Platelet pro-aggregatory effects of CD40L monoclonal antibody

Available online at www.sciencedirect.com

Molecular Immunology 45 (2008) 937–944

Platelet pro-aggregatory effects of CD40L monoclonal antibody Maribel Mirabet, Jos´e A. Barrab´es, Adoraci´on Quiroga, David Garcia-Dorado ∗ Servei de Cardiologia, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain Received 29 June 2007; received in revised form 1 August 2007; accepted 2 August 2007

Abstract An unexpected high incidence of thromboembolic complications has been described in patients with systemic autoimmune diseases treated with CD40L immunotherapy. Since activated platelets express CD40L, we aimed to investigate the effects of CD40L mAb in platelet aggregation induced by physiological stimuli. Optical aggregometry was performed on platelet-rich plasma and washed platelets obtained from systemic venous blood (0.38% citrate) of anesthetized pigs. CD40L mAb clone 5c8, used in clinical trials for autoimmune diseases, was used. In platelet-rich plasma, CD40L mAb neither induced platelet aggregation per se, nor significantly affected maximal aggregation or slope of ADP-induced aggregation curves. However, it dose-dependently inhibited spontaneous deaggregation observed in ADP-stimulated samples. This effect was not observed with an irrelevant isotype-matched immunoglobulin. The stabilizing effect on platelet aggregates was neither glycoprotein IIb/IIIa-mediated nor Ca2+ -dependent but was abolished by acetylsalicylic acid pretreatment. F(ab )2 fragments did not stabilize ADP-induced platelet aggregates but inhibited the stabilizing effect of CD40L mAb. Similar results were obtained with washed platelets, although higher amplification of ADP-induced aggregation was observed. In conclusion, CD40L expression produced by physiological or pathophysiological platelet activation can sustain a pro-aggregatory effect of CD40L mAb by a mechanism involving mAb Fc domain. These results could help to explain the mechanism of CD40L mAb-induced thromboembolic complications. © 2007 Elsevier Ltd. All rights reserved. Keywords: CD40L immunotherapy; Platelet aggregation; Thrombosis

1. Introduction CD40L is a trimeric, transmembrane protein of the tumor necrosis factor (TNF) family primarily found to be expressed on activated CD4+ T lymphocytes and to bind CD40, a member of the TNF receptor family, present on B lymphocytes. This CD40–CD40L interaction plays a critical role in T-cell-dependent humoral immunity (Schonbeck and Libby, 2001). A broader expression pattern of both CD40 and CD40L that extends to most leukocytes and cells in the vasculature has been described (Mach et al., 1997). Within this con-

Abbreviations: ASA, acetylsalicylic acid; Fc␥R, Fc gamma receptors; GP, glycoprotein; PPP, platelet-poor plasma; PRP, platelet-rich plasma; sCD40L, soluble CD40L; TNF, tumor necrosis factor; TXA2 , thromboxane A2 . ∗ Corresponding author at: Servicio de Cardiolog´ıa, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. Tel.: +34 934894038; fax: +34 934894032. E-mail address: [email protected] (D. Garcia-Dorado). 0161-5890/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2007.08.006

text, CD40–CD40L interactions have been demonstrated to be involved in cell-mediated immune responses and chronic inflammation (Schonbeck and Libby, 2001). The crucial role of CD40–CD40L interactions in immune and inflammatory responses made them a promising target for treatment of pathological immuno-inflammatory processes. Actually, blockade of CD40–CD40L interactions by means of specific CD40L monoclonal antibodies (mAbs) has been described to successfully prevent allograft rejection in primates (Kirk et al., 1999) and to be effective in animal models of autoimmune diseases (Kover et al., 2000; Howard et al., 1999) and atherosclerosis (Mach et al., 1998). In humans, two different humanized CD40L mAb clones have been used in clinical trials for treatment of different autoimmune diseases (Kalunian et al., 2002; Boumpas et al., 2003; Kuwana et al., 2004). Although in some cohorts of patients certain protocols of CD40L immunotherapy appeared to be potentially effective, an unusually high incidence of thromboembolic complications (Boumpas et al., 2003), also reported in CD40L mAb-treated

