Release of Soluble CD40L from Platelets Is Regulated by Glycoprotein Iib/Iiia and Actin Polymerization Mark I

Home , Eptifibatide, Tirofiban

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Journal of the American College of Cardiology providedVol. by 43,Elsevier No. 12,- Publisher 2004 Connector © 2004 by the American College of Cardiology Foundation ISSN 0735-1097/04/$30.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2003.12.055 Platelet GP IIb/IIIa Inhibition Release of Soluble CD40L From Platelets Is Regulated by Glycoprotein IIb/IIIa and Actin Polymerization Mark I. Furman, MD, FACC,*† Lori A. Krueger, BA, MLT, ART,* Matthew D. Linden, PHD,* Marc R. Barnard, MS,* Andrew L. Frelinger III, PHD,* Alan D. Michelson, MD* Worcester, Massachusetts

OBJECTIVES The purpose of this study was to examine the effects of glycoprotein (GP) IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) and other inhibitors on translocation of CD40L from intraplatelet stores to the platelet surface and on the release of soluble CD40L (sCD40L) from platelets. BACKGROUND CD40L is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. METHODS Platelet surface CD40L was measured by flow cytometry, and sCD40L was measured by enzyme-linked immunosorbent assay. RESULTS Translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by GP IIb/IIIa antagonists. However, release of sCD40L from the surface of activated platelets was inhibited by GP IIb/IIIa antagonists in a dose-dependent manner, in concert with inhibition of PAC1 binding to platelets (a surrogate marker for fibrinogen binding). Release of sCD40L from activated platelets was also markedly reduced in Glanzmann platelets (deficient in GP IIb/IIIa). Ethylenediaminetetraacetic acid was an effective inhibitor of sCD40L release, but only when added before platelet activation. Both cytochalasin D (an inhibitor of actin polymerization) and GM6001 (an inhibitor of matrix metalloproteinases [MMPs]) inhibited the release of sCD40L from platelets when added before, as well as 3 min after, platelet activation. However, neither cytochalasin D nor GM6001 affected translocation of CD40L to the platelet surface. CONCLUSIONS The GP IIb/IIIa antagonists inhibit release of sCD40L from activated platelets. Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. In addition to their well-characterized inhibition of platelet aggregation, GP IIb/IIIa antagonists may obviate the proinflammatory and prothrombotic effects of sCD40L. (J Am Coll Cardiol 2004;43:2319–25) © 2004 by the American College of Cardiology Foundation

In addition to their well-known function in hemostasis and preformed intraplatelet stores of CD40L to the platelet thrombosis, platelets play an important role in inflammation surface within seconds of activation in vitro and in the (1). Platelets contain a variety of inflammatory modula- presence of thrombus formation in vivo. Furthermore, tors—including CD40 ligand (CD40L, CD154), platelet- platelet surface CD40L interacts with endothelial cell derived growth factor, platelet factor 4, RANTES (regu- CD40 to induce a thromboinflammatory reaction of endo- lated upon activation, normal T-cell expressed and thelial cells (3). Activated platelets also release large amounts of soluble CD40L (sCD40L) over a period of See page 2326 minutes to hours (4–6). Indeed, platelets appear to be the predominant source of circulating sCD40L (5). The secreted), thrombospondin, and transforming growth factor sCD40L released from platelets during thrombosis has beta—that are released upon platelet activation. Atheroscle- three reported functions (7): 1) induction of the production rosis is a chronic inflammatory disease, and the CD40/ and release of proinflammatory cytokines from vascular cells CD40L interaction has a central role in the pathogenesis of and of matrix metalloproteinases (MMPs) from resident atherosclerosis (2). cells in the atheroma (although not all studies agree on this CD40L, a member of the tumor necrosis factor family, is point [5]); 2) stabilization of arterial thrombi (8); and 3) not expressed on the resting platelet surface. However, inhibition of re-endothelialization of injured blood vessels Henn et al. (3) demonstrated that platelets translocate (9). In the present study, we examined the effects of GP From the *Center for Platelet Function Studies and †Division of Cardiovascular IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) Medicine, Departments of Pediatrics and Medicine, University of Massachusetts and other inhibitors on the translocation of CD40L from Medical School, Worcester, Massachusetts. Supported in part by Eli Lilly & Co. Manuscript received July 14, 2003; revised manuscript received December 16, intraplatelet stores to the platelet surface and on the release 2003, accepted December 23, 2003. of sCD40L from platelets. 2320 Furman et al. JACC Vol. 43, No. 12, 2004 Regulation of CD40L Release From Platelets June 16, 2004:2319–25

