Thrombocyte Adhesion and Release of Extracellular Microvesicles

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Thrombocyte Adhesion and Release of Extracellular Microvesicles Correlate with Surface Morphology of Adsorbent Polymers for Lipid Apheresis René Weiss,† Andreas Spittler,‡ Gerd Schmitz,§ Michael B. Fischer,†,∥,⊥ and Viktoria Weber*,†,⊥ †

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Austria ‡ Core Facility Flow Cytometry and Surgical Research Laboratories and ∥Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Austria § Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany ABSTRACT: Whole blood lipid apheresis is clinically applied to reduce low density lipoprotein cholesterol in patients with homozygous familial hypercholesterolemia. Here, we studied the correlation between physicochemical parameters, in particular, surface roughness and blood compatibility, of two polyacrylate-based and a dextran sulfate-based polymer for lipid apheresis. The adsorbent surface roughness was assessed by atomic force microscopy. Freshly isolated human thrombocytes were circulated over adsorbent columns downscaled equivalent to clinical use to study thrombocyte adhesion and microvesicle generation. Quantification of thrombocytes and microvesicles in the flow-through of the columns revealed that both thrombocyte adhesion and microvesicle generation increased with increasing adsorbent surface roughness. Activation of thrombocytes with thrombin receptor-activating peptide-6 favored their adhesion to the adsorbents, as demonstrated by preferential depletion of CD62+ and PAC-1+ thrombocytes. Taken together, enhanced polymer surface roughness fostered cell adhesion and microvesicle release, underscoring the role of extracellular microvesicles as markers of cellular activation and of blood compatibility.

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INTRODUCTION The entry of low density lipoprotein (LDL) into the arterial wall and its retention through the binding of apolipoprotein B100 (apoB100) to proteoglycans of the extracellular matrix is regarded as a key initiator of atherogenesis.1−4 LDL undergoes enzymatic as well as nonenzymatic, free radical-mediated oxidation in vivo, in particular, under conditions of hyperlipidaemia.5−8 Oxidized LDL (oxLDL) acts as a damageassociated molecular pattern and is bound and internalized by scavenger receptors on macrophages, such as CD36 and SR-A. Unregulated uptake of oxLDL by macrophages stimulates the secretion of pro-inflammatory cytokines and matrix degrading enzymes and leads to the formation of foam cells, a major component of atherosclerotic plaques.9,10 Thrombocytes activated by oxidative stress and inflammation can adhere to the endothelium, induce further leukocyte recruitment, and promote the development of atherosclerotic lesions via Pselectin-dependent mechanisms, underscoring their contribution to the maintenance and propagation of artherosclerosis.11−14 Patients suffering from homozygous familial hypercholesterolemia (FH) are unresponsive to diet and drug therapy and have a markedly increased risk of developing atherosclerosis and its ischemic complications at an early age. Lowering of © 2014 American Chemical Society

LDL cholesterol levels in these patients can be achieved with LDL apheresis,15,16 This group of extracorporeal technologies can selectively or specifically remove LDL and other apoB100 containing lipoproteins, such as very low density lipoprotein, intermediate density lipoprotein, and lipoprotein (a), an independent risk factor of coronary artery disease.17,18 Six LDL-apheresis systems are currently available for clinical application. Four of these require the separation of plasma from blood cells prior to lipoprotein removal, while two systems, DALI and Liposorber D, have been developed for lipoprotein adsorption directly from whole blood.19 DALI is based on columns containing porous polyacrylamide beads functionalized with anionic polyacrylate, and Liposorber D consists of cellulose beads functionalized with dextran sulfate. Both systems remove positively charged apoB100 containing lipoproteins by electrostatic interactions with the negatively charged groups at the adsorbent surface. While these whole blood adsorption systems are easy to handle and highly efficient since they do not require plasma separation prior to adsorption, the direct contact of blood with artificial polymers demands a Received: April 3, 2014 Revised: May 17, 2014 Published: May 20, 2014 2648