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primates subjected to allotransplantation studies (Kawai et al., 2000), led to a halt in all ongoing trials. 1,2 Thromboembolic complications associated to CD40L mAb therapy could be related to the fact that CD40L is expressed on the membrane of activated platelets. CD40L is rapidly translocated to the platelet membrane from internal source upon platelet activation (Henn et al., 1998) and this transmembrane form is subsequently cleaved and secreted as soluble CD40L (sCD40L) (Jin et al., 2001; Henn et al., 2001). sCD40L can, in turn, bind to platelet membrane proteins such as CD40 (through its TNF structural domain) (Inwald et al., 2003; Henn et al., 2001), and glycoprotein (GP) IIb/IIIa (through its KGD domain) (Prasad et al., 2003). It is known that mAbs recognizing different platelet membrane proteins are able to induce platelet activation and aggregation by different mechanisms, mainly by activation of Fc gamma receptors (Fc␥R) (De Reys et al., 1993). It was recently described that incubation of platelets with preformed sCD40L-CD40L mAb immune complexes induced platelet activation and aggregation as a consequence of CD40 binding and subsequent Fc␥R activation (Langer et al., 2005). In this study, we aimed to investigate whether platelet CD40L expression achieved with physiological stimuli could support CD40L mAb pro-aggregatory effects by analyzing the influence of CD40L mAb clone 5c8, one of the clones used in clinical trials which cross-reacts with porcine CD40L (Lee et al., 2000), on macroscopic swine platelet aggregation in vitro. 2. Materials and methods All procedures were approved by the Ethics Committee on Animal Research of the Hospital Universitari Vall d’Hebron and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.1. Materials and antibodies Purified CD40L mAb clone 5c8 was produced by Bio Express Inc. (West Lebanon, NH). Mouse IgG2a isotype control antibody was from BD Biosciences Pharmingen (San Diego, CA), and CD32 mAb clone AT10 from Serotec Ltd. (Oxford, UK). ADP was purchased from Chrono-Log Corporation (Havertown, PA). Lamifiban was kindly provided by F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Human thrombin, ASA, fibrinogen, and all other products were purchased from Sigma (Saint Louis, MO). 2.2. Platelet-rich plasma (PRP) samples and platelet isolation Systemic venous blood was obtained from anesthetized (10 mg/kg IV thiopental) open-chest pigs. After discarding the 1 Biogen says it has stopped ongoing trials of anti-CD40L monoclonal antibody. Biogen Inc. Press Release, 2 November 1999. 2 IDEC Pharmaceuticals announces a clinical hold on ongoing clinical trials of its IDEC-131 antibody. IDEC Pharmaceuticals Press Release, 10 June 2002.