(Polysciences, Eppelheim, Germany) for 10 min at 22°C. Abbreviations and Acronyms Samples labeled with 24-31 were then washed by centrifu- EDTA ϭ ethylenediaminetetraacetic acid gation at 1,200 g for 7 min, and the formalin supernatant ELISA ϭ enzyme-linked immunosorbent assay was aspirated. The platelet pellet was then resuspended in ϭ FITC fluorescein isothiocyanate HT buffer containing the GP IIIa-specific FITC- GP ϭ glycoprotein ϭ conjugated monoclonal antibody Y2/51 (DAKO, Carpin- HT HEPES Tyrode’s buffer ␮ MMPs ϭ matrix metalloproteinases teria, California) 0.5 g/ml (or, for Glanzmann samples, PCI ϭ percutaneous coronary intervention the GPIX-specific PerCP-conjugated monoclonal antibody PE ϭ phycoerythrin Beb1 [Becton Dickinson] 1.25 ␮g/ml) and incubated at ϭ PerCP peridinin chlorophyll protein 22°C for 10 min. Samples were then diluted in 1% formalin PRP ϭ platelet-rich plasma sCD40L ϭ soluble CD40 ligand until analysis by flow cytometry. Flow cytometry was per- formed in a FACSCalibur (Becton Dickinson) with standard filter configuration and Cell Quest software. Platelets METHODS were identified by their characteristic forward and orthog- onal light scatter properties and by labeling with a platelet- Blood collection. This study was approved by the institu- specific monoclonal antibody (FITC-Y2/51, PerCP-RUU- tional review board of the University of Massachusetts 7F12, or PerCP-Beb1). Platelet surface CD40L and Memorial Health Care, and subjects gave written informed activated GP IIb/IIIa was measured by mean fluorescence consent. Peripheral blood was collected from healthy donors intensity (PE and FITC, respectively). Neither Y2/51 nor or patients with Glanzmann thrombasthenia (10) into RUU-7F12 block the binding of abciximab, eptifibatide, endotoxin-free 3.2% sodium citrate Vacutainer tubes (Bec- tirofiban, or PAC1. ton Dickinson, San Jose, California), after discarding the Enzyme-linked immunosorbent assay (ELISA). Platelet first 2 ml of drawn blood. No donor or patient had received counts in PRP were normalized to 300,000 platelets per ␮l. any drug known to affect platelet function (including aspi- The PRP was then incubated (22°C, 20 min) with various rin, cyclooxygenase inhibitors, statins) within the previous concentrations of a GP IIb/IIIa antagonist, EDTA 5 mM, 10 days. Platelet-rich plasma (PRP) was prepared by cen- GM6001 30 ␮M, cytochalasin D 60 ␮M, or HT buffer trifuging the blood at 150 g for 12 min at 22°C. alone. Samples were incubated (37°C, 1 h) with or without Flow cytometry. The PRP was diluted 1:5 (to prevent 50 ␮M isoTRAP. In some experiments, EDTA 5 mM, platelet aggregation) with modified HEPES Tyrode’s(HT) GM6001 30 ␮M, or cytochalasin D 60 ␮M was added 3 ␮ buffer (NaCl 137 mM, KCl 2.8 mM, MgCl2 1mM, min after the addition of the 50 M isoTRAP. After NaHCO3 12 mM, Na2HPO4 0.4 mM, glucose 5.5 mM, activation, the samples were centrifuged (8,000 g, 3 min, HEPES 10 mM, pH 7.4) and (to prevent Fc-mediated 22°C), the supernatants were transferred to a clean tube and effects) incubated at 22°C for 10 min with the Fc␥RIIa centrifuged again (8,000 g, 3 min, 22°C), and the subse- (CD32) blocking antibody IV.3 (Medarex, Princeton, New quent supernatants were transferred to a clean tube. These Jersey) 2.5 ␮g/ml. The PRP was then incubated at 22°C for supernatants were then immediately tested for sCD40L by 20 min with either HT buffer alone, abciximab 6.4 ␮g/ml, ELISA (Bender MedSystems, San Bruno, California). The eptifibatide 1.0 ␮g/ml, tirofiban 50 ng/ml, ethylenediamine- ELISA was set up according to the package insert except tetraacetic acid (EDTA) 5 mM, GM6001 (also known as that plasma samples were diluted 1:1 (50 ␮l:50 ␮l) with Galardin, an inhibitor of MMPs [11]) (Calbiochem, San assay buffer. The average of duplicate samples was recorded. Diego, California) 30 ␮M, or cytochalasin D (an inhibitor To determine the amount of released sCD40L, the of actin polymerization [12]) (Sigma, St. Louis, Missouri) sCD40L concentration of samples without added isoTRAP 60 ␮M. (Assays with GM6001 and cytochalasin D included was subtracted from the sCD40L concentration of samples 0.3% dimethyl sulfoxide.) Samples were then incubated with added isoTRAP. The range of 0.33 to 4.84 ng/ml in with or without 50 ␮M iso(S)FLLRN (iso-thrombin re- the background sCD40L of our normal donors and Glanz- ceptor activating peptide) (Multiple Peptide Systems, San mann thrombasthenia donors is consistent with the Diego, California) and with a saturating concentration of sCD40L range of 0.03 to 4.0 ng/ml obtained from a panel either the CD40L-specific phycoerythrin (PE)-conjugated of 40 healthy donors (Bender MedSystems, product insert). monoclonal antibody 24-31 (Ancell, Bayport, Minnesota) This normal variability in the background sCD40L of both (or the same concentration of PE-conjugated mouse isotype the normal donors and the Glanzmann thrombasthenia

