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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Influence of a preadsorbed terpolymer on human platelet accumulation, fibrinogen adsorption, and ex vivo blood activation in hemodialysis hollow fibers a
b
c
Feng Yan , Philippe Déjardin , Juliette N. Mulvihill , d
e
Jean-Pierre Cazenave , Thierry Crost , Michel Thomas & Christian Pusineri
f
g
a
Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083 Strasbourg, France b
Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083 Strasbourg, France c
Centre Régional de Transfusion Sanguine, INSERM U.311, 10 rue Spielmann, 67085 Strasbourg, France d
Centre Régional de Transfusion Sanguine, INSERM U.311, 10 rue Spielmann, 67085 Strasbourg, France e
Hospal-COT, 13 avenue de Lattre de Tassigny, 69881 Meyzieu, France f
Hospal-COT, 13 avenue de Lattre de Tassigny, 69881 Meyzieu, France g
Hospal-COT, 13 avenue de Lattre de Tassigny, 69881 Meyzieu, France Published online: 02 Apr 2012.
To cite this article: Feng Yan , Philippe Déjardin , Juliette N. Mulvihill , Jean-Pierre Cazenave , Thierry Crost , Michel Thomas & Christian Pusineri (1992) Influence of a preadsorbed terpolymer on human platelet accumulation, fibrinogen adsorption, and ex vivo blood activation in hemodialysis hollow fibers, Journal of Biomaterials Science, Polymer Edition, 3:5, 389-402, DOI: 10.1163/156856292X00204 To link to this article: http://dx.doi.org/10.1163/156856292X00204
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Influence
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accumulation, activation
of
a preadsorbed fibrinogen
in
hemodialysis
terpolymer adsorption, hollow
on and ex
human vivo
platelet blood
fibers
FENG YAN1, PHILIPPE DÉJARDIN1*, JULIETTE N. MULVIHILL2, JEAN-PIERRE CAZENAVE2, THIERRY CROST3, MICHEL THOMAS3 and CHRISTIAN PUSINERI3 'Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083Strasbourg, France 2CentreRégional de TransfusionSanguine, INSERM U.311, 10 rue Spielmann, 67085Strasbourg, France 3Hospal-COT,13 avenue de Lattre de Tassigny,69881Meyzieu,France Received2 October 1991;accepted 22 December 1991 Abstract-Results are presentedon kineticsof platelet accumulationin charged polyacrylonitrile(AN69) hollow fibers by continuous data recording under flow conditions (wall shear rate 108-1050s-1), using human plateletsin Tyrode's-albuminbuffer, containingwashed red suspensionsof washed 111In-labeled blood cells (0-40%). Preadsorption of a terpolymerof acrylonitrile,poly(ethyleneoxide)methacrylateand trimethylaminoethyl chloride methacrylateleads to very efficient passivation with respect to platelet accumulation and fibrinogen adsorption. In human ex vivo tests, evaluation of complement peptide C3a, platelet βthromboglobulin, leucocyte-polymorphonuclearneutrophile elastase and fibrinopeptide A shows no detectable activation. Furthermore, preadsorption appears to result in simultaneous improvement in hemocompatibilityof the blood lines leading to and from the dialysis module. This singlepretreatment of dialysismembranesshould allow injection of lower doses of anticoagulant to patients submitted to hemodialysis. Key words: Polymer adsorption; membrane; hemodialysis; hemocompatibility; poly(ethyleneoxide); polyacrylonitrile;hollow fiber. INTRODUCTION Blood contact with a foreign material is known to trigger a succession of interfacial events: adsorption of proteins, adhesion and aggregation of platelets and leukocytes and biochemical reactions of coagulation and complement activation induced by the high interfacial concentrations and conformational changes of the adsorbed species an undesired thrombus To [1]. Finally, may appear. prevent such phenomena, two lines of research are to complementary possible: (i) investigate what really occurs before thrombus formation; and (ii) to find an ad hoc treatment of the surface which minimizes such undesirable events. Due to their relatively small size, proteins are expected to contact the surface first, followed by cells like platelets, red cells and leukocytes. For many years, work has been performed on protein adsorption on synthetic foreign materials in order to understand the first steps occurring on blood arrival. For the purpose of fundamental analysis in simplified models, mono-protein solutions were used [2-8],
389
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390 although the authors were aware that blood is a very complex fluid which cannot be modeled by a simple buffer. Comparisons between experiments using a single protein and those using protein mixtures [9-10] or plasma [11-16] are often very fruitful. For example, fibrinogen adsorption from simple solutions and from plasma variations of interfacial concentration versus time differs sharply: non-monotonous or solution concentration were found in the presence of plasma and this was designated the Vroman effect [17]. Whatever the type of experiment performed, irreversible processes [18] enhance the difficulties of modeling and emphasize the importance of a dynamic analysis, especially for complex fluids. Dynamic studies were recently carried out of the adsorption of fibrinogen from radiolabeled solutions on polymer hollow fibers or on a glass tube from radiolabeled how a continuous recording of the interfacial consolutions which demonstrated centration versus time could reveal different successive regimes [19]. The same technique is applied here to platelet deposition. It is hoped that, on a wide range of supports, this method will give information about kinetic parameters analogous to that obtained from optical techniques like ellipsometry [20-23] or TIRF (Total Internal Reflection Fluorescence) [2, 7, 13] which requires planar transparent surfaces. and platelet accumulation Although limited, tests of fibrinogen adsorption a estimate of the of a surface. The term good hemocompatibility already represent in the sense that thrombus formation is avoided, could suggest 'hemocompatible', extreme strategies to transform raw synthetic materials into hemocompatible ones, from (i) initial complete coverage with biological species originating from the contacting blood, to (ii) a surface completely prepassivated with synthetic or biological materials, leading to an important decrease in the interfacial concentration of blood components. Following the second approach, an example illustrating that surface treatment with a synthetic polymer can lead to good passivation with respect to human platelet accumulation, and biochemical blood fibrinogen adsorption activation is presented. MATERIALSAND METHODS The values of wall shear rate in the following text were calculated from the measured flow rate, assuming a Poiseuille velocity profile in an impermeable tube. These figures are of course only indicative in the presence of red cells [24] or for porous fibers. Hollow fibers Modules of hemodialysis hollow fibers (48 fibers per module for in vitro and 340 fibers per module for ex vivo tests) were kindly provided by Hospal Industries (Meyzieu, France). The fibers have an internal diameter of 329 or 240,um and a wall thickness of 50 #m, the main constituent being a random copolymer of acrylonitrile and sodium methallyl sulfonate, known as AN69. To interpret the kinetics of platelet accumulation, the model of an impermeable tube may be considered as a good approximation of the system as we used fibers without pores. However, for the study of the influence of red cells on platelet accumulation porous hollow fibers were employed, as for fibrinogen adsorption and the ex vivo tests.