dx.doi.org/10.1021/bm500492r | Biomacromolecules 2014, 15, 2648−2655

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using a JEOL T330 microscope (JEOL Ltd., Tokyo, Japan) after sputtering the adsorbents with carbon. The surface roughness of the adsorbents was characterized by amplitude modulation atomic force microscopy with a Dimension 3100 Nanoscope IIIa (Veeco, Plainview, NY, U.S.A.) operated in the tapping mode. The following roughness parameters were determined: mean roughness, Ra, defined as arithmetic mean of the absolute values of the roughness profile ordinates; root-mean-square roughness, Rq, defined as the root-mean-square average of the roughness profile ordinates; and the total height of the roughness profile, Rt, defined as the distance between the highest peak and the lowest valley within the scanning length. Scanning length was 10 μm. Specific surface area and pore size distribution of the adsorbents were determined by recording nitrogen adsorption and desorption isotherms at 77 K (liquid nitrogen) using an ASAP 2010 surface area and porosimetry analyzer (Micromeritics Instrument Corporation, USA). Data analysis of the isotherms was performed using the BET method (Brunauer, Emmett, and Teller) for specific surface area determination. The average pore size diameter d(avg) was calculated using the total pore volume V(total) according to Gurwitsch’s rule [DIN 66134] and the specific surface area A as d(avg) = 4V(total)/A. Adsorption of Lipoproteins. Human plasma was standardized to an LDL concentration of 200−250 mg/dl, as described above, and aliquots of the adsorbents (10% v/v) were incubated with plasma with gentle shaking at 37 °C. Samples were taken at 30, 60, and 90 min, the adsorbents were removed by centrifugation (3 min, 10.000g), and the concentrations of LDL and HDL in the supernatant were determined using a Hitachi 902 Chemistry Analyzer and the reagent sets LDL-C plus and HDL-C plus (all from Roche Diagnostics, Risch, Switzerland). All experiments were carried out in triplicates. Release of Microvesicles from Thrombocyte Concentrates upon Circulation over Adsorbents. To determine the generation of microvesicles from nonactivated thrombocytes upon contact with adsorbent polymers, freshly isolated thrombocytes (3 × 105/μL in SSP+ anticoagulated with ACD-A 1:12; total volume 50 mL) were circulated over columns (3.5 × 1.8 cm, corresponding to a bed volume of 19.7 mL; downscaled equivalent to clinical use) packed with the respective adsorbent materials at a constant flow rate of 1.2 mL per min for 2 h. An empty column served as negative control. Samples of the column-flow through were taken every 30 min, thrombocytes were quantified using a blood cell counter (Sysmex KX-21N, Sysmex Europe, Neumünster, Germany), and microvesicles were determined by flow cytometry as described below. Flow Cytometric Characterization of Thrombocytes and Microvesicles. Flow cytometric analysis was performed with a Gallios Flow Cytometer (Beckman Coulter, Brea, California, U.S.A.) after staining of thrombocytes and thrombocyte-derived microvesicles with an R-phycoerythrin-cyanine (PC7)-conjugated anti-CD61 monoclonal antibody (mAb; Becton Dickinson, Franklin Lakes, New Jersey, U.S.A.). Samples of the column flow through were diluted 1:83 in PBS prior to analysis. Calibration of the refractory index of microvesicles was performed with fluorescent beads (Megamix, Biocytex, Marseille, France), as described in the manufacturer data sheet. The flow cytometer was adjusted to cover the microvesicle (0.5 and 0.9 μm) and the thrombocyte size ranges (0.9 and 3 μm). Activation of Thrombocytes. Thrombocytes (3 × 105/μL in SSP+ anticoagulated with ACD-A 1:12) were activated with increasing concentrations of TRAP-6 (0; 2.5; 5; 10; 50; 100 μM) for 10 min at room temperature in the dark and stained with a phycoerythrin (PE)conjugated anti-CD-41 mAb as thrombocyte marker, with a fluorescein isothiocyanate (FITC)-conjugated anti-CD62 mAb (Beckman Coulter, Brea, California, U.S.A.) and a FITC-conjugated antiPAC-1 mAb (Becton Dickinson, Franklin Lakes, New Jersey, U.S.A.) as thrombocyte activation markers, and with a FITC-conjugated Annexin V mAb (Becton Dickinson, Franklin Lakes, New Jersey, U.S.A.) as microvesicle marker. Samples were analyzed by flow cytometry as described above. To assess the binding of activated thrombocytes to the adsorbent polymers, freshly harvested thrombocytes (3 × 105/μL in SSP+ anticoagulated with ACD-A 1:12; total volume 50 mL) were activated