control IgG1 [Pharmingen, San Diego, California]) for 1 h donors does not detract from our conclusions, because each at 37°C or a cocktail of the activated GP IIb/IIIa-specific donor and patient acted as his or her own control. The PRP fluorescein isothiocyanate (FITC)-conjugated monoclonal incubated for 40 min in the absence of an agonist resulted in antibody PAC1 (Becton Dickinson) and the GP IIIa- no increase in sCD40L over the background sCD40L in the specific peridinin chlorophyll protein (PerCP)-conjugated same donor’s fresh platelet-poor plasma. monoclonal antibody RUU-7F12 (Becton Dickinson) for 3 Statistical analysis. Significance (p Ͻ 0.05) was deter- min at 22°C. Samples were then fixed with 1% formalin mined by two-tailed t test. JACC Vol. 43, No. 12, 2004 Furman et al. 2321 June 16, 2004:2319–25 Regulation of CD40L Release From Platelets

Figure 1. Translocation of CD40L to the platelet surface is not inhibited by glycoprotein IIb/IIIa receptor antagonists. The platelet-rich plasma diluted 1:5 in modified HEPES Tyrode’s buffer was incubated (22°C, 10 min) with an Fc␥RIIa blocking antibody, and then incubated (22°C, 20 min) with either buffer alone, abciximab (6.4 ␮g/ml), eptifibatide (1.0 ␮g/ml), or tirofiban (50 ng/ml). Samples were then activated (37°C,1h) with 50 ␮M isoTRAP (except the “no agonist” sample) and the phycoerythrin-conjugated CD40L-specific monoclonal antibody 24-31. Samples were then fixed with 1% formalin (22°C, 10 min), washed once, and the platelet pellet was gently resuspended in buffer containing the fluorescein isothiocyanate-conjugated CD61-specific monoclonal antibody Y2/51. Samples were analyzed for platelet surface CD40L by flow cytometry. Data are mean Ϯ SEM, n ϭ 4. As indicated by the asterisk, there was a significant difference between the buffer/isoTRAP sample and the buffer/no agonist sample. There were no significant differences between the buffer/isoTRAP sample and abciximab/isoTRAP, eptifibatide/ isoTRAP, and tirofiban/isoTRAP samples.