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391 Experimental apparatus The in vitro experimental apparatus consists of a microcomputer coupled to two syringe pumps, one containing buffer, the other a platelet suspension at concentration Cb. Buffer and suspension pass to the module of hollow fibers via teflon tubing and a three-way connector, which enables deviation to a waste vessel to eliminate air bubbles. The module is placed horizontally in front of a detector (Quartz et Silice, each France). Data are visualized on a screen and stored in the microcomputer, experimental point corresponding to the integration of radioactivity over 10 s. At the module exit, the liquid is collected on a balance and mass data as a function of time appear graphically on the screen, allowing instantaneous visualization of flow rates. Thermostation is ensured by circulation of buffer at 37°C in the compartment outside the fibers. 111 In-labeled washed human platelets Washed human platelets were prepared as described by Cazenave et al. [25] and resuspended in Tyrode's buffer containing 2 mM Ca2+, 1 mM Mg2+, 0.35% human albumin (Centre Regional de Transfusion Sanguine de Strasbourg), 0. l vo glucose and 20,uml/ml apyrase (Tyrode's albumin buffer), pH 7.30. Apyrase was included in the perfusion medium to prevent platelets becoming refractory and to inhibit major aggregate formation on the surface or in the fluid phase [26]. Platelet count was adjusted to 300000/,uml (Platelet Analyser 810, Baker Instruments, Allentown, USA). Radiolabeling of platelets was performed by incubation with 1 "In-oxine a 1'Energie Atomique, Saclay, France) for 15 min at (0.25,u1/ml, Commissariat 37°C, according to Eber et al. [27], labeling yield being of the order of 90% without modification of platelet aggregation to ADP, thrombin or collagen. Washed red blood cells from the same donor were prepared by the method of Cazenave et al. [26] and resuspended in Tyrode's albumin buffer, pH 9.0. Immediately before perfusion experiments, the red blood cell suspension was centrifuged for 4 min at 1600 g and packed erthrocytes were added to the suspension of " 1 In-labeled washed human platelets at the chosen volume ratio, corresponding in most experiments to a 40% hematocrit. Polymers Two types of polymers were synthesized. The first was obtained monomers and is denoted 'copolymer', while the second was derived monomers and is called 'terpolymer'.
from two from three
of acrylonitrile (AN) and monomethoxyCopolymer. Radical copolymerization nona(ethyleneglycol) methacrylate (MG,) was carried out at 60°C in dimethylas initiator. Extensive discussion of this sulfoxide with azobisisobutyronitrile can be found elsewhere The synthesis [28]. copolymers with a molar fraction of MG9 18% than are water soluble and of pH is performed with greater adjustment concentrated HCI.
392 on discs of flat hemodialysis membrane AN69 were performed Preadsorption from aqueous solutions (C = 0.7-0.8 mg/ml) at varying pH.
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of acrylonitile (AN), methoxypoly(ethyleneTerpolymer. Radical polymerization chloride methacrylate (MQ) and trimethylaminoethyl glycol) methacrylate (MG23) as was performed in water at 30°C over 3.25 h with the couple KC'03/NaHS03 initiator. The initial molar fractions of monomers were fAN = 0.92, fMG = 0.050 = 0.033. Elementary analysis gave through chloride and nitrogen contents and fMQ the following molar composition for the terpolymer: FAN = 0.746, FMG = 0.228 and = = = = FMQ 0.026, or in mass fractions WAN 0.125, WMG 0.819 and WMQ 0.018.
Viscometry yielded intrinsic viscosities varying between 44 ml/g in 10-2 M NaCI and 40 ml/g in 3 10-2 M and 1 M NaCI aqueous solutions. Refractive index increment (dn/dc) in 10-' M NaCI at 632 nm was 0.140 ml/g, while the molecular weight by light scattering was 3.85 106. In the present experiments, minimodules were pretreated prior to a test run by passage of a terpolymer solution (5 mg/ml in Tyrode's buffer), 5 ml at 6 ml/min and after which modules were rinsed with physiological saline then 17 ml at 0.1 ml/min, containing heparin (2.5 IU/ml). Pretreated hollow fibers minimodules were compared to line controls, untreated AN69 minidialysers and equivalent minimodules containing other commercial hollow fibers: cuprophan (CUP, ENKA, Germany) and Polysulfone (PS, Fresenius, Germany). Determination
of platelet
accumulation
and apparent
kinetic constants
Calibration of the in vitro system was carried out in situ by estimating the radioactivity increase due to filling of the fibers (radius R, length L) with a platelet suspension of known concentration Cb. Three-five minute alternate flows of delay which appeared to be too short to suspension and buffer were performed-a reach a stable state in the accumulation process. Given Ah the radioactivity increase while filling and AH the residual radioactivity after rinsing (Fig. 1) and assuming
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393
Figure 1. Example of a radioactivity recording in a three steps experiment. The rise Ah is directly proportional to the known solution (suspension)concentration, thus allowing the determination of the adsorbed (deposited)amount through AH. The slopedr/dt is measuredin the sameway through dHa/dt. the geometry of a perfectly
The apparent
smooth tube, the interfacial
platelet deposition
constant
concentration
is:
keXpis given by:
This procedure does not require an independent measurement of specific activity, since ratios of lengths on the graph lead directly to r and dr/dt when R is known. At the end of an experiment, as an additional control, the modules are cut into 2 cm long segments of which the radioactivity is measured in a well type gamma counter 1282, LKB, Turku, Finland) [29]. (Compugamma Purified
human albumin
and fibrinogen
Albumin and fibrinogen purified from human plasma were supplied by the Centre Regional de Transfusion Sanguine, Strasbourg, France. Both proteins were 99% while the pure by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, of to the method of 98 was and 95 0l0 clottability fibrinogen according Regoeczi [30] before and after radioactive labeling using the iodogen technique, respectively [31 ] . Ex vivo tests The ex vivo test to evaluate hemocompatibility of minimodule has been described in a previous publication [32]. Briefly, antecubital vein of a healthy volunteer is pumped through containing 340 fibers (internal diameter 240,um, external flow rate of 10 ml/min (wall shear rate 360 over a
hollow fiber dialysers blood drawn from the a minimodule dialyser diameter 340,um) at a test period of 30 min.
394 is assured by continuous on-line heparinization (= 0.14 IU/ml, Anticoagulation Roche, France) directly after the point of venous access. At the module exit, blood samples collected at 5 min intervals are analysed for markers of activation of coagulation (fibrinopeptide A, FPA), complement (anaphylatoxin C3a), platelets (flneutrophil (PMN) thromboglobulin, 6TG) and leukocytes (polymorphonuclear elastase). Analyses are performed using commercial kits: FPA and 6TG (ELISA, France) and PMN elastase Stago, France), complement C3a (RIA, Amersham, (ELISA, Merck, Germany). RESULTS AND DISCUSSION
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l. Platelet
accumulation
This section is divided into three subsections: firstly, the influence of red cells on platelet accumulation is examined, secondly, dependence on shear rate is analyzed, and finally ex vivo results obtained with polymer pretreated modules are discussed. Influence of red cells. Figure 2 shows curves of recorded radioactivity versus time during perfusion of hemodialysis modules at a shear rate of 510 s-1, with platelet (180000 pl/,ul) containing different concensuspensions of constant concentration trations of red cells. While the porous hemodialysis membrane shows almost no platelet accumulation without red cells, we observe a strong increase in accumulation between 20 and 30% red cells. This result is in accordance with observations described in the literature [24] and results from enhancement of the transport of platelets towards the surface in the presence of red cells. Another point is that the accumulation kinetics is linear with time over the first 5 min. Apparent kinetic constants were estimated to be 0.106, 0.77, 1.38, 5.7, and 7.6 10-5 cm S-1 for red
Figure 2. Kineticsof platelet accumulationon porous hollowfibers from platelet suspensionsof constant concentration (180000 pl/pl)containing different concentrations of red cells (0, 20, 30 and 40%).
