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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From Molecular Immunology, Robert
Koch-Institut, Berlin, Germany; and Rudolf-Buchheim-Institut
für Pharmakologie, Justus-Liebig-Universität,
Gießen, Germany.
Recently, we have demonstrated that human platelets carry preformed
CD40 ligand (CD154) molecules, which rapidly appear on the platelet
surface following stimulation by thrombin. Once on the surface,
platelet CD154 induces an inflammatory reaction of CD40-bearing
endothelial cells. This study shows that strong platelet agonists other
than thrombin also lead to the expression of CD154 on the platelet
surface. At the same time, several lines of evidence are presented that
together indicate that thrombotic events in the vasculature are
generally accompanied by activation of the inflammatory potential of
platelet CD154. This study also reports the constitutive expression of
CD40, the receptor for CD154, on platelets. The binding of CD154 to
coexpressed CD40 in the platelet aggregate leads within minutes to
hours to the cleavage of membrane-bound surface CD154 and the release
of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in
contrast to transmembrane CD154, can no longer induce an inflammatory
reaction of endothelial cells. These findings indicate that the
interaction of platelet CD154 with CD40 on neighboring cells is
temporally limited to prevent an uncontrolled inflammation at the site
of thrombus formation. Thus, similar to the very tight regulation of
the CD154-CD40 interaction in the immune system, an effective mechanism
controls the inflammatory potential of platelet CD154 in the vascular system.
(Blood. 2001;98:1047-1054) CD40, a 48-kd transmembrane protein homologous to
the tumor necrosis factor (TNF) receptor, was first described as a
constitutive cell-surface antigen on B cells.1,2 The CD40
ligand (CD40L, TRAP, gp39, CD154) is a 33-kd transmembrane homologue of
TNF- More recently, CD40 was identified on vascular endothelium and was
shown to mediate signals leading to de novo expression of adhesion
molecules and release of proinflammatory cytokines and chemokines by
endothelial cells.9-11 In these original experiments, it
was assumed that only activated T cells bearing CD154 can activate endothelial cells via CD40.9-11 This paradigm changed with
our recent finding that platelets carry preformed CD154 molecules and
rapidly express them on the cell surface after activation by
thrombin.12 Furthermore, we could demonstrate that the
interaction of CD154 on activated platelets with CD40 on endothelial
cells elicits an inflammatory reaction of the endothelium, which is characterized by expression of the adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 and the secretion of the chemokines IL-8 and MCP-1.12 These findings allowed the conclusion
that also in the vascular system, the action of platelet CD154
resembles the biologic effects of TNF- Although our work demonstrated the role of thrombin in the induction of
an "inflammatory platelet surface," the presented data did not
answer the question whether the expression of CD154 on the cell surface
is a general phenomenon accompanying platelet activation. In the
experiments presented here, we demonstrate that other strong platelet
agonists also induce the expression of CD154 on the platelet surface.
Given the abundance of platelets, the identification of CD154 on
activated platelets revealed a formidable inflammatory potential in the
vascular system. It remained unclear, however, which mechanisms restrict the action of CD154 in the circulation and thus prevent an
uncontrolled inflammation of the vasculature. Pertinent to this
question, we also report here the constitutive expression of CD40, the
receptor for CD154, on the surface of resting platelets. We describe
how the interaction of CD154 on activated platelets with coexpressed
CD40 temporally limits the duration of CD154 expression and thus
effectively curtails the inflammatory action of platelet CD154.