RESULTS Activation of platelets with 50 ␮M isoTRAP resulted in a marked increase in the platelet surface expression of CD40L (Fig. 1). This activation-dependent translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by pharmacologically relevant concentrations of the GP IIb/IIIa antagonists abciximab, eptifibatide, or tirofiban (Fig. 1). However, the release of sCD40L from activated platelets was inhibited in a dose-dependent man- Figure 2. The release of sCD40L from the surface of activated platelets is ner by abciximab, eptifibatide, and tirofiban (Fig. 2), in inhibited by glycoprotein (GP) IIb/IIIa antagonists. The platelet-rich concert with inhibition of PAC1 binding to platelets (a plasm (with platelet count adjusted to 300,000/␮l) was incubated (22°C, 20 surrogate marker for fibrinogen binding [13])(Fig. 2). min) with the indicated concentrations of GP IIb/IIIa antagonist (note log scale). A small aliquot was removed and incubated (22°C, 3 min) with 50 To further assess the role of GP IIb/IIIa in the redistri- ␮M isoTRAP, fluorescein isothiocyanate-conjugated activated GP IIb/ bution of CD40L molecules, we next examined the platelets IIIa-specific monoclonal antibody PAC1, and PerCP-conjugated GP of two patients with Glanzmann thrombasthenia (an inher- IIIa-specific monoclonal antibody RUU-7F12, then fixed with 1% formalin and analyzed by flow cytometry. The remaining aliquot was then ited deficiency of GP IIb/IIIa [10]). Consistent with our activated (37°C, 1 h) with 50 ␮M isoTRAP without stirring, centrifuged results with GP IIb/IIIa antagonists, the activation- twice, and the supernatants were tested in duplicate for sCD40L by enzyme-linked immunosorbent assay. Values for “Released sCD40L” were dependent translocation of CD40L from intraplatelet stores determined by subtracting sCD40L values obtained from samples that to the platelet surface was not reduced in Glanzmann were not isoTRAP-activated from values obtained from samples that were platelets (Fig. 3A), but the release of sCD40L from acti- iso-TRAP activated. Data are mean Ϯ SEM, n ϭ 3. Asterisks indicate vated platelets was markedly reduced in Glanzmann plate- significance as compared with samples with no GP IIb/IIIa antagonist. lets (Fig. 3B). ADAMs, both of which are inhibited by hydroxamate Because sCD40L is released from washed platelets (4,5), inhibitors of MMPs (11,14). Therefore, to further examine no plasma component is required for its cleavage. Members the mechanism of the redistribution of platelet CD40L and of the tumor necrosis factor family of molecules have been sCD40L release, we assessed the effects of EDTA (a strong reported to be cleaved by calcium-dependent MMPs and by calcium chelator) and GM6001 (a hydroxamate inhibitor of 2322 Furman et al. JACC Vol. 43, No. 12, 2004 Regulation of CD40L Release From Platelets June 16, 2004:2319–25

Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. 3) Translocation of CD40L from intraplatelet stores to the platelet surface is not inhibited by GP IIb/IIIa antagonists and is not regulated by GP IIb/IIIa or actin polymerization. Soluble CD40L ligand is prothrombotic via stabilization