great deal of blood compatibility to avoid or minimize activation of blood cells. Thrombocytes, in particular, undergo activation and adhesion during contact with foreign surfaces, potentially leading to activation of coagulation and thrombocytopenia. Microvesicles released from activated platelets are main culprits in the development of thrombotic events.20,21 They are intrinsically pro-coagulant due to their exposure of phosphatidylserine, which interacts with vitamin K-dependent coagulation factors and promotes coagulation. Microvesicles may also expose tissue factor, the primary initiator of the coagulation cascade. The morphology and physicochemical parameters of adsorbents used for whole blood lipid apheresis determine their adsorption characteristics, but also influence their blood compatibility. In this study, we assessed the impact of adsorbent morphology, in particular, surface roughness, on thrombocyte adhesion and on the generation of microvesicles. We demonstrate that increased adsorbent surface roughness coincides with enhanced lipoprotein adsorption, but also promotes thrombocyte adhesion and microvesiculation.

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EXPERIMENTAL SECTION

Chemicals and Reagents. Phosphate buffered saline (PBS) was purchased from Panreac, Barcelona, Spain. Priming solution (134 mM Na+, 4 mM K+, 1.75 mM Ca2+, 0.5 mM Mg2+, 106.5 mM Cl−, 36 mM HCO3−) and acid citrate dextrose solution A (ACD-A; 22.0 g/L trisodium citrate, 24.5 g/L glucose monohydrate, 7.3 g/L citric acid) were acquired from Fresenius Medical Care, Bad Homburg, Germany. Platelet storage medium SSP+ (3.18 g/L trisodium citrate dihydrate, 4.42 g/L sodium acetate trihydrate, 1.05 g/L sodium dihydrogen phosphate dihydrate, 3.05 g/L disodium hydrogen phosphate, 0.37 g/ L potassium chloride, 0.30 g/L magnesium chloride hexahydrate, 4.05 g/L sodium chloride; pH 7.2) was purchased from Macopharma, Tourcoing, France. Unfractionated heparin was obtained from Gilvasan Pharma, Vienna, Austria. Thrombin receptor-activating peptide-6 (TRAP-6) was purchased from Bachem, Bubendorf, Switzerland. Plasma and Thrombocyte Concentrates. Freshly drawn human plasma anticoagulated with heparin (2.5 IU/mL) was obtained from a local plasma donation center after informed consent of the donors. The plasma was concentrated using an AlbuFlow filter (Fresenius Medical Care, Bad Homburg, Germany) to obtain a concentration of low density lipoprotein (LDL) of 200−250 mg/dl. Thrombocyte concentrates (medical grade) were purchased from the Clinic for Blood Group Serology and Transfusion Medicine, Medical University Vienna, Austria. The concentrates were produced using a Trima Accel automated blood collection system (Version 5.0, Gambro BCT, Lund, Sweden) and stored for a maximum of 2 h at RT before use. Adsorbents. Two adsorbents which are approved for clinical application in lipid apheresis from whole blood, DALI (Fresenius Medical Care, Bad Homburg, Germany) and Liposorber D (Kaneka Pharma Europe, Wiesbaden, Germany), were used in this study. DALI consists of polyacrylamide beads functionalized with polyacrylate, and Liposorber D consists of cellulose beads functionalized with dextran sulfate. Both adsorbents are negatively charged and bind to apolipoprotein B100 (apoB100) containing lipoproteins via electrostatic interactions. A noncommercial polyacrylate-based resin functionalized with poly(acrylic acid) (ReliSorb) was tested in addition. Prior to use, adsorbent columns containing DALI and ReliSorb (6 × 1.8 cm; downscaled equivalent to clinical use) were rinsed with 2 × 20 mL of priming solution containing ACD-A (1:40), and 10 IU/mL of heparin was supplemented during the first rinsing step. Liposorber D was extensively washed with saline solution (0.9% w/v) prior to use. Characterization of Adsorbent Morphology, Surface Roughness, and Porosity. Scanning electron microscopy was performed 2649