Isolation and activation of platelets
Flow cytometry
Immunoprecipitation and Western blotting Peripheral blood mononuclear cells (PBMCs) were isolated from human blood of healthy donors by centrifugation over a Ficoll density gradient (Biochrom, Berlin, Germany). Platelets (1 × 109) and PBMCs (2 × 107) were lysed in NP-40 buffer (150 mM NaCl, 1 mM EDTA, 1% [vol/vol] Nonidet P-40, 50 mM Tris/HCl, pH 8.0) containing the protease inhibitors phenylmethylsulfonyl fluoride, leupeptin A, pepstatin, and aprotinin (all from Boehringer Mannheim). Samples were precleared using Sepharose-4B beads (Pharmacia), reacted with glycine, and immunoprecipitated using mAb TRAP216 or mAb G28-5 directly coupled to Sepharose-4B beads. To analyze the binding of sCD154 to CD40, supernatants of unstimulated and activated platelets (0.2 U/mL thrombin for 1 to 24 hours) were precleared 2 times with Protein A beads (Pharmacia), and sCD154 was immunoprecipitated using a chimeric CD40-Ig fusion protein17 (generously provided by Dr Kurrle, Behring-Werke, Marburg, Germany) and Protein A beads. Immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel) under reducing conditions, blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), and stained with specific antisera using the Western Light Plus-Kit and CDP-Star as substrate (Tropix, Bedford, MA). Generation of the anti-CD154 serum has been described before.18 The anti-CD40 serum was obtained by immunizing rabbits with the recombinant extracellular domain of human CD40.Determination of sCD154 release For the determination of sCD154 levels, samples of activated platelets were routinely centrifuged at 2000g for 5 minutes, after experiments with additional ultracentrifugation (4 hours at 250 000g) excluded any influence on the values obtained. sCD154 levels in the supernatants were determined using a specific enzyme-linked immunosorbent assay (ELISA).16 Briefly, 96-well plates (Maxisorb; Nunc, Roskilde, Denmark) were preincubated with mAb LL2 (kindly provided by Dr Brière, Schering-Plough, Dardilly, France), the wells were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20, and the samples were applied after dilution in Tyrodes/HEPES buffer (washed platelets) or PBS/1% BSA (PRP). Following incubation with peroxidase (POD)-labeled mAb TRAP1, the assay was developed with 3,3',5,5' tetramethylbenzidine. The signal obtained with 1 ng/mL of a soluble chimeric recombinant mIL-4/hCD154 reagent (kindly provided by Dr Kurrle, Behring-Werke) was arbitrarily defined as 1 U/mL of sCD154.Analysis of platelet phosphoproteins Platelets were prepared as described19 except that the buffers were phosphate free. Platelets (5 × 108/mL) were labeled with 0.5 mCi/mL 32P-orthophosphate (DuPont-NEN, Bad Homburg, Germany) and stimulated, and the phosphoproteins were analyzed as described.20Aggregation assay Aggregation assays were performed in a Kontron Uvikon 930 photometer (Kontron, Watford, United Kingdom) equipped with a stirring device, a heated cuvette holder, and time-driven recording of transmission values. Platelets were adjusted to 2 × 108 in plasma or Tyrodes/HEPES buffer, 0.3% BSA, and 2 mM CaCl2; both preparations gave identical results. Collagen (Nycomed, Munich, Germany), epinephrine, platelet-activating factor (PAF), and adenosine 5'-diphosphate (ADP) (all from Sigma) were used as agonists. Stimulation of platelets in buffered solution with ADP was performed in the presence of 0.5 mg/mL fibrinogen and 0.2 U/mL apyrase (both from Sigma). After the measurement of aggregation (10 minutes), the samples were further incubated for 50 minutes at 37°C without stirring to achieve optimal release of sCD154, then centrifuged at 2000g for 5 minutes, and the concentration of sCD154 was determined in the platelet-free supernatant. In some experiments, anti-CD40 mAb G28-5 or control mAb MOPC-21 was added to the platelet suspension at various concentrations (1 to 10 µg/mL) 20 minutes before or together with the above-mentioned agonists. In some experiments, goat anti-mouse Ig serum (Nordic Immunological Lab, Netherlands) was added at 1:150 dilution to induce cross-linking of mAb G28-5 on the platelet surface. Finally, all types of experiments were performed with platelets isolated by a different procedure19 and analyzed in an aggregometer (Platelet Aggregation Profiler, Model PAP-4; Bio/Data, Horsham, PA).Separation of blood components for sCD154 release Whole blood from healthy donors was collected into 4 mM EDTA, and cell counts were determined with a hemocytometer. Centrifugation at 150g for 20 minutes yielded PRP. Platelet-poor plasma was generated by centrifugation of PRP at 1300g, followed by centrifugation at 4000g, for 15 minutes each. Nucleated