of arterial thrombi by a beta3-integrin (GP IIIa)-dependent mechanism (8). The sCD40L has been reported to be proinflammatory via induction of leukocyte chemokine production (15) and endothelial cell adhesive proteins (16). Although the proinflammatory effects of recombinant sCD40L in vitro (15–17) and in vivo (18) are well described, Henn et al. (5) were unable to document such effects with natural sCD40L in vitro. Elevated plasma levels of sCD40L have been demonstrated in thrombotic and/or inflammatory states: unstable angina (4), percutaneous coronary intervention (PCI) (4), cardiopulmonary bypass (19), peripheral arterial occlusive disease (20), essential thrombocythemia (21), reactive thrombocytosis (21), and systemic lupus erythematosus (22). High plasma concentrations of sCD40L may be associated with increased cardiovascular risk in apparently healthy women (23). Platelets appear to be the predominant source of circulating sCD40L (5). The elevated plasma levels of sCD40L in unstable angina and during PCI in the absence of GP IIb/IIIa antagonist Figure 3. The activation-dependent translocation of CD40L to the plate- therapy (4), therefore, are of particular relevance to the let surface is not reduced in Glanzmann platelets (A), but the release of sCD40L from the surface of activated platelets is markedly reduced in presently described inhibition of sCD40L release from Glanzmann platelets (B). (A) Platelet surface CD40L, in the presence of platelets by GP IIb/IIIa antagonists (abciximab, eptifi- 50 ␮M isoTRAP, was measured as in Figure 1. (B) Released sCD40L in batide, tirofiban), because these agents are now standard platelet rich plasma was measured as in Figure 2. Data are mean Ϯ SEM, n ϭ 3 for normal control samples and mean of n ϭ 2 for Glanzmann therapy for unstable angina (24) and during PCI (25). The thrombasthenia. Asterisks indicate significance as compared with normal sCD40L is only released when platelets are activated (4–6), control samples. that is, at sites of local thrombosis. The presently described inhibitory effects of GP IIb/IIIa antagonists on sCD40L MMPs [11]). In addition, we assessed the effects of cy- release from activated platelets would therefore occur most tochalasin D (an inhibitor of actin polymerization [12]). prominently in vivo at the site or sites of greatest potential Neither EDTA, cytochalasin D nor GM6001 inhibited the benefit. Thus, the present findings suggest that GP IIb/IIIa activation-dependent translocation of CD40L from in- antagonist therapy, in addition to its well-known inhibition traplatelet stores to the platelet surface (Fig. 4). (In fact, of platelet aggregation, may be of benefit in patients with EDTA resulted in a modest increase in platelet surface unstable angina and during PCI via an additional mecha- CD40L.) The EDTA was an effective inhibitor of sCD40L nism of action: inhibition of the prothrombotic (8) and release, but only when present before platelet activation proinflammatory (15–18) effects of sCD40L. Because all (Fig. 5A). When EDTA was added 3 min after platelet three Food and Drug Administration-approved GP IIb/IIIa activation with 50 ␮M isoTRAP, no inhibition of sCD40L antagonists (abciximab, eptifibatide, and tirofiban) are able was observed (Fig. 5B). Both cytochalasin D and GM6001 to inhibit sCD40L release, our results do not differentiate markedly inhibited the release of sCD40L from platelets between these agents with respect to their ability to produce when added before, as well as 3 min after, platelet activation clinically relevant inhibition of inflammation. Heeschen et (Fig. 5). al. (26) recently demonstrated that measurement of DISCUSSION sCD40L in the first 12 h after the onset of ischemic symptoms in patients with unstable angina identifies a Our results demonstrated the following: 1) GP IIb/IIIa subgroup of patients that has a much greater clinical benefit antagonists (abciximab, eptifibatide, and tirofiban) inhibit from abciximab treatment. the release of sCD40L from activated platelets and there- Consistent with the present study, Nannizzi-Alaimo et fore, in addition to their well characterized inhibition of al. (27), in a study published after the completion of the platelet aggregation, they may obviate the proinflammatory present work, reported that the three Food and Drug (15–18) and prothrombotic effects (8) of sCD40L. 2) Administration-approved GP IIb/IIIa antagonists inhibit JACC Vol. 43, No. 12, 2004 Furman et al. 2323 June 16, 2004:2319–25 Regulation of CD40L Release From Platelets

Figure 4. The ethylenediaminetetraacetic acid (EDTA), cytochalasin D, and GM6001 do not inhibit the activation-dependent translocation of CD40L to the platelet surface. Platelet surface CD40L, in the presence of 50 ␮M isoTRAP and either no inhibitor, EDTA 5 mM, cytochalasin D 60 ␮M, or GM6001 30 ␮M, was measured as in Figure 1. Data are mean Ϯ SEM, n ϭ 4. The asterisk indicates signiﬁcance as compared with no inhibitor.