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Figure 1. Scanning electron micrographs and surface roughness parameters of adsorbents for whole blood lipid apheresis. Representative scanning electron micrographs of different adsorbent polymers (DALI, ReliSorb, Liposorber D) are shown (scale bars = 100, 30, and 3 μm). Surface roughness was determined by atomic force microscopy operated in the tapping mode. Ra, mean roughness (arithmetic mean of the absolute values of the roughness profile ordinates); Rq, root-mean-square roughness (root-mean-square average of the roughness profile ordinates); Rt, total height of the roughness profile (distance between the highest peak and the lowest valley within the scanning length). Scanning length was 10 μm. with TRAP-6 (50 μM) and circulated over a column packed with the DALI adsorbent at a flow rate of 1.2 mL per min for 2 h. A control experiment was performed under identical conditions using nonactivated thrombocytes. Sampling, quantification of thrombocytes, and flow cytometric analysis were performed as described above. Statistical Analysis. Statistical analysis was performed using the software package SPSS Statistics for Windows, version 18.0 (SPSS Inc., Chicago, Illinois, U.S.A.). Data were analyzed using the Student’s t test. Data are expressed as means ± standard error of the mean. Significance was accepted at P ≤ 0.05.

study were characterized morphologically by scanning electron microscopy and atomic force microscopy (Figure 1). The two polyacrylate-based polymers had a specific surface area of 26 m2/g for ReliSorb and 49 m2/g for DALI. The pore volume was 1.4 and 1.7 mL per g dry adsorbent for ReliSorb and DALI, respectively, and their charge density was 300 and 530 μequiv COOH per g dry adsorbent. While the two adsorbents exhibited a comparably structured inner surface, they showed clear differences with respect to their outer surface, which appeared open and porous on scanning electron micrographs for ReliSorb, while it had a closed and smooth appearance for DALI.

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RESULTS Morphological Characterization of the Adsorbents. The adsorbents for whole blood lipid apheresis used in this 2650


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Figure 2. Adsorption of low density lipoprotein and high density lipoprotein by adsorbents for whole blood lipid apheresis. Adsorption was assessed in batch experiments in human plasma with an initial LDL concentration of 200−250 mg/dl using an adsorbent-to-plasma ratio of 1:9 (n = 3); *p < 0.05.

Figure 3. Determination of microvesicles using flow cytometry. Fluorescent beads with diameters of 0.5, 0.9, and 3 μm, respectively, were used for calibration according to the protocol of the supplier (Megamix, Biocytex, Marseille, France; left); the microvesicle gate (F) was set around the 0.9 μm bead cloud (middle); example of microvesicles in a fresh thrombocyte concentrate (right); microvesicles were quantified in gate F relative to the number of total events.

Amplitude modulation atomic force microscopy was used to characterize the surface roughness of the adsorbent polymers. In consistence with the morphological data obtained by scanning electron microscopy, surface roughness was lower for DALI as compared to ReliSorb and Liposorber D with a mean roughness Ra of 84.8 versus 251 versus 148.9 nm, respectively, and a maximum height of the profile Rt of 777 versus 1964 versus 1054 nm, respectively. Adsorption of LDL and HDL. The binding of low and high density lipoproteins was quantified in batch experiments using human plasma which was standardized to initial LDL concentrations of 200−250 mg/dl (Figure 2). DALI and Liposorber D bound comparable amounts of LDL over time (3.2 vs 3.5 mg LDL per mL adsorbent after 90 min), while ReliSorb showed significantly higher LDL binding (6.3 mg LDL per mL adsorbent after 90 min). Binding of HDL was negligible for all three adsorbents. Identification and Characterization of Microvesicles by Flow Cytometry. Microvesicles in the flow-through of columns containing the different adsorbent polymers were identified and characterized by flow cytometry. Calibration was performed using a mix of fluorescent beads with diameters of 0.5 and 0.9 μm to cover the microvesicle size range and 0.9 and 3 μm to cover the thrombocyte size range (Figure 3). The microvesicle gate F was set around the cloud of the 0.9 μm