cells were separated from whole blood by Ficoll-Hypaque centrifugation. The interphase containing lymphocytes and monocytes and the pellet containing granulocytes and erythrocytes were collected separately and washed 3 times at 150g to remove contaminating platelets. Cells were resuspended in the original concentrations in platelet-poor plasma. Samples of each cell preparation were incubated at 37°C, and clotting was induced by the addition of 20 mM calcium or 0.2 U/mL human thrombin. After 1 hour, cell-free supernatants were collected and analyzed for sCD154 by ELISA.Isolation and activation of endothelial cells HUVECs were isolated by collagenase treatment.21 Cells were cultured in endothelial cell growth medium with endothelial cell growth supplement (PromoCell, Heidelberg, Germany). HUVECs were used after the second and third passage and were cocultured with supernatants of activated platelets (0.2 U/mL thrombin for 1 hour). Furthermore, HUVECs were incubated with CD154 myeloma transfectant P3xTB.A74 at a ratio of 1:5, or reacted with sCD154 present in serum-free culture supernatant of the myeloma transfectant P3xTB.A7,4 or a combination of both CD154 transfectant and sCD154. In the sCD154-containing supernatants, any residual membrane fragments were removed by centrifugation at 1000g for 30 minutes and at 250 000g for 4 hours. After coculture, HUVECs were dislodged with PBS containing 5 mM EDTA and analyzed by flow cytometry.Immunohistology Frozen sections (8 µm) of fresh postmortem human venous thrombi were fixed with acetone for 10 minutes, incubated with the mAb TRAP1, G28-5, 142.11, WAPS 12.2 (anti-P-selectin; American Type Culture Collection), or isotype control mAb MOPC-21, and stained using the alkaline phosphatase antialkaline phosphatase (APAAP) technique (Dako, Glostrup, Denmark) with new fuchsin as chromogen. Counterstaining was performed with Mayer hematoxylin (Sigma). Artificial thrombi were generated in parallel by activating PRP with 0.2 U/mL human thrombin, immediately followed by vortexing. After incubation for up to 24 hours at 37°C, the artificial thrombi were snap-frozen, and cryosections were stained for CD154, gpIb, P-selectin, or CD40 using the tyramide signal amplification system (NEN Life Science Products, Boston, MA). AEC (3-amino-9-ethylcarbazole) was used as chromogen.
CD40 is constitutively expressed on the surface of platelets In the course of our studies on platelet activation, we noticed that platelets express substantial amounts of CD40 on their cell surface. Figure 1A shows flow cytometry profiles of unstimulated platelets stained with the CD40-specific mAb G28-5 and, for comparison, with mAb 142.11, which recognizes the platelet marker gpIb. To assess the levels of CD40 expression on activated platelets, we stimulated platelets with thrombin for up to 4 hours and analyzed them by flow cytometry using expression of CD154 as a positive control. As demonstrated earlier,12 CD154 is expressed on the platelet surface within seconds of activation, after which its levels gradually decline (Figure 1B). In contrast to this activation-dependent expression of CD154, no significant changes in CD40 levels were noticed in a series of experiments (Figure 1B). Similar results for CD40 expression on resting or activated platelets were obtained using mAb 89, another CD40-specific reagent (results not shown). To compare the CD40 molecule expressed on platelets with CD40 on B cells and monocytes, we immunoprecipitated CD40 from lysates of platelets or PBMCs and subjected them to Western blotting using a CD40-specific antiserum. An identical protein band of the expected size of 48 kd was detected in both samples (Figure 1C). Taken together, these data demonstrate that human platelets constitutively express CD40.
Interaction of CD154 with CD40 on the cell surface of activated platelets leads to cleavage of CD154 On T cells, the interaction of CD154 with CD40 on B cells or dendritic cells induces a rapid down-regulation of membrane-bound CD154,22,23 and this process is accompanied by the release of a soluble 18-kd trimeric form of CD154 (sCD15423), which can be measured using a specific ELISA.16 To determine the mechanism of the observed down-regulation of CD154 following its initial expression on platelets (Figure 1B), we measured whether sCD154 is released into the supernatant. Platelets were incubated with 0.2 U/mL thrombin for up to 6 hours, and the amount of sCD154 detectable in the supernatants was determined. A substantial release of sCD154 (up to 50 U/mL of sCD154/2 × 108 platelets in 1 mL) could be measured within 1 to 2 hours of platelet activation, after which the levels of detectable sCD154 gradually declined (Figure 2A).