first 5 ml, 20 ml of blood was collected through a catheter inserted in the femoral vein in a syringe containing 2 ml of 3.8% citrate anticoagulant, and immediately centrifuged at 140 × g for 15 min at room temperature to obtain PRP. Platelet-poor plasma (PPP) was obtained by centrifugation of the remaining cell pellet at 1500 × g for 15 min. For platelet isolation, PRP was centrifuged at 900 × g for 10 min, and the platelet pellet was washed at 700 × g for 10 min with modified HEPES/Tyrode’s buffer (in mM: 137 NaCl, 2.8 KCl, 1 MgCl2 , 12 NaHCO3 , 0.4 Na2 HPO4 , 5.5 glucose, 10 HEPES, 0.35% BSA, pH 7.4). Platelets were finally resuspended in this buffer and counted with a LH 750 Coulter cell counter. 2.3. CD40L expression CD40L mAb clone 5c8 cross-reacts with porcine CD40L (Lee et al., 2000). mAb titering for immunofluorescence studies was performed with thrombin-activated pig platelets. Washed platelets were diluted to 4 × 1010 l−1 with HEPES/Tyrode’s buffer supplemented with 1 mM CaCl2 and incubated for 10 min at 37 ◦ C with 0.1 U/ml thrombin (final concentration) or saline. P-selectin and CD40L surface expression in platelets were determined by immunofluorescence and flow cytometry. The KO 2.9 anti-P-selectin monoclonal antibody (culture supernatant) (Massaguer et al., 2000) and the purified CD40L mAb clone 5c8 (0.1–100 ␮g/ml) were used for P-selectin and CD40L detection, respectively, and F(ab )2 fragments of a goat anti-mouse FITCconjugated antibody (Caltag Laboratories, California) were used as secondary antibody. The platelet population was analyzed in a FACScaslibur flow cytometer (Becton Dickinson Inc., San Jose, CA) calibrated weekly for fluorescence and light scatter using fluorescent beads. Platelets were identified on the basis of forward and sideward scatter parameters in the logarithmic mode. For each sample 20,000 platelets were collected. Data were analyzed with CELLQuestTM software (Becton Dickinson). The optimal titer obtained for CD40L mAb clone 5c8 was 5–10 ␮g/ml (n = 3), similar as described for human CD40L. 2.4. Aggregometry and treatments Optical aggregometry was performed in a four-channel 570 SV aggregometer (Chrono-log Corporation) at 1000 rpm and 37 ◦ C. Aggregation was induced in PRP (uncorrected for platelet count) by addition of 2.5–20 ␮M ADP and measured as the maximal increase in light transmittance as a percentage of that observed with PPP. PRP samples were stirred for 1–5 min at 37 ◦ C with PBS, IgG2a , CD40L mAb clone 5c8 or its F(ab )2 fragments before addition of ADP. Lamifiban was preincubated in the same conditions for 1 min prior to ADP addition. EDTA (4 mM, final concentration) was added at the time of ADPinduced maximal aggregation. For acetylsalicylic acid (ASA) treatment, PRP was preincubated unstirred with 100 ␮g/ml ASA (in 10 mM HEPES, 150 mM NaCl, pH 7.4) for 10 min at room temperature before aggregation experiments. For aggregometry studies, washed platelets were resuspended at a final concentration of 2 × 1011 l−1 in modified HEPES/Tyrode’s buffer. Ca2+ (2 mM) and porcine fibrinogen (0.5 mg/ml) were added to sam-

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ples previously to ADP or to the different treatments, which were performed in an analogous manner as for PRP. Modified HEPES/Tyrode’s buffer was used instead of PPP for normalization. All experiments were performed with PRP or washed platelets from blood obtained from at least three different animals and representative tracings are shown in figures. 2.5. Production of F(ab )2 F(ab )2 fragments of CD40L mAb clone 5c8 were produced using the ImmunoPureTM F(ab )2 preparation kit from Pierce (Rockford, IL) according to the manufacturer’s instructions. Integrity and purity of the fragments were assessed by polyacrylamide gel electrophoresis (SDS-PAGE) under both reducing and non-reducing conditions. Functionality of F(ab )2 fragments was assessed by immunofluorescence and flow cytometry with thrombin-activated platelets. 2.6. Statistical analysis Dose-effect of CD40L mAb on absorbance measurements of aggregation curves 9 min after ADP addition were analyzed by one-way analysis of variance (ANOVA) followed by Dunett test by using SPSS software. P values <0.05 were considered significant. All values are expressed as mean ± S.E.M.

Fig. 1. CD40L mAb stabilizes ADP-induced platelet aggregates in PRP. (A) Effect of PBS, 100 ␮g/ml CD40L mAb or 100 ␮g/ml IgG2a on 10 ␮M ADPinduced aggregation. (B) Absorbance values 1.5 min and 9 min after 20 ␮M ADP addition in samples pretreated with 100 ␮g/ml irrelevant IgG2a or 1–100 ␮g/ml CD40L mAb (n = 5). * P < 0.05 respect to IgG2a -treated samples.

