom each donor was incubated in three loops at each AT

BLOOD VELOCITY, HEPARIN, AND BLOOD COMPATIBILITY 459

binding capacity. The different velocities converted to linearvelocities are shown in Table I.

Analyses

In all assays below except platelet count, factor H and ATbinding capacity citrate plasma was used as sample.

Platelet count

Platelet number in the ethylenediaminetetraacetic acidchelated whole blood was counted using a Coulter� AC � Tdiff™ Analyzer from Coulter Corporation, Miami, FL. Eachsample was analyzed twice.

�-TG

�-TG was measured using the Asserachrome™ �-TG en-zyme immunoassay (EIA) kit from Diagnostica Stago,Asnieres-sur-Seine, France. �-TG was captured in wellscoated with specific rabbit anti-human �-TG F(ab�)2 frag-ments. Horseradish peroxidase (HRP)-coupled rabbit anti-human �-TG antibody was used for detection. The valuesare given as international units/milliliter.

TAT

TAT was measured using the Enzygnost� TAT micro EIAkit from Behringwerke AG, Warburg, Germany. TAT wascaptured in wells coated with rabbit anti-human thrombin.HRP-coupled rabbit anti-human AT antibody was used fordetection. The values are given as micrograms/liter.

Other EIAs

Phosphate-buffered saline (PBS) containing 1% (w/v) bo-vine serum albumin and 0.1% (v/v) TWEEN 20 was used asworking buffer and PBS containing 0.1% TWEEN 20 and0.02% Antifoam™ (Pharmacia, Uppsala, Sweden) as wash-ing buffer.

C3a fragments

Plasma diluted 1:100 was incubated in wells coated withmonoclonal antibody 4SD17.3 that served as capture anti-

Figure 1. Assembly of the heparin layer. A conditioning layer of polymeric amine was first adsorbed to the surface ontowhich a macromolecular conjugate of heparin then was attached. Arrows mark the time of addition. This procedure was thenrepeated two more times. A buffer exchange took place between the additions of the polymer denoted PAV and the heparinconjugate.

TABLE IStandardized platelet loss as a function of both linear

flow velocity and AT binding capacity

Speed(rpm)

Linear Flow(cm/s)

AT(pmol/cm2)

Platelet Loss(%)a

15 12.8 12 4.1 � 1.733 28.1 6 8.2 � 1.3

12 5.1 � 1.519 5.2 � 1.1

50 42.5 12 5.2 � 1.2aMean � SEM.

460 ANDERSSON ET AL.

body. As previously described, C3a was detected using abiotinylated anti-C3a followed by HRP-conjugated strepta-vidin (Amersham Biosciences, Little Chalfort, UK).33 Zymo-san-activated serum calibrated against a solution of purifiedC3a served as standard and the values are given in nano-grams/milliliter.

sC5b-9

The complement activation product sC5b-9 was measuredusing a modification of the previously described method byMollnes et al.4,34 Plasma samples diluted 1:5 were added towells coated with anti-neoC9 monoclonal antibody McaE11which was a kind gift from Prof. Tom-Eirik Mollnes, Uni-versity of Tromsø, Norway. Polyclonal anti-C5 antibodies(Dako, Glostrup, Denmark) diluted 1:500 were used for de-tection. Zymosan-activated serum defined as containing40,000 AU/mL served as standard.

Factor H binding capacity

Factor H was first eluted from the heparin-coated tubingusing PBS containing 1M NaCl. The factor H content of theeluates was measured using a sandwich EIA. The wells werecoated with polyclonal anti-factor H (The Binding Site, Bir-mingham, UK). The samples were diluted 1:32. Factor H wasdetected using a biotinylated anti-human factor H (TheBinding Site) followed by HRP-conjugated streptavidin(Amersham Biosciences). Factor H, purified as described byHammer et al.,35 served as standard, and values are ex-pressed as nanograms/square centimeter.

Assay using chromogenic substrate

AT binding capacity

The binding capacity of AT was assessed as described byKodama et al.36 This method is based on the fact that AT inthe presence of heparin inhibits factor Xa. The heparin-coated tubing was first rotated with normal citrate plasma.After rinsing with Tris-NaCl [50 mmol/L Tris HCl (pH 7.8)and NaCl to a final ionic strength of 0.15], bound AT waseluted by Tris-NaCl containing 150 IU/mL heparin (Bioi-berica, Barcelona, Spain). The AT activity in the eluate wasdetermined in a factor Xa assay in which factor Xa wasadded to the sample. After addition of the chromogenicsubstrate S-2765 (Chromogenics, Molndal, Sweden) and ter-mination by addition of citric acid, remaining factor Xaactivity was measured at 405 nm.

Statistics

To avoid the effect of individual variations, platelet losswas calculated as percentage of the values of the samplestaken before the experiment. All other parameters weregiven as absolute values. Data are presented as mean �

SEM. Statistical significance was calculated with Mann-Whitney using StatView� for Macintosh (Abacus ConceptsInc., Berkeley, CA).