the release of sCD40L from platelets in vitro. These investigators (27) also reported that suboptimal doses of these three antagonists (that resulted in between 0% and 50% platelet aggregation) potentiated the release of sCD40L from platelets in vitro, raising the possibility that the proinflammatory effects of sCD40L could explain the negative results of the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Arteries Figure 5. The ethylenediaminetetraacetic acid (EDTA), cytochalasin D, (GUSTO IV) trial of abciximab (28). However, our exper- and GM6001 inhibit the release of sCD40L from the surface of activated platelets. Released sCD40L in platelet rich plasma, in the presence of 50 iments used doses of GP IIb/IIIa antagonist that under our ␮M isoTRAP and either no inhibitor, EDTA 5 mM, cytochalasin D 60 experimental conditions resulted in a similar degree of ␮M, or GM6001 30 ␮M, was measured as in Figure 2. (A) Inhibitors inhibition of platelet aggregation yet did not potentiate added before isoTRAP. (B) Inhibitors added 3 min after isoTRAP. Data are mean Ϯ SEM, n ϭ 3. Asterisks indicate significance as compared with sCD40L release (Fig. 2). This apparent discrepancy be- no inhibitor. tween our results and those of Nannizzi-Alaimo et al. (27) may result from technical differences, including anticoagu- sCD40L in patients after in vivo treatment with abciximab lants (citrate vs. D-phenylalanyl-prolyl-arginine), agonists or eptifibatide, measured when receptor occupancy was low, ␮ ␮ (50 M isoTRAP vs. 5 M thrombin receptor activating was not elevated compared with pretreatment levels (30). peptide), and unstirred versus stirred conditions. The en- In contrast to both our findings and those of Nannizzi- hanced inhibition of sCD40L release by eptifibatide and Alaimo et al. (27) with intravenous GP IIb/IIIa antagonists, tirofiban compared with abciximab observed in the study by Zondlo et al. (31) reported that oral GP IIb/IIIa antagonists Nannizzi-Alaimo et al. (27) was likely due to the fact that (roxifiban and orbofiban) in pharmacologically relevant these investigators initiated platelet activation and GP doses do not affect the release of sCD40L from platelets in IIb/IIIa inhibition simultaneously by adding PRP to a vitro. The resultant lack of inhibition of the proinflamma- mixture of platelet agonist and GP IIb/IIIa antagonist. tory (15–18) and prothrombotic effects (8) of sCD40L may Because abciximab has a slower on rate than the small contribute to the reported lack of clinical effectiveness of molecule inhibitors eptifibatide and tirofiban (29), processes oral GP IIb/IIIa antagonists (32). Clopidogrel, a thienopy- mediating the release of sCD40L may already be initiated ridine, completely abolishes adenosine diphosphate-induced before complete receptor blockade by abciximab. In our expression of platelet surface CD40L (33), but the mecha-