3. Results 3.1. Effect of CD40L mAb clone 5c8 on ADP-induced platelet aggregation In untreated swine PRP samples, ADP-induced aggregation was reversible with time even for ADP concentrations provoking maximal effect (Fig. 1A). CD40L mAb clone 5c8 did not induce platelet aggregation per se at any concentration tested (1–100 ␮g/ml, not shown). However, preincubation with the mAb inhibited spontaneous deaggregation observed in control ADP-stimulated samples, without significantly affecting maximal amplitude or slope values of aggregation curves (Fig. 1A). The stabilizing effect was statistically significant when using CD40L mAb concentrations higher than 25 ␮g/ml on aggregation induced by 20 ␮M ADP (Fig. 1B). These effects were not reproduced by an irrelevant isotype-matched immunoglobulin. 3.2. Effect of GP IIb/IIIa blockade and Ca2+ chelation on stabilization of platelet aggregates by CD40L mAb Previous GP IIb/IIIa blockade with 1 ␮g/ml lamifiban completely prevented ADP-induced platelet aggregation. In the absence of primary aggregation, CD40L mAb had no effect on ADP-treated PRP. In the presence of lamifiban concentrations inducing only partial GP IIb/IIIa blockade, the stabilizing effect of CD40L mAb on the residual ADPinduced aggregation was maintained and scarcely affected as compared to primary aggregation (Fig. 2A). Since ADPinduced primary aggregation was required for CD40L mAb

effects, GP IIb/IIIa blockade was performed after maximal ADP-induced aggregation and prior to adding CD40L mAb. Lamifiban added after maximal ADP-induced aggregation neither significantly modified deaggregation rate in control samples nor inhibited the stabilizing effect of CD40L mAb (Fig. 2B). To further explore the mechanisms involved in the stabilizing effect of CD40L mAb on platelet aggregates, we investigated its dependence on extracellular [Ca2+ ]. It is known that although GP IIb/IIIa-mediated platelet aggregation is Ca2+ -dependent, once release reaction has occurred and secondary aggregation has been produced, Ca2+ chelation is not able to induce platelet deaggregation (Cattaneo et al., 1987). Addition of EDTA at the moment of maximal ADP-induced aggregation in control samples increased the initial deaggregation rate. However, Ca2+ chelation with EDTA did not affect the stabilizing effect of CD40L mAb on platelet aggregates formed in response to ADP (Fig. 2C). 3.3. Involvement of thromboxane A2 synthesis in platelet aggregate stabilization by CD40L mAb Thromboxane A2 (TXA2 ) has been described as an autocrine mediator involved in platelet secondary aggregation (Meyers et al., 1979). In coherence with this, the reversible ADP-induced aggregation in citrated swine PRP was not sensitive to ASA. Pretreatment with ASA, however, abolished the stabilizing effect of CD40L mAb on platelet aggregates formed in response to ADP (Fig. 2D).

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Fig. 2. The stabilizing effect of CD40L mAb on ADP-induced aggregation was neither mediated by GP IIb/IIIa nor dependent on Ca2+ , but was prevented by ASA. (A) PRP was preincubated with lamifiban and 100 ␮g/ml CD40L mAb or PBS for 1 min prior to 20 ␮M ADP addition. (B) PBS or lamifiban were added after maximal 20 ␮M ADP-induced aggregation. After 1 min incubation, 100 ␮g/ml CD40L mAb or PBS were added. (C) PRP was preincubated with PBS or 100 ␮g/ml CD40L mAb. At maximal 20 ␮M ADP-induced aggregation, PBS or EDTA was added. (D) Samples pretreated or not with ASA were incubated with PBS or 100 ␮g/ml CD40L mAb before 20 ␮M ADP addition.