RESULTS

The heparin surface

The assembly of three layers of macromolecularheparin conjugate was monitored using QCM-D tech-nique.26 During preparation of the first layer, therewas a frequency decrease, indicating a mass increase,when the surface was exposed first to the polymericamine and then to the heparin conjugate. In contrast,there was a frequency increase, indicating mass loss,and a major dissipation decrease, suggesting stiffen-ing of the layer, during incubation of the polymericamine after the first and second layer of heparin. In thethree cycles, the binding of heparin conjugate showeda similar shape but the binding kinetics were gradu-ally slower with each cycle (Fig. 1). The AT bindingcapacity of tubing with one, two, and three layers ofheparin (incubated at 28 cm/s) was 6, 12, and 19 pmolAT/cm2, respectively (Table I). Measured as describedpreviously,37,38 no release of heparin into plasmacould be detected at any of the surface concentrationsof heparin. All surfaces bound factor XII to a similardegree during plasma incubation, but no conversionof factor XII into factor XIIa could be detected usingpreviously described methods (not shown).38,39

The relation between the heparin surfaceconcentration and blood compatibility

The three different surface concentrations of hepa-rin displaying different binding capacity of AT weretested at a blood velocity of 28 cm/s for 60 min. Theincrease in AT binding capacity did not significantlydecrease the platelet loss (Table I). Both �-TG releaseand TAT generation decreased with increasingamounts of surface heparin and subsequent AT bind-ing capacity [Fig. 2(A,B)]. �-TG was significantlylower at both 12 (p � 0.0130) and 19 pmol AT/cm2

(p � 0.0027) compared with 6 pmol AT/cm2. This wasalso true for TAT generation which was lower at both12 (p � 0.0234) and 19 pmol AT/cm2 (p � 0.0130)compared with 6 pmol AT/cm2. The generation ofcomplement fragment C3a decreased with increasingamounts of surface heparin. At both 12 (p � 0.0012)and 19 pmol AT/cm2 (p 0.0001), the level wassignificantly lower compared with 6 pmol AT/cm2

(Fig. 2). There was a decrease in generation of sC5b-9at the surfaces with AT binding capacity of 12 (p �


0.0022) and 19 pmol AT/cm2 (p � 0.0018) comparedwith 6 pmol AT/cm2 [Fig. 2(C,D)]. None of the mea-sured parameters were significantly improved whenthe AT binding capacity was increased from 12 to 19pmol AT/cm2.

Linear blood velocity and blood compatibility ofthe heparin surface

The blood compatibility of heparin surfaces with anAT binding capacity of 12 pmol AT/cm2 was com-pared at three different blood velocities for 60 min(12.8, 28.1, and 42.5 cm/s; Table I). No difference inplatelet loss or �-TG release was detected [Table I, Fig.3(A)]. The coagulation marker TAT was significantlylower at 12.8 cm/s than at both 28.1 (p � 0.0002) and42.5 cm/s (p � 0.0006) [Fig. 3(B)]. The complementactivation product C3a was increased, as the velocitywas increased [Fig. 3(C)]. The activation at 28.1 cm/swas significantly higher than at 12.8 cm/s (p � 0.0129).Additionally, the generation of C3a at 42.5 cm/s wassignificantly higher than at both 28.1 (p � 0.0007) and12.8 cm/s (p � 0.0001). No significant difference wasobserved in the generation of sC5b-9 [Fig. 3(D)].

Factor H binding capacity

To elucidate the inhibitory effects of the heparinsurface on complement activation, the factor H bind-ing capacity of the heparin surfaces was investigated.Tubing with different AT binding capacities of hepa-rin coat were incubated with non-anticoagulatedblood in the Chandler loop model at 28 cm/s for 60min. The factor H binding did not correlate with thesurface concentration of heparin (Fig. 4).

DISCUSSION

The heparin surface

Modification of surfaces with immobilized heparinis an established method to improve blood biocom-patibility.40 In this study, the step-by-step process ofpreparing a multilayered CHS surface was monitoredusing QCM-D. During the build-up of the first twolayers, there was a frequency decrease, indicatingmass binding, during incubation of first the polymericamine and then the heparin conjugate, but the kinetics

Figure 2. Release of �-TG (A) and generation of TAT (B), C3a (C), and sC5b-9 (D) after 60 min of incubation of blood at 37°Cin the Chandler loop. The loop was rotated at 28.1 cm/s and the results were compared with the AT binding capacity. Dataare presented as mean � SEM from experiments using blood from four different donors. Experiments were performed intriplicate with the blood from each donor.


of the two binding events were different. When thenext layer of amine was added, there was, however,a frequency increase, but more interestingly, a mas-sive decrease of dissipation occurred. This indicatesthe formation of an interpenetrating network be-tween the polymeric amine and the previouslybound heparin and that water is expelled from thelayer upon binding of amine to heparin, wherebythe surface layer becomes stiffer.29 The binding ofheparin conjugate followed the same pattern at allthree incubations, but the kinetics became slowerfor every cycle. The dissipation after the binding ofthe heparin conjugate increased only slightly aftereach cycle indicating that the underlying layers re-mained stiff and rigid and that the top layer domi-nated the information acquired by QCM-D. This istrue even if the last layer should be expected to be asflexible as the first layer of heparin conjugate. Aspresented in Results, we show that by building upmultiple layers of immobilized heparin on the sur-face, the available amount of immobilized heparinand AT binding capacity of the surface can be mod-ulated to match a specific application. This isachieved relying on identical chemistry in all cases.