When the same experiment was performed in the presence of anti-CD40 mAb G28-5, the release of sCD154 was essentially abrogated, whereas the presence of a control antibody was without effect (Figure 2A). These experiments indicate that the interaction of CD154 with CD40 triggers the release of CD154 from the cell surface, probably by the action of a membrane-bound protease. The release of CD154 from the platelet surface is the sole mechanism for the down-regulation of CD154 The presence of membrane-bound CD154 and sCD154 in platelets and supernatants was determined in the same experimental setup as used for the measurement of sCD154 release, except that platelet aggregation was induced by stirring. With unstimulated platelets, immunoprecipitation followed by Western blotting revealed the presence of transmembrane CD154 (33 kd and 28 kd) in the platelet lysate and gave no signal for the 18-kd sCD154 in the supernatant (Figure 2B). Upon activation of platelets by 0.2 U thrombin for 1 hour, the signal for the transmembrane form of CD154 almost fully disappeared and sCD154 became detectable in the supernatant (Figure 2B). The presence of blocking antibodies against CD40 (mAb G28-5) or against CD154 (mAb TRAP1) largely prevented the transformation of the membrane-form of CD154 to sCD154 after thrombin activation (Figure 2B and not shown). These results confirm the conclusions reached from the experiments measuring sCD154 in the supernatants. The complete disappearance of the transmembrane form of CD154 in activated platelets indicates that the CD154 molecule is not partly internalized, as observed in T cells, but is fully released as sCD154 into the extracellular milieu. The released sCD154 can bind to CD40, as determined by immunoprecipitation with a recombinant soluble CD40-Ig reagent (Figure 2C). The amount of immunoprecipitable sCD154 in the platelet supernatants decreased over time with an approximate half-life of 6 hours (Figure 2C), probably because of the inherent instability of the trimeric sCD154 (see "Discussion").Agonists inducing sCD154 release In the next series of experiments, we asked whether platelet agonists other than thrombin lead to cell-surface CD154 expression and sCD154 release. With the exception of thrombin, these agonists require stirring of platelets to exert their activation potential, and some of them irreversibly aggregate platelets. The aggregated platelets cannot be analyzed by flow cytometry, and therefore we previously could not determine whether CD154 is expressed on the platelet surface following activation by these agents. The discovery of the sCD154 release phenomenon allowed us to examine this issue. To this end, platelets were activated in the standard aggregometry assay, and the levels of sCD154 in the sample supernatants were determined 1 hour later. Addition of epinephrine, which did not induce aggregation, or addition of PAF, which induced only a slight and reversible aggregation, did not lead to release of sCD154 above the background levels observed with unstimulated platelets (Figure 3). In clear contrast, the addition of collagen or thrombin to stirred platelets triggered strong aggregation and resulted in a substantial release of sCD154 (Figure 3). The response to ADP and ADP plus epinephrine showed substantial donor variability; in responding platelet preparations, a release of sCD154 could be observed (Figure 3). Although strong platelet aggregation in the experiments was usually accompanied by the release of sCD154, the 2 events appear not to be causally linked because thrombin activation of platelets without stirring gave no overt aggregation but nevertheless resulted in CD154 expression and substantial sCD154 release (Figures 1 and 2A). In summary, the aggregation experiments indirectly determined that not only thrombin, but also other strong platelet agonists, lead to the expression of CD154 on the platelet surface.