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395 cells concentrations of 0, 10, 20, 30, and 40% respectively. Concerning the rinsing steps, we observed very good stability of the signal at long times, showing no desorption in presence of buffer. However, the drop of the signal when flushing with buffer was much less, especially when accumulation was high, than the initial rise when introducing a platelet suspension after buffer. A clear explanation for this phenomenon is not yet available, a possibility being that the absence of albumin in the buffer could activate some platelets leading to a short but non negligible enhancement of the deposition. For the two highest red cell concentrations of 30 and 40%, the platelet deposition at the end of the perfusion step was 70 and 80% respectively of the total accumulation at the end of the rinsing step. Maximal coverage obtained after rinsing was 41000 pl/mm2. Thus a linear increase of radioactivity versus time was observed to a coverage of 33 000 PI/MM2, which represents about half the maximal coverage (60 000 pl/mm2, see Fig. 5). As it was clear that platelet accumulation was significant in the presence of red cells and in order to remain as close as possible to physiological conditions, subsequent experiments were carried out with suspensions containing 40% red cells and a platelet concentration of 180 000 pl/#I. Contrary to the above studies concerning the effect of red cells, non porous hollow fibers were used. Influence of shear rate. Figure 3 shows the influence of wall shear rate y on the kinetics of platelet accumulation recorded at 10 cm from the module entrance, using 1 suspensions prepared from blood provided by a single human donor. At 1050 s-1 the activity rise occurs more rapidly than at lower shear rates. The initial slopes do not appear to be the highest ones: this is particularly evident at high shear
Figure 3. Kinetics of platelet accumulation at various Poiseuille's wall shear rates from platelet suspensionsof constant concentration (180000pl/?1) containing 40Vored blood cells.
396
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rates. Slow lateral transport of platelets and/or red cells could be responsible for a relatively long time lag before obtaining a steady state profile and linear variation of Nor can we exclude a possible slight initial overthe interfacial concentration. adhesion during the change from buffer to platelet suspension. We again observed that the drop Ah' was smaller than the rise Ah (see Fig. 1), the difference being larger for higher shear rates. The L6v?que constant may be estimated from:
With an assumed platelet diffusion coefficient in plasma D = 1.58 10-9 CM2 s-1 at 37°C [28], bulk concentration Cb = 180000 pl/,ul and distance to the fiber entrance z = 10 cm, kLe? is calculated as 1.61, 2.24 and 3.46 10-6 cm s-1 for the shear rates of 108, 285 and 1050 s-1, respectively. keXpis larger than the maximum mass transfer to corresponding by a factor which increases with Poiseuille's shear rate: kexpIkL,, = 22, 32 and 52 for 108, 285 and 1050 s-1, respectively. One can argue that: (i) red cells significantly enhance lateral platelet transport; and (ii) this enhancement, with respect to the y113 law, increases with flow rate. Such behavior has been previously described for rabbit blood [33]. Without red cells (bottom curve in Fig. 2) and at a shear rate of 510 keXp is 1.06 10-6 cm s-1 and kLev is 2.71 10-6 cm s-' . Through the relations [34] :
it is estimated that an intrinsic accumulation constant ka = 1.74 10-6 cm sec? ?and a ratio of the steady state concentration near the wall Cst(0) to the bulk concentration Cb close to 0.6. However, this estimation does not include a possible effect of the transmembrane pressure through the porous membrane. The variation of platelet deposition versus distance from the fiber entrance is presented in Fig. 4. Let us recall that the foregoing kinetic analysis was performed at 10 cm from the entrance. We can see that at 1050 çl there is almost no variation along the fiber: after 5 min the surface should be almost entirely covered with platelets. This is in accordance with the kinetic curve, where we observe a decreasing rate of accumulation towards 4 to 5 min. Rapid completion of the surface occupation probably occurs during the change from suspension to albumin free Tyrode's buffer. At lower shear rates, a gradient of interfacial platelet concentration along the fiber is clearly observed, measurements at 280 and 285 s-1 showing remarkable reproducibility given the complexity of the fluid involved. As linear variation of accumulation versus time was observed until the end of the perfusion step, platelet of these curves should be approximated by a transport quantitative interpretation limited model. If we assume that the volume concentration near the wall has a steady state value, a modified L6v?que model should apply [30]:
Since r varies linearly with time during the perfusion step, Ar/At, where Or is the platelet deposition over a time period such a representation of the data. As expected, we do not find the two highest shear rates, as the saturation value of 60000
we have (dr /dt) = At. Figure 4 shows a linear relation for pl/mm2 (Fig. 5) is
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397
Figure 4. Inverseof the apparent kinetic accumulationconstant versus (Z/Y)1/3from data obtained after the rinsing step. z (cm) is the distance from the fiber entrance and y (s-') the Poiseuillewall shear rate. Insert: Platelet accumulationversus distance from the fiber entrance at various shear rates: 110 ç (+), 280 s-1(circle), 1050s-' (square).
Figure 5. Influenceof terpolymerpretreatment of fibers on the kineticsof platelet accumulationat 10cm from the fiber entrance. Lower curve: pretreated module; upper curve: untreated module. Insert: Platelet accumulationalong the fibers for pretreated module (bottom curve) and untreated module (top curve). z = distance from the fiber entrance.
398
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almost reached within 5 min at 4 cm from the module entrance and therefore a steady state assumption is not valid. From the slope at 110 s-1 we estimate an apparent platelet diffusion coefficient of 57 10-9 CM2s-', which would demonstrate again the importance of red cells. However, the ordinate at the origin is negative and does not allow a coherent interpretation from Eq. (5). Two phenomena may play a role in this behavior: firstly the entrance length to establish a steady velocity profile was neglected and secondly the increase in deposition when changing from suspension to buffer (leading to Ah' smaller than Ah) was observed to be an increasing function of the flow rate or, in other words, of the inverse of the Nernst diffusion of Cb (dr/dt)-' in the perfusion step, layer. This should lead to an underestimation an underestimation more important at smaller distance from the fiber entrance. Reduction of platelet accumulation. We now briefly present some results obtained with porous hollow fibers to illustrate than an appropriate surface treatment, in this case preadsorption with the terpolymer AN - MG23 - MQ, can considerably reduce 5 accumulation. shows the kinetic curves recorded during 10 min platelet Figure of a treated module and an one (upper curve), untreated perfusion (bottom curve) while the insert on the same figure gives the variation of platelet deposition with distance from the module entrance determined by measurement of the radioactivity of 2 cm long segments at the end of the experiment. The efficiency of the treatment is clear: average platelet accumulation drops from 54000 to 1500 pl/mm2. Fundamental analysis of this complex process would however be somewhat hazardous given that, although we know the flow rate in the dialysate compartment (15 ml/min), we do not know the transmembrane pressure. 2. Albumin The effect