beads and microvesicles were quantified relative to the number of total events. Thrombocyte Adhesion and Release of Microvesicles. To study thrombocyte adhesion to the adsorbent polymers (Figure 4, upper panel), freshly isolated thrombocytes were circulated over columns packed with DALI, ReliSorb, and Liposorber D, as described in the Experimental Section. Thrombocyte adhesion was significantly higher for ReliSorb as compared to DALI as calculated from the number of thrombocytes remaining in the pool after 30, 60, 90, and 120 min (195 ± 30 vs 246 ± 29 vs 300 ± 22 × 103/μL after 120 min for ReliSorb vs DALI vs control without adsorbent). DALI and Liposorber D showed similar levels of thrombocyte adhesion (223 ± 41 vs 245 ± 46 vs 285 ± 42 × 103/μL after 120 min for DALI vs Liposorber D vs control without adsorbent). Likewise, passage of thrombocytes over ReliSorb resulted in significantly higher levels of microvesicles (Figure 4, lower panel) as compared to DALI (5.7% vs 4.3% vs 4% of total events for ReliSorb vs DALI vs control without adsorbent after 120 min). Again, Liposorber D and DALI showed comparable microvesicle release (4.4% vs 4.1% vs 3.8% for DALI vs Liposorber D vs control without adsorbent). Activation of Thrombocytes with TRAP-6. Treatment of thrombocytes with increasing amounts of TRAP-6 (0; 2.5; 5; 10; 50; 100 μM) resulted in concentration-dependent 2651


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Figure 4. Generation of microvesicles and adhesion of thrombocytes upon circulation of thrombocyte concentrates over adsorbents for whole blood lipid apheresis. Freshly isolated thrombocytes (3 × 105/μL in SSP+) were circulated over columns, as described in the Experimental Section. At the indicated time points, thrombocytes (upper panel) and microvesicles (lower panel) in the flow-through were quantified using a blood cell counter and flow cytometry, respectively (n = 5); *p < 0.05.

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DISCUSSION The morphology and physicochemical characteristics of adsorbent polymers determine their blood compatibility next to their adsorption capacity. Therefore, we studied the correlation between physicochemical parameters, in particular, surface roughness, and blood compatibility of three adsorbents for whole blood lipid apheresis. DALI and Liposorber D are clinically approved and applied to selectively remove the apoB100-containing lipoproteins LDL and Lp(a) from blood of patients suffering from familial hypercholesterolemia. Selective binding to their target substances is achieved by electrostatic interactions between the negatively charged adsorbent surface (polyacrylate and dextran sulfate, respectively) and positively charged moieties of apoB100. As a third polymer, we included ReliSorb, a polyacrylate-based adsorbent, which is not yet in clinical application. The two polyacrylate-based polymers, DALI and ReliSorb, exhibited clear differences in the structure of their outer surface, which appeared smooth and closed on scanning electron micrographs for DALI, but open and porous for ReliSorb. This finding correlated with atomic force microscopy, where ReliSorb exhibited higher surface roughness than DALI in terms of all tested parameters. Due to its different chemical composition, cellulose functionalized with dextran sulfate, Liposorber D is not directly comparable to DALI and ReliSorb, but in consistence with the appearance of its outer surface, its roughness was higher than for DALI, but considerably lower than for ReliSorb. DALI and Liposorber D bound equal amounts of LDL, which is in accordance with clinical data.22 Despite a lower charge density, ReliSorb adsorbed significantly more LDL than DALI and Liposorber D, reflecting a better accessibility of the