The central function of CD40 on the surface of platelets is the down-regulation of expressed CD154 Our experiments demonstrated that interaction of CD154 with CD40 is required for cleavage of CD154 (Figure 2). Thus, CD40 mediates a signal leading to the proteolytic cleavage of CD154. In a series of experiments, we sought to determine whether CD40 has additional effects on the function of platelets. Experiments designed to detect any blocking effects of mAb G28-5 on platelet aggregation triggered by various platelet agonists (for the setup of these experiments, see Figure 3) failed to reveal any effects (data not shown). In addition, we examined the phosphorylation pattern of 32P-labeled platelet proteins after incubation with mAb G28-5, which is known to induce protein phosphorylation in B cells,24 either in the presence or absence of optimal amounts of thrombin. Again, we could not observe any significant effects. These experiments led us to the conclusion that the interaction between CD154 and CD40, apart from inducing CD154 cleavage, does not participate in platelet activation and is not required for platelet aggregation.Platelets express CD154 in the physiologic blood-clotting reaction Our experiments predicted that aggregating platelets in clotting blood would release sCD154. To test this prediction, blood samples collected in EDTA were coagulated by the addition of calcium or thrombin, and sCD154 was measured in aggregate-free serum. Up to 110 U/mL sCD154 was found within 1 hour after induction of blood coagulation, whereas virtually no sCD154 was detected without addition of the agonists (Figure 4). Essentially the same amounts of sCD154 were released when PRP was prepared from the identical whole-blood samples and assayed in parallel (Figure 4). Only background sCD154 signals were obtained with platelet-poor plasma or with physiologic concentrations of PBMCs, erythrocytes, or granulocytes suspended in platelet-poor plasma (Figure 4). In addition to the clotting experiments, we immunostained cytocentrifuged PBMCs of healthy donors for the presence of intracellular or extracellular CD154 and obtained no signal (data not shown). Taken together, these data allow us to conclude that CD154 is expressed and later released by platelets in clotting blood and that platelets are the only source of sCD154 in this reaction. Inherently, these results also reveal that platelets are the predominant source of CD154 in the vascular system.
Soluble CD154 does not elicit an inflammatory response in endothelial cells We have previously demonstrated that the membrane-bound form of CD154 on platelets induces a significant inflammatory reaction of endothelial cells expressing CD40.12 Because sCD154 is capable of binding to CD40 (Figure 2C), we sought to determine whether sCD154 has similar biologic properties. To this end, HUVECs were cultured for 4 hours in medium alone, in supernatants from thrombin-activated platelets, or in supernatants from a CD154-expressing transfectant containing high amounts of sCD154 (1000 U/mL). In parallel, HUVECs were cocultured with a CD154 transfectant (Figure 5). As reported previously,12 the transmembrane form of CD154 potently induced the expression of E-selectin and VCAM-1 in HUVECs and up-regulated ICAM-1 (Figure 5). In contrast, supernatants containing sCD154, either alone (Figure 5) or in combination with suboptimal amounts of TNF- (not shown), did not exert any effects on the
expression of the adhesion molecules (Figure 5), and also failed to
induce the synthesis of IL-8 (not shown). Furthermore, coculture of the CD154 transfectant with HUVECs in the presence of 1000 U/mL sCD154 did
not modulate the biologic effects of membrane-bound CD154 (Figure 5),
suggesting a substantially lower affinity of sCD154 to CD40. Taken
together, these experiments demonstrate that the soluble form of CD154,
in contrast to the membrane-bound form, does not elicit an inflammatory
response of endothelial cells.
Intravital expression of CD154 and CD40 in human thrombi To assess the intravital expression of CD154 and CD40 in thrombi, we stained serial sections of human venous thrombi for CD154, CD40, platelet marker gpIb, and platelet activation marker P-selectin (Figure 6A). Nuclear staining revealed numerous leukocytes in the thrombus. The staining for gpIb clearly marked the area of platelet accumulation in the thrombus and coincided with the expression of P-selectin and CD40 (Figure 6A). In contrast, the number of CD154+ platelets was substantially lower. In many areas of the thrombus, CD154 expression was in fact restricted to single platelets (Figure 6A). To assess the expression of CD154 in a thrombus under controlled conditions, we generated artificial thrombi in vitro and analyzed sections from these thrombi at various time points. Sections obtained after 3 minutes gave significant signals when staining for CD154 (Figure 6B). Over the next few hours, the signals decreased substantially, and only relatively few CD154+ platelets were observed after 6 hours (Figure 6B). Thus, the histology results obtained with intravitally formed thrombi and also with in vitro generated thrombi support the concept of a quick initial expression and function of platelet CD154, followed by gradual inactivation through coexpressed CD40 in the later phases of thrombus formation.