and fibrinogen
adsorption
on albumin and fibrinogen adsorption of preadsorption with the of and copolymer acrylonitrile monomethoxy-nona(ethyleneglycol) methacrylate was also investigated. Adsorbance of radiolabeled albumin and fibrinogen on membrane discs was measured by counting directly the radioactivity on the discs. Figure 6 shows the results, compared to untreated membranes, for various compositions of the copolymer and preadsorption conditions. Adsorption of this copolymer has been the subject of previous studies [35]. It can be seen that whatever the pH of passivation efficiency is zero for copolymers with a high content of preadsorption poly(ethyleneoxide) (PEO) side chains, while a maximum reduction in adsorbance of 60?Io is attained with a copolymer containing a molar fraction of 79?/o acrylonitrile, near the limit of solubility of these copolymers in water. This 6007oreduction is however less than the passivation efficiencies obtained on silica or glass with PEO based polymers [36, 37]. We conclude that a higher content of acrylonitrile in the copolymer may improve anchoring of the polymer on the membrane surface and therefore prevent protein adsorption more effectively than in the case of copolymers with a lower content of acrylonitrile, more readily displaced from the surface by proteins. a small amount of a third monomer positively Following this interpretation, was added into the reaction medium. The terwith a ammonium quaternary charged polymer obtained (Materials and Methods) which bears positive charges, should increase the stability of the adsorbed polymer layer on the membrane by electrostatic
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399
Figure 6. Relativeadsorbance of albumin (open symbols)and fibrinogen (closed symbols)on flat discs of AN69 membrane, with respect to untreated membrane, after preadsorption with copolymer solution (C = 0.7-0.8 mg/ml) at pH 3 (squares, pH 4.5 (circles)and pH 6 (triangles). FMG= molar fraction of methacrylate). MG9(monomethoxy-nonaethyleneglycol interactions with sulfonate groups. Moreover, a macromonomer with a longer side chain of methoxypoly(ethyleneoxide) was chosen, 23 units instead of 9, as in the work of Miyama [38]. Terpolymer preadsorption was performed from solutions at 5 mg/ml in Tyrode's buffer, the same buffer as used to prepare concentration fibrinogen solutions. A reduction of at least 87?7o in fibrinogen adsorbance was obtained. Although this water-soluble terpolymer might be leached out during these experiments, it seems that, if any, such a leaching should have been low enough to keep prevention of protein adsorption. In view of the high efficiency of this terpolymer in preventing fibrinogen adsorption and platelet accumulation in vitro, the effect on blood activation of this new composite material was further evaluated by means of ex vivo tests in humans using dialysis modules of porous hollow fibers. 3. Ex vivo tests A deeper insight into the hemocompatibility of dialysis membranes preadsorbed with the terpolymer should include studies of the interactions of this new material with whole blood. Therefore ex vivo tests with dialysis modules were performed to evaluate: (i) activation of coagulation according to the generation of FPA; (ii) complement activation as indicated by the formation of C3a; (iii) platelet activation as determined by measurement of 8TG release; and (iv) neutrophil activation as estimated from release of PMN elastase. Figure 7 shows the results, as compared to untreated AN69 and other hemodialysis membranes. Although further tests will be required to draw definitive conclusions,
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400
Figure 7. Plasma concentrations of fibrinopeptide A (FPA), p-thromboglobulin complement fragment C3a and PMN elastase measured at the exit of dialysis modules in ex vivo tests. Polysulfone (dotted line, symbol S, n = 5); cuprophane (dotted line, symbolC, n = 5); untreated AN69 (A, n = 5); terpolymer preadsorbed AN69 (1, test 1; 2, test 2); line control (dotted line, symbol L, n = 5).
401 it is remarkable that systematically the levels of activation of blood components by the pretreated AN69 membranes are lower than those of the other supports. Moreover, especially for elastase release, they are similar or even less than control values. It is probable that polymer adsorption on the blood lines during module pretreatment led to additional surface passivation, in particular towards neutrophils. CONCLUSIONS in vitro of kinetic new experimental system allows the determination parameters of molecular adsorption and cellular deposition. Use of the technique with platelet suspensions shows the importance of red cells in platelet accumulation, in accordance with previous results [24]. In the absence of red cells, the experimental accumulation constant is of the same order of magnitude as the value deduced from a Leveque model: the deposition process is partially transport controlled. As red cell content increases enhancement of the accumulation rate occurs, since hydrodynamic conditions are drastically modified [24]. When varying the flow, we find that the platelet deposition rate varies strongly with wall shear rate over the range studied and at 1050 s-1 complete surface coverage is attained within 5 min. In all these experiments, an unexplained phenomenon occurring when replacing platelet suspension may be tentatively ascribed to the absence of albumin in the buffer, which could However this point remains to be checked. lead to some platelet activation. could occur when replacing Although less obvious a similar type of perturbation buffer by platelet suspension. More trivial, an abrupt short overpressure when switching the electric valves could produce such a phenomenon. Further studies of platelet accumulation and fibrinogen adsorption, demonstrate of a passivation of the polyacrylonitrile dialysis membrane AN69 by preadsorption PEO rich terpolymer bearing a small number of quaternary ammonium charges. The in vitro passivation tests seem to be a good guide to the hemocompatibility check of new materials, since subsequent ex vivo tests in humans using heparinized blood show minimal levels of cellular and biochemical blood activation. These promising results could open the way to the development of composite dialysis obtained by simple surface treatment membranes of improved hemocompatibility in patient therapy. and thus enable the use of lower levels of anticoagulant level by adsorption from aqueous Furthermore, passivation at a (macro)molecular solutions, contrary to thicker coatings, should not greatly modify the permeability of the original membranes.
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This
Acknowledgements We are indebted to Hospal Industries for providing hollow fiber modules. Efficient technical assistance of J. Iss was greatly appreciated and we acknowledge the technical contribution of G. Maennel and F. Woehl for two syringe pumps. REFERENCES 1. E. W. Salzman and E. W. Merrill, in: Haemostasis and Thrombosis.Basic Principles and Clinical Practice, pp. 1335-1347,2nd ed. R. W. Colman, J. Hirsch, V. J. Marder and E. W. Salzman(Eds). J. B. Lippincott Company, Philadelphia (1987). 2. B. K. Lok, Y.-L. Cheng and C. R. Robertson, J. Colloid Interface Sci. 91, 87 (1983). 3. J. C. Voegel, N. de Baillou, J. Sturm and A. Schmitt, Colloids Surfaces 10, 9 (1984).
.