3.4. Involvement of Fc domain of CD40L mAb in platelet aggregate stabilization As shown in Fig. 3A, F(ab )2 fragments of CD40L mAb did not stabilize platelet aggregates formed in response to ADP in swine citrated PRP. However, they blocked the stabilizing effect of the native mAb (Fig. 3B). The effective F(ab )2 /IgG ratio needed to inhibit the stabilizing effect of CD40L mAb varied among PRP from different animals and was dependent on the magnitude of primary ADP-induced aggregation and on the potency of the stabilizing effect of the mAb, possibly indicating differences in CD40L expression in response to ADP among different PRP samples. 3.5. Effect of CD40L mAb on ADP-induced aggregation of washed platelets CD40L mAb did not induce aggregation of washed platelets per se. However, the magnitude and the potency of the effects

of CD40L mAb on ADP-induced aggregation were higher on washed platelets than on PRP (Fig. 4). Not only stabilization of platelet aggregates, but also an increase in maximal amplitude and slope of the aggregation curves was observed, specially at high concentrations. Although in some samples an increase of aggregation was also produced by 100 ␮g/ml irrelevant IgG2a , the potentiating effects of 100 ␮g/ml CD40L mAb were usually observed in the absence of effect of the isotype-matched IgG (Fig. 4A). CD40L mAb at 10 ␮g/ml also increased platelet aggregation, although less potently and with a prolonged lag time with respect to the 100 ␮g/ml concentration (Fig. 4B). The effects of CD40L mAb could be partially reversed by preincubation with ASA, specially for the lowest mAb concentrations (Fig. 4A and B) and were totally inhibited by GP IIb/IIIa blockade with lamifiban previous to ADP addition (not shown). As observed in PRP, F(ab )2 fragments of 5c8 mAb did not affect aggregation of ADP-stimulated washed platelets, but reversed the pro-aggregatory effects of the mAb (Fig. 4C and D).

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Fig. 3. Involvement of Fc domain of CD40L mAb in its pro-aggregatory effects. (A) PRP was preincubated with PBS, CD40L mAb (50 ␮g/ml) or its F(ab )2 fragments (80 ␮g/ml) prior to 20 ␮M ADP addition. (B) PRP was preincubated with PBS or 10 ␮g/ml CD40L mAb, in the presence or the absence of 80 ␮g/ml F(ab )2 fragments, prior to 20 ␮M ADP addition.

Fig. 4. CD40L mAb enhanced ADP-induced aggregation in washed platelets. (A) Platelets were preincubated with PBS, 100 ␮g/ml CD40L mAb or 100 ␮g/ml IgG2a prior to 20 ␮M ADP addition. (B) Treatment as in (A) but using 10 ␮g/ml CD40L mAb or IgG2a . (C) Platelets were pretreated with PBS, 80 ␮g/ml F(ab )2 fragments or 50 ␮g/ml CD40L mAb before 20 ␮M ADP addition. (D) Platelets were preincubated with PBS or 10 ␮g/ml CD40L mAb, in the presence or the absence of 40 ␮g/ml F(ab )2 fragments, prior to 20 ␮M ADP addition.