We have shown earlier that human platelets express preformed CD154 on the cell surface within a very short period of time after activation by thrombin.12 Furthermore, we have demonstrated that platelet-bound CD154 induces an inflammatory reaction of endothelial cells via CD40.12 Our present work indicates that not only thrombin, but also other strong platelet agonists, can trigger platelets to express CD154 on the cell surface. Together with observations on CD154 expression in intravital thrombi and in vitro generated thrombi, our data indicate a link between the formation of a thrombotic plug and the immediate generation of a local inflammatory signal in the affected vessel in vivo. Another essential finding of our present study was the detection of CD40, the receptor for the CD154 molecule, on the surface of resting platelets. Quite conspicuously, platelets are thus capable of coexpressing CD154 and its receptor CD40. CD40 has been identified on several cell types in the immune and vascular systems, and its ligation by CD154 usually induces a marked activation of these cells.8 Although CD40 on platelets is indistinguishable from CD40 on other cells by a number of criteria, we found no evidence for a direct involvement of CD40 in the activation or aggregation of platelets. Apparently, binding of CD40 by its ligand is not essential for the hemostatic function of platelets, and this conclusion is indirectly supported by the absence of an overt bleeding disorder in "hyper-IgM" patients lacking functional CD154 molecules. We observed that the interaction of CD154 on stimulated platelets with the coexpressed CD40 receptor leads within minutes to hours to a proteolytic cleavage of CD154 and the release of sCD154, the soluble form of this molecule. The generated 18-kd sCD154 molecule is similar to sCD154 released from activated T cells16 and can also bind CD40 (Figure 2C). Interestingly, even high levels of sCD154 were not capable of inducing any inflammatory reaction of endothelial cells. These results fundamentally differ from the data with recombinant forms of soluble CD154 obtained by various investigators using endothelial cells in vitro9,10 or performing experiments in vivo.25 These stabilized forms of recombinant sCD154 clearly do not reflect the biology of natural sCD154. Natural sCD154, although capable of binding to CD40-Ig reagents, is relatively unstable. This conclusion reached from experiments using physical methods26 is supported by our finding of a relatively rapid decline of detectable sCD154 in our ELISA measurements, which is paralleled by the decrease of immunoprecipitable sCD154 (Figure 2). Taken together, our data indicate that cleavage of CD154 from the surface of activated platelets is a central mechanism limiting the inflammatory action of CD154 in the vascular system. We have attempted to use the generation of sCD154 as a diagnostic indicator of platelet activation in vivo. To date, plasma samples from patients undergoing balloon angioplasty and patients with active systemic lupus erythematosus (SLE) were examined without positive results, and only patients receiving extracorporeal circulation exhibited increased sCD154 levels in the plasma (Henn et al, unpublished data). Our findings contradict a recent report on the presence of substantial amounts of sCD154 in the plasma of SLE patients,27 but the discrepancy can easily be explained by the failure of the authors to remove platelets from the plasma samples. The difficulty in measuring sCD154 levels in platelet-free plasma in various pathologic conditions may result from a high dilution of generated sCD154 in the circulation, which potentially could be compensated for by a very sensitive detection system. In vitro, however, the measurement of sCD154 allows easy monitoring of platelet activation, and this could be exploited in transfusion-medicine research. Given our findings on the coexpression of CD154 and CD40 on platelets, the following scenario in vivo can be assumed. Once platelets are strongly activated in the vascular system, CD154 is rapidly expressed on the cell surface of aggregating platelets. As a result, platelet CD154 interacts with CD40 on the neighboring endothelial cells and at the same time with CD40 on monocytes trapped in the thrombus (Figure 6). In both endothelial cells and monocytes, this interaction probably immediately triggers an inflammatory response. These CD154 molecules, which are not engaged by