402 J. L. Brash, S. Uniyal, C. Pusineri and A. Schmitt, J. Colloid Interface Sci. 95, 28 (1983). N. de Baillou, J. C. Voegeland A. Schmitt, Colloids Surfaces 16, 271 (1985). J. C. Voegel, N. de Baillou and A. Schmitt, Colloids Surfaces 16, 289 (1985). B. K. Lok, Y.-L. Cheng and C. R. Robertson, J. Colloid Interface Sci. 91, 104 (1983). A. Schmitt, R. Varoqui, S. Uniyal, J. L. Brash and C. Pusineri, J. Colloid Interface Sci. 92, 25 (1983). 9. B. R. Young, W. G. Pitt and S. L. Cooper, J. Colloid Interface Sci. 124, 28(1988);ibid. 125, 246. 10. J. L. Brash and S. Uniyal, J. Polym. Sci., Polym. Symp. 66, 377 (1979). 11. T. A. Horbett, Thromb. Haemostas. 66, 377 (1984). 12. S. M. Slack and T. A. Horbett, J. Colloid Interface Sci. 124, 535 (1988). 13. V. Hlady, D. R. Reineckeand J. D. Andrade, J. Colloid Interface Sci. 111, 555(1986). 14. J. L. Brash and P. ten Hove, Thromb. Haemostas. 51, 326 (1984). 15. P. Wojciewchowski,P. ten Hove and J. L. Brash, J. Colloid Interface Sci. 111, 455(1986). 16. S. Uniyal and J. L. Brash, Thromb. Haemostas. 47, 285 (1982). 17. J. D. Andrade and V. Hlady, J. BiomaterialsSci. 2, 161-172(1991). 18. J. D. Andrade and V. Hlady, Adv. Polym. Sci. 79, 1 (1986). 19. F. Yan and Ph. Déjardin, Langmuir 7, 2230-2235(1991). F. Boumaza, Ph. Déjardin, F. Yan, F. Bauduin and Y. Holl, Biophys. Chem. 42, 87-92 (1992). 20. U. Jönsson, B. Ivarsson, 1. Lundström and L. Berghem, J. Colloid Interface Sci. 90, 148 (1982). 21. I. Arnebrant, B. Ivarsson, K. Larsson, I. Lundström and T. Nylander, Prog. Colloid Polymer Sci. 70, 62 (1985). 22. U. Jöhnsson, M. Malmqvist,I. Rönnbergand L. Berghem,Prog. ColloidPolymer Sci. 70, 96(1985). 23. J. W. Corsel, G. M. Willems, J. M. M. Kop, P. A. Cuypers and W. Th. Hermens, J. Colloid Interface Sci. 111, 544 (1986). 24. H. L. Goldsmith and V. T. Turitto, Thromb. Haemost. 55, 415-435. 25. J.-P. Cazenave,S. Hemmendiger,A. Beretz,A. Sutter-Bay, J. Launay, Ann. Biol. Clin. 41, 167-79 (1983). 26. J. P. Cazenave, D. Blondowska, M. Richardson, R. L. Kinlough-Rathbone,M. A. Packam and J. F. Mustard, J. Lab. Clin. Med. 93, 60-70 (1979). 27. M. Eber, J.-P. Cazenave,J.-C. Grob, J. Abecassis,G. Methlin, in: Blood Cellsin Nuclear Medicine, Part I. Cell Kineticsand Bio-distribution,pp. 29-43, M. R. Hardemen and Y. Najean (Eds). Martinus Nijhoff Publishers, Boston (1984). 28. F. Yan, Ph. Déjardin and J.-C. Galin, Polymer 31, 736-742 (1990). 29. J. N. Mulvihill,H. G. Huisman, J.-P. Cazenave, J. A. van Mourik and W. G. van Aken, Thromb. Haemostas. 58, 724-731 (1987). 30. E. Regoeczi,J. Nucl. Biol. Med. 15, 37-41 (1971). 31. E. Regoeczi,Iodine-labeledPlasma Proteins, pp. 49-56. CRC Press, Florida (1984). 32. J. N. Mulvihill, J.-P. Cazenave, J.-P. Mazzucotelli, T. Crost, C. Collier, J.-L. Renaux and C. Pusineri, Biomaterials, in press. 33. V. T. Turitto and H. R. Baumgartner, MicrovascularRes. 17, 38-54 (1979). 34. Ph. Déjardin, J. Colloid Interface Sci. 133, 418 (1989). 35. F. Yan, Ph. Déjardin, J.-C. Galin and A. Schmitt, Colloid Polymer Sci. 269, 1021-1025(1991). 36. C. Orgeret-Ravanat,Ph. Gramain, Ph. Déjardin and A. Schmitt, Colloids Surfaces33, 109(1988). 37. C. Maechling-Strasser,Ph. Déjardin, J.-C. Galin, A. Schmitt; V. Housse-Ferrari, B. Sébille; J. N. Mulvihilland J.-P. Cazenave, J. Biomed. Mater. Res. 23, 1395(1989). 38. H. Miyama, H. Shimada, N. Fujii, Y. Nasaka and Y. Noishiki, J. Appl. Polym. Sci. 35, 115-125 (1988).
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4. 5. 6. 7. 8.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Influence of a preadsorbed terpolymer on human platelet accumulation, fibrinogen adsorption, and ex vivo blood activation in hemodialysis hollow fibers a
b
c
Feng Yan , Philippe Déjardin , Juliette N. Mulvihill , d
e
Jean-Pierre Cazenave , Thierry Crost , Michel Thomas & Christian Pusineri
f
g
a
Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083 Strasbourg, France b
Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083 Strasbourg, France c
Centre Régional de Transfusion Sanguine, INSERM U.311, 10 rue Spielmann, 67085 Strasbourg, France d
Centre Régional de Transfusion Sanguine, INSERM U.311, 10 rue Spielmann, 67085 Strasbourg, France e
Hospal-COT, 13 avenue de Lattre de Tassigny, 69881 Meyzieu, France f
Hospal-COT, 13 avenue de Lattre de Tassigny, 69881 Meyzieu, France g
Hospal-COT, 13 avenue de Lattre de Tassigny, 69881 Meyzieu, France Published online: 02 Apr 2012.
To cite this article: Feng Yan , Philippe Déjardin , Juliette N. Mulvihill , Jean-Pierre Cazenave , Thierry Crost , Michel Thomas & Christian Pusineri (1992) Influence of a preadsorbed terpolymer on human platelet accumulation, fibrinogen adsorption, and ex vivo blood activation in hemodialysis hollow fibers, Journal of Biomaterials Science, Polymer Edition, 3:5, 389-402, DOI: 10.1163/156856292X00204 To link to this article: http://dx.doi.org/10.1163/156856292X00204
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Influence
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accumulation, activation
of
a preadsorbed fibrinogen
in
hemodialysis
terpolymer adsorption, hollow
on and ex
human vivo
platelet blood
fibers
FENG YAN1, PHILIPPE DÉJARDIN1*, JULIETTE N. MULVIHILL2, JEAN-PIERRE CAZENAVE2, THIERRY CROST3, MICHEL THOMAS3 and CHRISTIAN PUSINERI3 'Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083Strasbourg, France 2CentreRégional de TransfusionSanguine, INSERM U.311, 10 rue Spielmann, 67085Strasbourg, France 3Hospal-COT,13 avenue de Lattre de Tassigny,69881Meyzieu,France Received2 October 1991;accepted 22 December 1991 Abstract-Results are presentedon kineticsof platelet accumulationin charged polyacrylonitrile(AN69) hollow fibers by continuous data recording under flow conditions (wall shear rate 108-1050s-1), using human plateletsin Tyrode's-albuminbuffer, containingwashed red suspensionsof washed 111In-labeled blood cells (0-40%). Preadsorption of a terpolymerof acrylonitrile,poly(ethyleneoxide)methacrylateand trimethylaminoethyl chloride methacrylateleads to very efficient passivation with respect to platelet accumulation and fibrinogen adsorption. In human ex vivo tests, evaluation of complement peptide C3a, platelet βthromboglobulin, leucocyte-polymorphonuclearneutrophile elastase and fibrinopeptide A shows no detectable activation. Furthermore, preadsorption appears to result in simultaneous improvement in hemocompatibilityof the blood lines leading to and from the dialysis module. This singlepretreatment of dialysismembranesshould allow injection of lower doses of anticoagulant to patients submitted to hemodialysis. Key words: Polymer adsorption; membrane; hemodialysis; hemocompatibility; poly(ethyleneoxide); polyacrylonitrile;hollow fiber. INTRODUCTION Blood contact with a foreign material is known to trigger a succession of interfacial events: adsorption of proteins, adhesion and aggregation of platelets and leukocytes and biochemical reactions of coagulation and complement activation induced by the high interfacial concentrations and conformational changes of the adsorbed species an undesired thrombus To [1]. Finally, may appear. prevent such phenomena, two lines of research are to complementary possible: (i) investigate what really occurs before thrombus formation; and (ii) to find an ad hoc treatment of the surface which minimizes such undesirable events. Due to their relatively small size, proteins are expected to contact the surface first, followed by cells like platelets, red cells and leukocytes. For many years, work has been performed on protein adsorption on synthetic foreign materials in order to understand the first steps occurring on blood arrival. For the purpose of fundamental analysis in simplified models, mono-protein solutions were used [2-8],
389