Figure 4. The amount of factor H bound to the heparinsurface in tubing incubated with non-anticoagulated blood at37°C for 60 min at 12.8 cm/s. Factor H binding capacity wascompared with the amount of available heparin on the surface.The data are presented as mean � SEM from three experimentsusing blood from different donors. Experiments were per-formed in triplicate with the blood from each donor.

Figure 3. Release of �-TG (A) and generation of TAT (B), C3a (C), and sC5b-9 (D) after 60 min of incubation of blood at 37°Cin the Chandler loop. Tubing with an AT binding capacity of 12 pmol AT/cm2 was used and the results were compared withthe linear flow rate. Data are presented as mean � SEM from experiments using blood from four different donors, except at12.8 cm/s in which one donor was excluded. Experiments were performed in triplicate with the blood from each donor.


The chandler loop model

Testing biomaterials in circulating blood using theChandler loop model is a simple and straightforwardmethod,25,41 whereby whole human blood can beused. When testing ordinary polymeric materials, it isusually necessary to add heparin to the blood to avoidclotting.4 In the current study, the surfaces were allexpected to be low-activating, and therefore, bloodsampled without any addition of anticoagulant wasused to achieve maximal sensitivity. Recirculation ofblood for 1 h was deemed clinically relevant becausethe duration of open-heart surgery is 1–2 h.

Biological performance and heparin surfaceconcentration

Different surface concentrations of heparin resultingin different capacity to bind AT were compared in awhole blood loop model using a linear blood velocityof 28 cm/s. Coagulation and complement parameterswere improved when the binding capacity was in-creased from 6 to 12 pmol AT/cm2. Further increasedid not add any significant improvement in this testmodel. This has previously only been shown inplasma.20 No significant difference in platelet loss wasdetected, but still platelet activation, as measured byrelease of �-TG, was lower when higher heparin sur-face concentrations were used. The generation of TATwas also less pronounced when the AT binding capac-ity was increased from 6 to 12 pmol AT/cm2 with nofurther improvement beyond that level. Complementcompatibility mediated by the heparin surface is sup-posed to depend on binding of complement regulatorsrather than AT.42 Generation of C3a decreased whenthe surface concentration of heparin and subsequentlythe AT binding capacity was increased from 6 to 12pmol AT/cm2. This showed that the platelet, coagu-lation, and platelet activation were inhibited in thesame surface concentration interval. Attempts to cor-relate the inhibition of complement activation with thebinding of factor H did not yield conclusive results,which points to other explanations for the inhibition.Complement activation requires a surface that allowscovalent binding of C3b to the surface. On a materialsurface, adsorbed proteins such as albumin and im-munoglobulin G provide these acceptor sites. We hy-pothesize that the previously reported altered proteinbinding pattern to the heparin surface43 explains thelower complement activating properties of the coatingrather than factor H alone.

Heparin surfaces and the effect of velocity

The activation products C3a and sC5b-9 are gener-ated on the biomaterial surface in extracorporeal cir-

cuits during cardiopulmonary bypass and hemodial-ysis. In addition, C3a is generated on gas surfaces thatare huge in oxygenators. A similar phenomenon hasbeen reported in vivo during hemodialysis44 becauseof increased blood flow. To test the effect of surface-bound heparin on this type of complement activation,different blood flow rates were investigated in theChandler loop model using tubing with an AT bind-ing capacity of 12 pmol AT/cm2. At this surface con-centration, platelet loss and release of �-TG were notinfluenced by flow rate. Significantly higher TAT val-ues were recorded at the two highest flow rates com-pared with the lowest, but there was no differencebetween the highest and the intermediate flow rate.This is in accordance with the finding by Pasche etal.45 that the amount of surface-bound AT decreaseswith increased velocity in plasma. The complementactivation product C3a, but not sC5b-9, increased asthe linear flow rate was increased, showing significantdifferences between all velocities. This showed thatcomplement activation due to a more vigorous mixingof blood and air was not affected by the heparincoating.

CONCLUSIONS

Immobilization of heparin is an excellent way torender biomaterial surfaces blood compatible. Here,we demonstrate that the biocompatibility of a surfacewith regard to coagulation, complement, and plateletactivation can be further improved by increasing theheparin surface concentration to within the 6–12 pmolAT/cm2 concentration interval. The binding of factorH is not influenced by the increased binding of hepa-rin suggesting that this factor is not the primary reg-ulator of complement on heparin surfaces. In addition,the heparin coating has no effect on the complementactivation that occurs on gas surfaces in extracorporealcircuits. Further studies to elucidate the mechanismbehind the control on complement activation medi-ated by immobilized heparin are required.

The authors thank Ms. Lillemor Funke for excellent tech-nical assistance including development of the factor H bind-ing capacity assay.

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optimal heparin surface concentration and antithrombin binding capacity as evaluated with human...

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