CD40 on other cell types, bind to CD40 coexpressed in the platelet aggregate. All of these interactions result over time in the transformation of the proinflammatory form of transmembrane CD154 into the biologically inactive, soluble form of the molecule. The generation of sCD154 takes minutes to hours in vitro, and a similar time scale can be assumed in vivo. The scant expression of CD154 observed in intravitally formed thrombi as well as the time course of the decline of CD154 detection in experimentally formed thrombi (Figure 6) seem to support the conclusions reached. The tightly restricted action of platelet CD154 remarkably resembles the regulation of the molecule in activated T cells. There, CD154 is located intracellularly most of the time and, when translocated to the cell surface following a major histocompatibility complex-restricted stimulus, only briefly interacts with CD40 on partner cells (eg, B cells). On ligation to CD40, T-cell CD154 is either internalized22 or shed in a soluble form, with a major sCD154 product resulting from the cleavage between E112 and M113 (single-letter amino acid code).16 Despite the short duration of CD154 interaction with CD40 on cells of the immune system, the biologic effects of this interaction are impressive, one example being its pivotal role in the switch from IgM to IgG production by B cells.6 In fact, a very tight regulation of CD154 in the immune system is required because unduly prolonged exposure to a biologically effective form of CD154 leads to a massive inflammation or even triggers the development of lymphomas.28-30 The basic rules observed with T-cell CD154 are apparently also valid for platelet CD154. Thus, it can be assumed that the limited duration of CD154 expression on platelets prevents an excessive inflammation of the endothelium and of CD40+ cells in the vicinity of a hemostatic plug. When compared with its structural and functional homologue TNF- The mechanism limiting CD154 function on platelets may not suffice in chronic vascular diseases such as atherosclerosis. There, aggregation of platelets on altered endothelium and plaque rupture are ever-recurring events in the formation of progressive lesions. Atherosclerotic plaques contain aggregated platelets expressing CD15412 in the vicinity of CD40-bearing macrophages and fibroblasts, cell types capable of synthesizing proinflammatory mediators and matrix proteases when triggered via CD40.32-34 It thus seems likely that platelet CD154 contributes to the inflammatory component of atherosclerosis, in which CD40 was shown to play a prominent role.35 Apart from the postulated involvement in chronic vascular disease, it can be assumed that platelet CD154 also participates in the generation of local inflammatory signals in acute thrombotic events such as heart and brain infarction. The involvement of platelet CD154 in predominantly inflammatory conditions of the vascular system cannot be assessed as yet. Given the high incidence of thrombosis in inflamed vessels, it seems likely that platelet CD154 interacts with CD40 on endothelial cells also under these conditions.
We thank Dr Michael Gräfe for the culture of endothelial cells and Dr Ioannis Anagnostopoulos for providing us with specimens of venous thrombi.
Submitted June 22, 2000; accepted April 9, 2001.
Supported by Deutsche Forschungsgemeinschaft grant Kr827/10-2 (R.A.K.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Richard A. Kroczek, Molecular Immunology, Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany; e-mail: kroczek{at}rki.de.
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L. F. Brass, L. Zhu, and T. J. Stalker Novel Therapeutic Targets at the Platelet Vascular Interface Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): s43 - s50. [Abstract] [Full Text] [PDF]
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P. C. Ng, K. Li, T. F. Leung, R. P.O. Wong, G. Li, K. M. Chui, E. Wong, F. W.T. Cheng, and T. F. Fok Early Prediction of Sepsis-Induced Disseminated Intravascular Coagulation with Interleukin-10, Interleukin-6, and RANTES in Preterm Infants Clin. Chem., June 1, 2006; 52(6): 1181 - 1189. [Abstract] [Full Text] [PDF]
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S. Fujimi, M. P. MacConmara, A. A. Maung, Y. Zang, J. A. Mannick, J. A. Lederer, and P. H. Lapchak Platelet depletion in mice increases mortality after thermal injury Blood, June 1, 2006; 107(11): 4399 - 4406. [Abstract] [Full Text] [PDF]