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390 although the authors were aware that blood is a very complex fluid which cannot be modeled by a simple buffer. Comparisons between experiments using a single protein and those using protein mixtures [9-10] or plasma [11-16] are often very fruitful. For example, fibrinogen adsorption from simple solutions and from plasma variations of interfacial concentration versus time differs sharply: non-monotonous or solution concentration were found in the presence of plasma and this was designated the Vroman effect [17]. Whatever the type of experiment performed, irreversible processes [18] enhance the difficulties of modeling and emphasize the importance of a dynamic analysis, especially for complex fluids. Dynamic studies were recently carried out of the adsorption of fibrinogen from radiolabeled solutions on polymer hollow fibers or on a glass tube from radiolabeled how a continuous recording of the interfacial consolutions which demonstrated centration versus time could reveal different successive regimes [19]. The same technique is applied here to platelet deposition. It is hoped that, on a wide range of supports, this method will give information about kinetic parameters analogous to that obtained from optical techniques like ellipsometry [20-23] or TIRF (Total Internal Reflection Fluorescence) [2, 7, 13] which requires planar transparent surfaces. and platelet accumulation Although limited, tests of fibrinogen adsorption a estimate of the of a surface. The term good hemocompatibility already represent in the sense that thrombus formation is avoided, could suggest 'hemocompatible', extreme strategies to transform raw synthetic materials into hemocompatible ones, from (i) initial complete coverage with biological species originating from the contacting blood, to (ii) a surface completely prepassivated with synthetic or biological materials, leading to an important decrease in the interfacial concentration of blood components. Following the second approach, an example illustrating that surface treatment with a synthetic polymer can lead to good passivation with respect to human platelet accumulation, and biochemical blood fibrinogen adsorption activation is presented. MATERIALSAND METHODS The values of wall shear rate in the following text were calculated from the measured flow rate, assuming a Poiseuille velocity profile in an impermeable tube. These figures are of course only indicative in the presence of red cells [24] or for porous fibers. Hollow fibers Modules of hemodialysis hollow fibers (48 fibers per module for in vitro and 340 fibers per module for ex vivo tests) were kindly provided by Hospal Industries (Meyzieu, France). The fibers have an internal diameter of 329 or 240,um and a wall thickness of 50 #m, the main constituent being a random copolymer of acrylonitrile and sodium methallyl sulfonate, known as AN69. To interpret the kinetics of platelet accumulation, the model of an impermeable tube may be considered as a good approximation of the system as we used fibers without pores. However, for the study of the influence of red cells on platelet accumulation porous hollow fibers were employed, as for fibrinogen adsorption and the ex vivo tests.
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391 Experimental apparatus The in vitro experimental apparatus consists of a microcomputer coupled to two syringe pumps, one containing buffer, the other a platelet suspension at concentration Cb. Buffer and suspension pass to the module of hollow fibers via teflon tubing and a three-way connector, which enables deviation to a waste vessel to eliminate air bubbles. The module is placed horizontally in front of a detector (Quartz et Silice, each France). Data are visualized on a screen and stored in the microcomputer, experimental point corresponding to the integration of radioactivity over 10 s. At the module exit, the liquid is collected on a balance and mass data as a function of time appear graphically on the screen, allowing instantaneous visualization of flow rates. Thermostation is ensured by circulation of buffer at 37°C in the compartment outside the fibers. 111 In-labeled washed human platelets Washed human platelets were prepared as described by Cazenave et al. [25] and resuspended in Tyrode's buffer containing 2 mM Ca2+, 1 mM Mg2+, 0.35% human albumin (Centre Regional de Transfusion Sanguine de Strasbourg), 0. l vo glucose and 20,uml/ml apyrase (Tyrode's albumin buffer), pH 7.30. Apyrase was included in the perfusion medium to prevent platelets becoming refractory and to inhibit major aggregate formation on the surface or in the fluid phase [26]. Platelet count was adjusted to 300000/,uml (Platelet Analyser 810, Baker Instruments, Allentown, USA). Radiolabeling of platelets was performed by incubation with 1 "In-oxine a 1'Energie Atomique, Saclay, France) for 15 min at (0.25,u1/ml, Commissariat 37°C, according to Eber et al. [27], labeling yield being of the order of 90% without modification of platelet aggregation to ADP, thrombin or collagen. Washed red blood cells from the same donor were prepared by the method of Cazenave et al. [26] and resuspended in Tyrode's albumin buffer, pH 9.0. Immediately before perfusion experiments, the red blood cell suspension was centrifuged for 4 min at 1600 g and packed erthrocytes were added to the suspension of " 1 In-labeled washed human platelets at the chosen volume ratio, corresponding in most experiments to a 40% hematocrit. Polymers Two types of polymers were synthesized. The first was obtained monomers and is denoted 'copolymer', while the second was derived monomers and is called 'terpolymer'.
from two from three
of acrylonitrile (AN) and monomethoxyCopolymer. Radical copolymerization nona(ethyleneglycol) methacrylate (MG,) was carried out at 60°C in dimethylas initiator. Extensive discussion of this sulfoxide with azobisisobutyronitrile can be found elsewhere The synthesis [28]. copolymers with a molar fraction of MG9 18% than are water soluble and of pH is performed with greater adjustment concentrated HCI.
392 on discs of flat hemodialysis membrane AN69 were performed Preadsorption from aqueous solutions (C = 0.7-0.8 mg/ml) at varying pH.
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of acrylonitile (AN), methoxypoly(ethyleneTerpolymer. Radical polymerization chloride methacrylate (MQ) and trimethylaminoethyl glycol) methacrylate (MG23) as was performed in water at 30°C over 3.25 h with the couple KC'03/NaHS03 initiator. The initial molar fractions of monomers were fAN = 0.92, fMG = 0.050 = 0.033. Elementary analysis gave through chloride and nitrogen contents and fMQ the following molar composition for the terpolymer: FAN = 0.746, FMG = 0.228 and = = = = FMQ 0.026, or in mass fractions WAN 0.125, WMG 0.819 and WMQ 0.018.
Viscometry yielded intrinsic viscosities varying between 44 ml/g in 10-2 M NaCI and 40 ml/g in 3 10-2 M and 1 M NaCI aqueous solutions. Refractive index increment (dn/dc) in 10-' M NaCI at 632 nm was 0.140 ml/g, while the molecular weight by light scattering was 3.85 106. In the present experiments, minimodules were pretreated prior to a test run by passage of a terpolymer solution (5 mg/ml in Tyrode's buffer), 5 ml at 6 ml/min and after which modules were rinsed with physiological saline then 17 ml at 0.1 ml/min, containing heparin (2.5 IU/ml). Pretreated hollow fibers minimodules were compared to line controls, untreated AN69 minidialysers and equivalent minimodules containing other commercial hollow fibers: cuprophan (CUP, ENKA, Germany) and Polysulfone (PS, Fresenius, Germany). Determination
of platelet
accumulation
and apparent
kinetic constants
Calibration of the in vitro system was carried out in situ by estimating the radioactivity increase due to filling of the fibers (radius R, length L) with a platelet suspension of known concentration Cb. Three-five minute alternate flows of delay which appeared to be too short to suspension and buffer were performed-a reach a stable state in the accumulation process. Given Ah the radioactivity increase while filling and AH the residual radioactivity after rinsing (Fig. 1) and assuming
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393
Figure 1. Example of a radioactivity recording in a three steps experiment. The rise Ah is directly proportional to the known solution (suspension)concentration, thus allowing the determination of the adsorbed (deposited)amount through AH. The slopedr/dt is measuredin the sameway through dHa/dt. the geometry of a perfectly
The apparent
smooth tube, the interfacial
platelet deposition
constant
concentration
is:
keXpis given by:
This procedure does not require an independent measurement of specific activity, since ratios of lengths on the graph lead directly to r and dr/dt when R is known. At the end of an experiment, as an additional control, the modules are cut into 2 cm long segments of which the radioactivity is measured in a well type gamma counter 1282, LKB, Turku, Finland) [29]. (Compugamma Purified
human albumin
and fibrinogen
Albumin and fibrinogen purified from human plasma were supplied by the Centre Regional de Transfusion Sanguine, Strasbourg, France. Both proteins were 99% while the pure by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, of to the method of 98 was and 95 0l0 clottability fibrinogen according Regoeczi [30] before and after radioactive labeling using the iodogen technique, respectively [31 ] . Ex vivo tests The ex vivo test to evaluate hemocompatibility of minimodule has been described in a previous publication [32]. Briefly, antecubital vein of a healthy volunteer is pumped through containing 340 fibers (internal diameter 240,um, external flow rate of 10 ml/min (wall shear rate 360 over a
hollow fiber dialysers blood drawn from the a minimodule dialyser diameter 340,um) at a test period of 30 min.