thrombocyte activation, as monitored by expression of the activation marker CD62. The levels of CD62 + CD41 + thrombocytes increased from 28% for untreated thrombocytes to 89% for thrombocytes treated with 50 and 100 μM TRAP-6 (Figure 5a). A concentration of 50 μM TRAP-6 was used for all further experiments. Adhesion of Activated Thrombocytes to DALI. Activated and nonactivated thrombocytes were circulated over DALI as described in Materials and Methods. Activated thrombocytes adhered to DALI to a significantly higher extent than nonactivated thrombocytes as calculated from the number of thrombocytes remaining in the pool after 30, 60, 90, and 120 min (126 ± 51 vs 232 ± 54 × 103/μL after 120 min for activated vs nonactivated thrombocytes; Figure 5b). Activated thrombocytes were preferentially bound by DALI, as reflected by a decrease of CD41+ CD62+ thrombocytes from 96.2 to 85.2% after 120 min (Figure 5c) and a decrease of CD41+ PAC-1+ thrombocytes from 42.4 to 17.7% after 120 min (Figure 5d). The number of CD41+ AnnexinV+ events in the thrombocyte region increased from 3.3% to 12.9% upon circulation of activated thrombocytes over DALI, while no such increase was seen for nonactivated thrombocytes (Figure 5e, corresponding to region O in Figure 5f). We postulate that the two distinct populations in region O represent thrombocytemicrovesicle aggregates (T) and aggregates of thrombocytederived microvesicles (A), which are characterized by their high AnnexinV expression. Consequently, the relative amounts of CD41+ AnnexinV+ microvesicles decreased during circulation of activated thrombocytes over DALI (Figure 5g) due to their adhesion to either the adsorbent material, to thrombocytes, or due to aggregation with other thrombocyte-derived microvesicles. 2652


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Figure 5. Binding of activated thrombocytes to DALI. Thrombocytes were activated with TRAP-6, circulated over columns containing DALI, and samples of the flow-through were analyzed by flow cytometry as described in the Experimental Section. CD41 was used as thrombocyte marker, and CD62 and PAC-1 served as markers of thrombocyte activation: (a) correlation between TRAP-6 concentration and thrombocyte activation; (b) adhesion of activated and nonactivated thrombocytes to DALI as assessed by blood cell counting; (c, d) binding of activated and nonactivated thrombocytes to DALI as assessed by flow cytometry using CD62 or PAC-1 as activation markers; (e) binding of microvesicles to thrombocytes reflected by CD41+ AnnexinV+ events in the thrombocyte region, corresponding to region O (upper right quadrant) in (f). The two populations T and A represent thrombocyte-microvesicle aggregates (T) and aggregates of thrombocyte-derived microvesicles (A). All experiments shown in (b−f) were performed with 50 μM TRAP-6. (g) Relative amounts of CD41+ AnnexinV+ events (thrombocyte-derived microvesicles), CD41+ AnnexinV− events, CD41− AnnexinV+ events (microvesicles of nonthrombocyte origin, most likely, erythrocyte-derived), and CD41− AnnexinV− events (n = 5); *p < 0.05. 2653


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may boost the interaction of activated thrombocytes with both, negatively charged adsorbents and with microvesicles exposing negatively charged phosphatidylserine residues on their surface. Consistently, LDL apheresis with Liposorber D and DALI has been shown to reduce PF4 on the platelet surface,26 which was explained as a re-equilibration mechanism following depletion of plasma-derived PF4 by LDL adsorbent columns. Lipoprotein (a) may be another player in the setting of platelet activation induced by LDL apheresis. Although its physiological function remains to be fully elucidated, there are strong indications for its pro-thrombotic role. It has been shown to promote platelet activation via thrombin-receptoractivated hexapeptide, and it induces production and surface exposure of tissue factor on monocytes.27,28 Taking these data together, Lp(a) attached to anionic adsorbents via its positively charged apoB100 moiety may promote tissue factor upregulation on platelets, resulting in local thrombin generation and inducing a positive feedback loop toward further thrombocyte activation and adhesion. Our study was performed with thrombocyte concentrates, while we are aware that proteins which rapidly coat a surface following its exposure to whole blood profoundly influence cell adhesion. Among the numerous proteins, which may be adsorbed to a biomaterial surface, fibrinogen has been identified as a main inducer of platelet activation. Noteworthy, it has been demonstrated that platelet adhesion is strongly correlated with the degree of adsorption-induced protein unfolding.29,30