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4. Discussion Since the description of thromboembolic complications in nonhuman primates and humans receiving CD40L mAb immunotherapy, different authors have speculated with the possibility that direct platelet activating effects of the mAb, including Fc␥R activation, could be involved (Boumpas et al., 2003; Kuwana et al., 2004; Koyama et al., 2004; Ferrant et al., 2004; Roth et al., 2004). The first data in favor of this possibility were described by Langer et al. (2005), who showed that preformed immune complexes between sCD40L and CD40L mAb activated human platelets by a mechanism dependent on CD40 binding followed by Fc␥R activation. The present study addressed the effects of CD40L mAb on ADP-induced aggregation of swine platelets. The mAb clone used was 5c8, which produced thromboembolic complications in patients enrolled in clinical trials for treatment of autoimmune diseases (Boumpas et al., 2003) and in nonhuman primates subjected to allotransplantation studies (Kawai et al., 2000). Results obtained show that this CD40L mAb clone has specific pro-aggregatory effects both on citrated PRP and on washed platelets, effects that can be attained by CD40L expression levels achieved with physiological or pathophysiological platelet activation. These effects were mediated by mAb Fc domain-dependent interactions subsequent to CD40L binding and were prevented by ASA. These findings may help to explain the increased rate of thromboembolic events associated to treatment with CD40L mAb and to define strategies for their prevention. CD40L mAb did not elicit any aggregatory stimulus per se in citrated PRP, as expected due to the lack of CD40L expression on the surface of resting platelets, but produced a specific dosedependent stabilizing effect on aggregates previously formed in response to ADP. Not only ADP-induced CD40L expression but also ADP-aggregatory trigger stimulus was necessary for the effects of the mAb, as shown by the fact that GP IIb/IIIa blockade, which has been shown not to inhibit CD40L membrane expression (Furman et al., 2004), completely prevented any proaggregatory effect of CD40L mAb. Once platelet aggregation had occurred in response to ADP, the stabilizing effect of CD40L mAb did not involve new fibrinogen–GP IIb/IIIa interactions, since it was not altered by lamifiban and was not affected by Ca2+ chelation with EDTA. The lack of sensitivity of aggregate stabilization to Ca2+ chelation probably indicates the occurrence of new interactions that render platelet aggregates irreversible. These interactions have been proposed to be the consequence of platelet release reaction and to mediate secondary aggregation (Cattaneo et al., 1987), and their occurrence in our experiments is supported by the observation that COX-1 inhibition with ASA, which has been described not to affect ADP-induced sCD40L production (Nannizzi-Alaimo et al., 2003), completely abrogated the effect of CD40L mAb on ADP-induced aggregation. Activation of TXA2 synthesis appears thus to be a crucial step in the mechanism of aggregate stabilization induced by CD40L mAb. In isolated washed platelets amplification of ADP-induced aggregation was observed in the presence of CD40L mAb. The pro-aggregatory effects were seen at lower mAb concentrations

and were also totally dependent on GP IIb/IIIa-mediated initial aggregation by ADP, discarding agglutination as the mechanism involved. Blockade of TXA2 synthesis inhibited mAb effects in washed platelets, although only partially. The differences on the effects of CD40L mAb in citrated PRP and washed platelets could be explained, at least in part, by the different Ca2+ concentration in both platelet preparations. These differences probably influence the TXA2 response and the release reaction generated in each conditions (Zurbano et al., 1998), powerful enough to induce additional GP IIb/IIIa activation in swine washed platelets but not in citrated PRP. If this is the case, it is possible that studies performed in citrated PRP underestimate the effects of CD40L mAb on swine platelet aggregation. Different mAbs have been described to induce macroscopic platelet aggregation. Mechanisms involved include mAb Fab domain-mediated activation of the target protein by conformational changes or by clusterization, and mAb Fc domain interaction with platelet Fc␥R or with complement subsequent to Fab domain-dependent binding (Horsewood et al., 1991). Oligomeric antigens that allow interaction with more than one mAb simultaneously are susceptible to clusterization. This is the case of CD40L, which forms a homotrimeric structure (Hsu et al., 1997). Both CD40L mAb clone 5c8 and its bivalent F(ab )2 fragments have been shown to induce clusterization of CD40L on the surface of Jurkat cells (Ferrant et al., 2002). However, F(ab )2 fragments of CD40L mAb clone 5c8 had no effect on swine platelet aggregation, indicating that CD40L clusterization, including that of GP IIb/IIIa-sCD40L bound form, has no effect per se on swine platelet aggregation, and that Fc- instead of Fab-mediated interactions are responsible for the proaggregating effects of the mAb. Our results also indicate that these Fc domain-dependent interactions require initial binding of the mAb to its target protein CD40L, since its F(ab )2 fragments blocked the pro-aggregatory effects of the intact IgG. Most Fc-dependent effects of mAbs on platelets are mediated by Fc␥R. Activation of Fc␥R in human platelets requires clusterization after IgG binding and induces initial phospholipase C activation followed by cytosolic Ca2+ increase, phospholipase A2 activation, and synthesis of TXA2 (Anderson and Anderson, 1990), that is considered the main mediator of Fc␥R-induced platelet activation (Kang et al., 1993). The results of this study, showing that CD40L mAb pro-aggregatory effects are dependent on its Fc domain and on TXA2 synthesis, could thus be consistent with a CD40L mAb-mediated activation of Fc␥R. It is likely that the spatial arrangement of Fc domains resulting from the high-ordered assemblies of complexes that 5c8 CD40L mAb is expected to form with CD40L on the platelet surface (Ferrant et al., 2002; Karpusas et al., 2001) are able to clusterize platelet Fc␥R. On the other hand, the dependence of CD40L effects on ADP-induced primary aggregation suggests that this agonist is required as a cofactor for Fc␥R-mediated platelet activation, as it has been previously described (Gratacap et al., 2000). Although antibody Fc-induced platelet activation can also occur by a complement-dependent mechanism (Tomiyama et al., 1992), CD40L mAb effects on ADP-induced aggregation are maintained and even amplified in experiments with washed platelets, indicating that at least under these experimental con-