394 is assured by continuous on-line heparinization (= 0.14 IU/ml, Anticoagulation Roche, France) directly after the point of venous access. At the module exit, blood samples collected at 5 min intervals are analysed for markers of activation of coagulation (fibrinopeptide A, FPA), complement (anaphylatoxin C3a), platelets (flneutrophil (PMN) thromboglobulin, 6TG) and leukocytes (polymorphonuclear elastase). Analyses are performed using commercial kits: FPA and 6TG (ELISA, France) and PMN elastase Stago, France), complement C3a (RIA, Amersham, (ELISA, Merck, Germany). RESULTS AND DISCUSSION
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l. Platelet
accumulation
This section is divided into three subsections: firstly, the influence of red cells on platelet accumulation is examined, secondly, dependence on shear rate is analyzed, and finally ex vivo results obtained with polymer pretreated modules are discussed. Influence of red cells. Figure 2 shows curves of recorded radioactivity versus time during perfusion of hemodialysis modules at a shear rate of 510 s-1, with platelet (180000 pl/,ul) containing different concensuspensions of constant concentration trations of red cells. While the porous hemodialysis membrane shows almost no platelet accumulation without red cells, we observe a strong increase in accumulation between 20 and 30% red cells. This result is in accordance with observations described in the literature [24] and results from enhancement of the transport of platelets towards the surface in the presence of red cells. Another point is that the accumulation kinetics is linear with time over the first 5 min. Apparent kinetic constants were estimated to be 0.106, 0.77, 1.38, 5.7, and 7.6 10-5 cm S-1 for red
Figure 2. Kineticsof platelet accumulationon porous hollowfibers from platelet suspensionsof constant concentration (180000 pl/pl)containing different concentrations of red cells (0, 20, 30 and 40%).
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395 cells concentrations of 0, 10, 20, 30, and 40% respectively. Concerning the rinsing steps, we observed very good stability of the signal at long times, showing no desorption in presence of buffer. However, the drop of the signal when flushing with buffer was much less, especially when accumulation was high, than the initial rise when introducing a platelet suspension after buffer. A clear explanation for this phenomenon is not yet available, a possibility being that the absence of albumin in the buffer could activate some platelets leading to a short but non negligible enhancement of the deposition. For the two highest red cell concentrations of 30 and 40%, the platelet deposition at the end of the perfusion step was 70 and 80% respectively of the total accumulation at the end of the rinsing step. Maximal coverage obtained after rinsing was 41000 pl/mm2. Thus a linear increase of radioactivity versus time was observed to a coverage of 33 000 PI/MM2, which represents about half the maximal coverage (60 000 pl/mm2, see Fig. 5). As it was clear that platelet accumulation was significant in the presence of red cells and in order to remain as close as possible to physiological conditions, subsequent experiments were carried out with suspensions containing 40% red cells and a platelet concentration of 180 000 pl/#I. Contrary to the above studies concerning the effect of red cells, non porous hollow fibers were used. Influence of shear rate. Figure 3 shows the influence of wall shear rate y on the kinetics of platelet accumulation recorded at 10 cm from the module entrance, using 1 suspensions prepared from blood provided by a single human donor. At 1050 s-1 the activity rise occurs more rapidly than at lower shear rates. The initial slopes do not appear to be the highest ones: this is particularly evident at high shear
Figure 3. Kinetics of platelet accumulation at various Poiseuille's wall shear rates from platelet suspensionsof constant concentration (180000pl/?1) containing 40Vored blood cells.
396
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rates. Slow lateral transport of platelets and/or red cells could be responsible for a relatively long time lag before obtaining a steady state profile and linear variation of Nor can we exclude a possible slight initial overthe interfacial concentration. adhesion during the change from buffer to platelet suspension. We again observed that the drop Ah' was smaller than the rise Ah (see Fig. 1), the difference being larger for higher shear rates. The L6v?que constant may be estimated from:
With an assumed platelet diffusion coefficient in plasma D = 1.58 10-9 CM2 s-1 at 37°C [28], bulk concentration Cb = 180000 pl/,ul and distance to the fiber entrance z = 10 cm, kLe? is calculated as 1.61, 2.24 and 3.46 10-6 cm s-1 for the shear rates of 108, 285 and 1050 s-1, respectively. keXpis larger than the maximum mass transfer to corresponding by a factor which increases with Poiseuille's shear rate: kexpIkL,, = 22, 32 and 52 for 108, 285 and 1050 s-1, respectively. One can argue that: (i) red cells significantly enhance lateral platelet transport; and (ii) this enhancement, with respect to the y113 law, increases with flow rate. Such behavior has been previously described for rabbit blood [33]. Without red cells (bottom curve in Fig. 2) and at a shear rate of 510 keXp is 1.06 10-6 cm s-1 and kLev is 2.71 10-6 cm s-' . Through the relations [34] :
it is estimated that an intrinsic accumulation constant ka = 1.74 10-6 cm sec? ?and a ratio of the steady state concentration near the wall Cst(0) to the bulk concentration Cb close to 0.6. However, this estimation does not include a possible effect of the transmembrane pressure through the porous membrane. The variation of platelet deposition versus distance from the fiber entrance is presented in Fig. 4. Let us recall that the foregoing kinetic analysis was performed at 10 cm from the entrance. We can see that at 1050 çl there is almost no variation along the fiber: after 5 min the surface should be almost entirely covered with platelets. This is in accordance with the kinetic curve, where we observe a decreasing rate of accumulation towards 4 to 5 min. Rapid completion of the surface occupation probably occurs during the change from suspension to albumin free Tyrode's buffer. At lower shear rates, a gradient of interfacial platelet concentration along the fiber is clearly observed, measurements at 280 and 285 s-1 showing remarkable reproducibility given the complexity of the fluid involved. As linear variation of accumulation versus time was observed until the end of the perfusion step, platelet of these curves should be approximated by a transport quantitative interpretation limited model. If we assume that the volume concentration near the wall has a steady state value, a modified L6v?que model should apply [30]:
Since r varies linearly with time during the perfusion step, Ar/At, where Or is the platelet deposition over a time period such a representation of the data. As expected, we do not find the two highest shear rates, as the saturation value of 60000
we have (dr /dt) = At. Figure 4 shows a linear relation for pl/mm2 (Fig. 5) is
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397
Figure 4. Inverseof the apparent kinetic accumulationconstant versus (Z/Y)1/3from data obtained after the rinsing step. z (cm) is the distance from the fiber entrance and y (s-') the Poiseuillewall shear rate. Insert: Platelet accumulationversus distance from the fiber entrance at various shear rates: 110 ç (+), 280 s-1(circle), 1050s-' (square).