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B. D. Elzey, J. F. Grant, H. W. Sinn, B. Nieswandt, T. J. Waldschmidt, and T. L. Ratliff Cooperation between platelet-derived CD154 and CD4+ T cells for enhanced germinal center formation J. Leukoc. Biol., July 1, 2005; 78(1): 80 - 84. [Abstract] [Full Text] [PDF]
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D. D. Wagner New Links Between Inflammation and Thrombosis Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1321 - 1324. [Abstract] [Full Text] [PDF]
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M. Ishikawa, T. Vowinkel, K. Y. Stokes, T. V. Arumugam, G. Yilmaz, A. Nanda, and D. N. Granger CD40/CD40 Ligand Signaling in Mouse Cerebral Microvasculature After Focal Ischemia/Reperfusion Circulation, April 5, 2005; 111(13): 1690 - 1696. [Abstract] [Full Text] [PDF]
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E. E. Gardiner, J. F. Arthur, M. L. Kahn, M. C. Berndt, and R. K. Andrews Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase Blood, December 1, 2004; 104(12): 3611 - 3617. [Abstract] [Full Text] [PDF]
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C. Heeschen, S. Dimmeler, C. W. Hamm, M. J. van den Brand, E. Boersma, A. M. Zeiher, M. L. Simoons, and the CAPTURE Study Investigators Soluble CD40 Ligand in Acute Coronary Syndromes N. Engl. J. Med., March 20, 2003; 348(12): 1104 - 1111. [Abstract] [Full Text] [PDF]
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L. Nannizzi-Alaimo, V. L. Alves, and D. R. Phillips Inhibitory Effects of Glycoprotein IIb/IIIa Antagonists and Aspirin on the Release of Soluble CD40 Ligand During Platelet Stimulation Circulation, March 4, 2003; 107(8): 1123 - 1128. [Abstract] [Full Text] [PDF]
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M. Kuwana, Y. Kawakami, and Y. Ikeda Suppression of autoreactive T-cell response to glycoprotein IIb/IIIa by blockade of CD40/CD154 interaction: implications for treatment of immune thrombocytopenic purpura Blood, January 15, 2003; 101(2): 621 - 623. [Abstract] [Full Text] [PDF]
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P. Andre, L. Nannizzi-Alaimo, S. K. Prasad, and D. R. Phillips Platelet-Derived CD40L: The Switch-Hitting Player of Cardiovascular Disease Circulation, August 20, 2002; 106(8): 896 - 899. [Full Text] [PDF]
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L. Nannizzi-Alaimo, M. H. Rubenstein, V. L. Alves, G. Y. Leong, D. R. Phillips, and H. K. Gold Cardiopulmonary Bypass Induces Release of Soluble CD40 Ligand Circulation, June 18, 2002; 105(24): 2849 - 2854. [Abstract] [Full Text] [PDF]
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J.-F. Viallard, A. Solanilla, B. Gauthier, C. Contin, J. Dechanet, C. Grosset, J.-F. Moreau, V. Praloran, P. Nurden, J.-L. Pellegrin, et al. Increased soluble and platelet-associated CD40 ligand in essential thrombocythemia and reactive thrombocytosis Blood, April 1, 2002; 99(7): 2612 - 2614. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) or were activated
with 0.2 U/mL thrombin for the indicated times, and the concentration
of sCD154 in the supernatants was determined using a specific ELISA.
The data are shown as mean values ± SD of duplicates and are
representative of 4 independent experiments. The low background levels
of sCD154 observed in unstimulated platelets probably reflect a minimal
activation of platelets through the isolation and incubation
procedures. 
, activated; 
, activated plus mAb MOPC-21; 
,
activated plus mAb G28-5. (B) CD154 was immunoprecipitated from lysates
and supernatants of unstimulated or activated (0.2 U/mL thrombin for 1 hour) platelets with mAb TRAP2 and detected by Western blotting with a
CD154-specific antiserum. In parallel, platelet activation was
performed in the presence of anti-CD40 mAb G28-5 (10 µg/mL). Unstim
indicates unstimulated; Lys, lysate; Sup, supernatant. (C) Platelets
were either left unstimulated or activated for the indicated time
periods, and sCD154 was immunoprecipitated with a chimeric CD40-Ig
reagent. The immunoprecipitate was subjected to SDS-PAGE and detected
by Western blotting with a CD154-specific antiserum. Unstim indicates
unstimulated.
) or 0.2 U/mL human thrombin (
) for 1 hour at 37°C. sCD154 concentrations were determined in cell-free
supernatants by ELISA. 
, unstimulated. Experiments were performed in
triplicate; data are shown as mean ± SD and are representative of
3 independent experiments.