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ditions Fc␥R activation by CD40L mAb clone 5c8 is likely to occur. Human platelets express exclusively the Fc␥RIIA (CD32) isoform (Tomiyama et al., 1992), but the Fc␥R subtype expressed in swine platelets is unknown. Since CD32 mAb clone AT10 blocks porcine Fc␥RII (Balmelli et al., 2005), its influence on the effects of aggregated IgG on swine platelets was assessed. CD32 mAb clone AT10 was not able to block platelet aggregation induced by human aggregated IgG neither in porcine PRP nor in porcine washed platelets (not shown), which precluded studies on the effects of CD40L mAb. Supporting the lack of efficacy of AT10 mAb on Fc␥R blockade on swine platelets, a recent report has shown that Fc␥RII expression in swine is limited to the inhibitory ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc␥RIIB subclass (Qiao et al., 2006). This leaves the possibility open that an IgG2a -activatable ITAM (immunoreceptor tyrosine-based activation motif)-associated Fc␥R could be involved in the effects of 5c8 mAb on swine platelets. Despite differences existing between pig and human platelets (Zurbano et al., 1998), our findings on the pro-aggregatory effects of CD40L mAb clone 5c8 could help to explain the higher incidence of thromboembolic complications associated with its administration in clinical trials. The results obtained regarding the mechanisms involved in the pro-aggregatory effects of the mAb could also help to develop therapeutic strategies to avoid its undesired effects. In this regard, the use of functional F(ab )2 fragments of CD40L blocking mAbs could be a therapeutic alternative to avoid the IgG-associated thromboembolic effects. However, the efficacy of CD40L mAbs seems not to be mediated only by blockade of signaling through CD40 but also by Fc␥R-dependent mechanisms in immune cells. These Fc effector mechanisms of 5c8 CD40L mAb are required for inhibition of immune responses in transplanted mice, although not in experimental models of systemic autoimmunity (Ferrant et al., 2004). On the other hand, anti-platelet therapy targeted to TXA2 synthesis could be an effective approach for reducing thromboembolic events in patients receiving CD40L immunotherapy. In agreement with this, studies evaluating several antithrombotic strategies against CD40L mAb-associated thrombophilia in nonhuman primate models of allograft transplantation have suggested that inhibition of aggregation with ketorolac tromethamine, an agent that inhibits collagen- or arachidonic acid-induced platelet aggregation (Conrad et al., 1988), is more effective than anticoagulation with heparin in preventing thromboembolic events (Koyama et al., 2004; Kawai et al., 2000). The relevance of the present observations to clinical situations cannot be directly established, and would require the demonstration of the effects of CD40L mAb on human platelets. However, due to the low binding affinity of Fc␥RIIA for gamma 2 IgG (Parren et al., 1992), it is possible that a pro-aggregatory effect of CD40L mAb clone 5c8 could not be observed on human washed platelets. Experiments with the IgG1 humanized form of 5c8 mAb, which has been administered in clinical trials, would be needed to assess the relevance of the present findings in humans.

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Acknowledgments This work was partially supported by a grant from the Sociedad Espa˜nola de Cardiolog´ıa (2005), and by Red de Enfermedades Cardiovasculares (RECAVA). We thank Dr. Rosa Ma Lid´on for her help with aggregometry studies.

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