Figure 5. Influenceof terpolymerpretreatment of fibers on the kineticsof platelet accumulationat 10cm from the fiber entrance. Lower curve: pretreated module; upper curve: untreated module. Insert: Platelet accumulationalong the fibers for pretreated module (bottom curve) and untreated module (top curve). z = distance from the fiber entrance.
398
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almost reached within 5 min at 4 cm from the module entrance and therefore a steady state assumption is not valid. From the slope at 110 s-1 we estimate an apparent platelet diffusion coefficient of 57 10-9 CM2s-', which would demonstrate again the importance of red cells. However, the ordinate at the origin is negative and does not allow a coherent interpretation from Eq. (5). Two phenomena may play a role in this behavior: firstly the entrance length to establish a steady velocity profile was neglected and secondly the increase in deposition when changing from suspension to buffer (leading to Ah' smaller than Ah) was observed to be an increasing function of the flow rate or, in other words, of the inverse of the Nernst diffusion of Cb (dr/dt)-' in the perfusion step, layer. This should lead to an underestimation an underestimation more important at smaller distance from the fiber entrance. Reduction of platelet accumulation. We now briefly present some results obtained with porous hollow fibers to illustrate than an appropriate surface treatment, in this case preadsorption with the terpolymer AN - MG23 - MQ, can considerably reduce 5 accumulation. shows the kinetic curves recorded during 10 min platelet Figure of a treated module and an one (upper curve), untreated perfusion (bottom curve) while the insert on the same figure gives the variation of platelet deposition with distance from the module entrance determined by measurement of the radioactivity of 2 cm long segments at the end of the experiment. The efficiency of the treatment is clear: average platelet accumulation drops from 54000 to 1500 pl/mm2. Fundamental analysis of this complex process would however be somewhat hazardous given that, although we know the flow rate in the dialysate compartment (15 ml/min), we do not know the transmembrane pressure. 2. Albumin The effect
and fibrinogen
adsorption
on albumin and fibrinogen adsorption of preadsorption with the of and copolymer acrylonitrile monomethoxy-nona(ethyleneglycol) methacrylate was also investigated. Adsorbance of radiolabeled albumin and fibrinogen on membrane discs was measured by counting directly the radioactivity on the discs. Figure 6 shows the results, compared to untreated membranes, for various compositions of the copolymer and preadsorption conditions. Adsorption of this copolymer has been the subject of previous studies [35]. It can be seen that whatever the pH of passivation efficiency is zero for copolymers with a high content of preadsorption poly(ethyleneoxide) (PEO) side chains, while a maximum reduction in adsorbance of 60?Io is attained with a copolymer containing a molar fraction of 79?/o acrylonitrile, near the limit of solubility of these copolymers in water. This 6007oreduction is however less than the passivation efficiencies obtained on silica or glass with PEO based polymers [36, 37]. We conclude that a higher content of acrylonitrile in the copolymer may improve anchoring of the polymer on the membrane surface and therefore prevent protein adsorption more effectively than in the case of copolymers with a lower content of acrylonitrile, more readily displaced from the surface by proteins. a small amount of a third monomer positively Following this interpretation, was added into the reaction medium. The terwith a ammonium quaternary charged polymer obtained (Materials and Methods) which bears positive charges, should increase the stability of the adsorbed polymer layer on the membrane by electrostatic
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399
Figure 6. Relativeadsorbance of albumin (open symbols)and fibrinogen (closed symbols)on flat discs of AN69 membrane, with respect to untreated membrane, after preadsorption with copolymer solution (C = 0.7-0.8 mg/ml) at pH 3 (squares, pH 4.5 (circles)and pH 6 (triangles). FMG= molar fraction of methacrylate). MG9(monomethoxy-nonaethyleneglycol interactions with sulfonate groups. Moreover, a macromonomer with a longer side chain of methoxypoly(ethyleneoxide) was chosen, 23 units instead of 9, as in the work of Miyama [38]. Terpolymer preadsorption was performed from solutions at 5 mg/ml in Tyrode's buffer, the same buffer as used to prepare concentration fibrinogen solutions. A reduction of at least 87?7o in fibrinogen adsorbance was obtained. Although this water-soluble terpolymer might be leached out during these experiments, it seems that, if any, such a leaching should have been low enough to keep prevention of protein adsorption. In view of the high efficiency of this terpolymer in preventing fibrinogen adsorption and platelet accumulation in vitro, the effect on blood activation of this new composite material was further evaluated by means of ex vivo tests in humans using dialysis modules of porous hollow fibers. 3. Ex vivo tests A deeper insight into the hemocompatibility of dialysis membranes preadsorbed with the terpolymer should include studies of the interactions of this new material with whole blood. Therefore ex vivo tests with dialysis modules were performed to evaluate: (i) activation of coagulation according to the generation of FPA; (ii) complement activation as indicated by the formation of C3a; (iii) platelet activation as determined by measurement of 8TG release; and (iv) neutrophil activation as estimated from release of PMN elastase. Figure 7 shows the results, as compared to untreated AN69 and other hemodialysis membranes. Although further tests will be required to draw definitive conclusions,
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Figure 7. Plasma concentrations of fibrinopeptide A (FPA), p-thromboglobulin complement fragment C3a and PMN elastase measured at the exit of dialysis modules in ex vivo tests. Polysulfone (dotted line, symbol S, n = 5); cuprophane (dotted line, symbolC, n = 5); untreated AN69 (A, n = 5); terpolymer preadsorbed AN69 (1, test 1; 2, test 2); line control (dotted line, symbol L, n = 5).
401 it is remarkable that systematically the levels of activation of blood components by the pretreated AN69 membranes are lower than those of the other supports. Moreover, especially for elastase release, they are similar or even less than control values. It is probable that polymer adsorption on the blood lines during module pretreatment led to additional surface passivation, in particular towards neutrophils. CONCLUSIONS in vitro of kinetic new experimental system allows the determination parameters of molecular adsorption and cellular deposition. Use of the technique with platelet suspensions shows the importance of red cells in platelet accumulation, in accordance with previous results [24]. In the absence of red cells, the experimental accumulation constant is of the same order of magnitude as the value deduced from a Leveque model: the deposition process is partially transport controlled. As red cell content increases enhancement of the accumulation rate occurs, since hydrodynamic conditions are drastically modified [24]. When varying the flow, we find that the platelet deposition rate varies strongly with wall shear rate over the range studied and at 1050 s-1 complete surface coverage is attained within 5 min. In all these experiments, an unexplained phenomenon occurring when replacing platelet suspension may be tentatively ascribed to the absence of albumin in the buffer, which could However this point remains to be checked. lead to some platelet activation. could occur when replacing Although less obvious a similar type of perturbation buffer by platelet suspension. More trivial, an abrupt short overpressure when switching the electric valves could produce such a phenomenon. Further studies of platelet accumulation and fibrinogen adsorption, demonstrate of a passivation of the polyacrylonitrile dialysis membrane AN69 by preadsorption PEO rich terpolymer bearing a small number of quaternary ammonium charges. The in vitro passivation tests seem to be a good guide to the hemocompatibility check of new materials, since subsequent ex vivo tests in humans using heparinized blood show minimal levels of cellular and biochemical blood activation. These promising results could open the way to the development of composite dialysis obtained by simple surface treatment membranes of improved hemocompatibility in patient therapy. and thus enable the use of lower levels of anticoagulant level by adsorption from aqueous Furthermore, passivation at a (macro)molecular solutions, contrary to thicker coatings, should not greatly modify the permeability of the original membranes.
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