inner adsorbent surface of ReliSorb for the large low density lipoprotein complex. Binding of high density lipoprotein was negligible for all three adsorbents, since HDL does not contain apoB100 and therefore lacks the positively charged moieties that mediate adsorption to anionic polymers. Under identical experimental conditions, cellulose beads functionalized with phenyl groups (Cellufine Phenyl, JNC Corporation, Japan) exhibited excellent LDL adsorption due to hydrophobic interactions between LDL and the phenyl groups on the polymer surface (data not shown). These resins, however, resulted in a reduction of HDL levels by almost 50%, which precludes their application in lipid apheresis, since HDL represents an important atheroprotective factor11 due to its ability to mediate reverse cholesterol transport23 and due to its antioxidative as well as anti-inflammatory properties.24 Owing to their direct contact with blood during lipid apheresis, the blood compatibility of the adsorbents is of critical importance to minimize activation and adhesion of blood cells and inflammatory response. We hypothesized that activation and adhesion of blood cells, in particular, thrombocytes, might correlate with the polymer surface roughness. Affirming this assumption, we observed significantly higher thrombocyte adhesion for ReliSorb as compared to DALI and Liposorber D during circulation of freshly isolated thrombocytes over columns containing the different adsorbent polymers. Our hypothesis was further substantiated by flow cytometric detection of thrombocyte-derived microvesicles in the flowthrough of the adsorbent columns. Again, microvesicle release correlated with surface roughness and was significantly higher for ReliSorb as compared to DALI, adding evidence to the relevance of microvesicles as markers of cellular activation. There is emerging evidence for thrombocyte-derived microvesicles as players in thrombotic and inflammatory diseases25 apart from their participation in homeostatic multicellular processes such as hemostasis, maintenance of vascular health, and immunity, and our findings suggest that they can also serve as markers for blood compatibility of polymers. Treatment of thrombocytes with thrombin receptor-activating peptide-6 resulted in a dose dependent up-regulation of thrombocyte activation markers CD62 (platelet surface Pselectin) and PAC-1 (activated GP IIb/IIIa). The activation of thrombocytes correlated with their adhesion to DALI, and flow cytometric analysis revealed preferential adhesion of activated vs nonactivated thrombocytes as suggested by the preferential depletion of CD62+ and PAC-1+ thrombocytes. The appearance of significantly increased numbers of AnnexinV + thrombocytes in the flow through of the DALI column for activated vs nonactivated thrombocytes provided a strong indication of microvesicle adhesion to thrombocytes. It is thus to be assumed that microvesicles do not only adhere to the adsorbent polymers, but also to activated platelets and to each other, forming strongly AnnexinV-positive aggregates in the thrombocyte size range. Changes in thrombocyte shape may simply be held responsible for their enhanced interaction with adsorbent polymers and microvesicles, but it seems likely that there are other mechanisms fostering this interaction. Platelet factor 4 (PF4), a platelet-specific chemokine, is released in large amounts by activated platelets and expressed on the platelet surface. Under physiological conditions, PF4 is positively charged, as reflected by its interaction with glycosaminoglycans of the endothelial layer. It is therefore tempting to assume that the expression of positively charged PF4 on the platelet surface

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CONCLUSIONS The physicochemical parameters and the morphology of adsorbents for extracorporeal blood purification determine their binding of target factors and their blood compatibility. The results of this study indicate that the adhesion of thrombocytes as well as the release of AnnexinV+ microvesicles correlate with morphology and surface roughness of adsorbents for whole blood lipid apheresis. Thrombocyte activation fostered their adhesion to adsorbent polymers, and the appearance of AnnexinV+ thrombocytes provided evidence for the adhesion of microvesicles to activated thrombocytes. Binding of activated thrombocytes to adsorbents and microvesicles may be mediated by positively charged proteins, such as platelet factor 4, which is released from activated thrombocytes. While the influence of adsorbent morphology on thrombocyte adhesion and microvesicle release was a major topic of this investigation, chemical parameters such as the effective charge density, the distribution, and the accessibility of functional groups on the adsorbent may have additional impact, which will be elucidated in further studies using whole blood. This will also allow to determine the cellular origin of the microvesicles released during contact of blood with the adsorbents and to study the influence of deposited plasma proteins on the adhesion of blood cells.

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AUTHOR INFORMATION

Corresponding Author

*Phone: ++43 2732 893 2632. Fax: ++43 2732 893 4600. Email: [email protected]. Author Contributions ⊥

Both authors contributed equally to this work (M.B.F. and V.W.).

Notes

The authors declare no competing financial interest. 2654


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ACKNOWLEDGMENTS The excellent technical support by Ingrid Linsberger is gratefully acknowledged. The authors are grateful to Tanja Eichhorn for support with preparation of figures. This work was funded by the Christian Doppler Society (Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis).

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REFERENCES

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