experiments, GP IIb/IIIa antagonists were preincubated nism is via blockade of the P2Y12 adenosine diphosphate with platelets for 20 min at room temperature before receptor, not via a direct effect of clopidogrel on CD40L. platelet activation, thereby allowing adequate time for all Our results also document a heretofore unrecognized GP IIb/IIIa antagonists to reach equilibrium. This latter defect in patients with Glanzmann thrombasthenia: a lack approach may be more relevant to the clinical situation in of activation-dependent release of sCD40L from platelets which GP IIb/IIIa antagonists are dosed at high levels (Fig. 3B). The biologic and/or clinical significance of this before procedures that activate platelets. Thus, plasma finding remains to be determined. Patients with Glanzmann 2324 Furman et al. JACC Vol. 43, No. 12, 2004 Regulation of CD40L Release From Platelets June 16, 2004:2319–25 thrombasthenia appear to have higher platelet surface ex- vent GP IIb/IIIa on one platelet from binding to the KGD pression of CD40L (Fig. 3A). A trivial explanation for this sequence in uncleaved CD40L on another platelet. would be that the platelet surface CD40L is more accessible Platelet activation results in fibrinogen binding to the GP to the detecting antibody owing to the lack of platelet IIb/IIIa complex (37), which results in activation of the surface GP IIb/IIIa in Glanzmann thrombasthenia. An cytoskeleton via actin polymerization (37) and, we hypoth- alternate explanation is that, because translocation of esize based on our results with cytochalasin D (Fig. 5), CD40L from intraplatelet stores to the platelet surface and trafficking of the putative CD40L-cleaving protease to the proteolysis of platelet surface CD40L is probably occurring platelet surface. Consistent with this hypothesis, EDTA, concurrently, the higher platelet surface expression of when added before the platelet agonist, inhibits the initial CD40L in patients with Glanzmann thrombasthenia re- binding of fibrinogen, the subsequent signaling to the flects decreased proteolysis. This latter explanation may also cytoskeleton, movement of the protease to the platelet account for the modest increase in the platelet surface surface, and therefore release of sCD40L (Fig. 5A). When expression of CD40L in experiments with EDTA (Fig. 4) EDTA was added after fibrinogen binding (i.e., 3 min after and the statistically insignificant increases in the platelet agonist addition), EDTA was ineffective in inhibiting the surface expression of CD40L in experiments with abcix- release of sCD40L (Fig. 5B), suggesting that: 1) signals imab and eptifibatide (Fig. 1). initiating trafficking of the protease had already occurred In human T cells, CD40L is processed into its soluble and/or 2) the putative protease is not divalent cation- form in intracellular microsomes (34). However, as inde- dependent. Because most metalloproteinases are divalent pendently demonstrated by three groups of investigators cation-dependent, an alternate possibility is that, although (5,27,35), platelet lysates, unlike T-cell lysates, demonstrate EDTA inhibits the activation of the putative CD40L- full length but not cleaved forms of CD40L. Thus, the cleaving metalloproteinase, once the enzyme has been acti- intracellular pool of CD40L contains no sCD40L. Al- vated and engages CD40L at the cleavage site, removal of though the release of sCD40L from platelets is activation- divalent cations cannot prevent proteolysis. As predicted by our hypothesis, actin polymerization is still required post- dependent, sCD40L release is not the direct result of ␣ activation because addition of cytochalasin D 3 min after the granule secretion because the time frames are different. The agonist (like addition of cytochalasin D before the agonist) release of CD40L is much slower (maximal at approxi- still effectively blocked sCD40L release (Fig. 5). Irrespective mately 45 min [5,6,27]) than alpha granule release (maximal of whether it was added before or after the agonist, at approximately 1 min) (36). Henn et al. (5) reported that GM6001 inhibited the release of sCD40L (Fig. 5) because CD40 is constitutively expressed on platelets, and that the GM6001 is a direct inhibitor of the metalloproteinase that interaction of CD40 with CD40L on the surface of acti- putatively cleaves CD40L. Consistent with these findings, vated platelets is required for proteolysis of sCD40L. The Jin et al. (6) reported that the simultaneous addition of present study suggests that the GP IIb/IIIa complex (inte- ␣ ␤ thrombin and KB8301, another metalloproteinase inhibitor, grin IIb 3) augments the release of sCD40L. Thus, Glanz- inhibited the thrombin-induced release of sCD40L from mann thrombasthenia platelets and normal platelets treated platelets. with fibrinogen-blocking GP IIb/IIIa antagonists have reduced activation-dependent sCD40L release (Figs. 2 and Reprint requests and correspondence: Dr. Alan D. Michelson, 3B). These findings may reflect the lack of the normal Director, Center for Platelet Function Studies, University of “outside-in” signaling initiated by fibrinogen binding to Massachusetts Medical School, Room S5-846, 55 Lake Avenue activated GP IIb/IIIa (37). Alternately, maximal release of North, Worcester, Massachusetts 01655. E-mail: michelson@ sCD40L may require cell-cell contact, and this is disrupted platelets.org. in Glanzmann thrombasthenia and by GP IIb/IIIa antagonist therapy. Although sCD40L has been shown to bind to REFERENCES the activated GP IIb/IIIa complex via the lysine-arginine- glutamic acid (KGD) peptide sequence of CD40L (8), 1. Klinger MHF. Inflammation. In: Michelson AD, editor. Platelets. uncleaved platelet membrane CD40L is unlikely to bind to New York, NY: Academic Press/Elsevier Science, 2002:459–67. 2. Lutgens E, Daemen MJ. CD40-CD40L interactions in atherosclero- GP IIb/IIIa by this mechanism because the KGD sequence sis. Trends Cardiovasc Med 2002;12:27–32. of CD40L is located immediately adjacent to the trans- 3. Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature membrane domain (34), whereas the RGD (and KGD) 1998;391:591–4. binding site appears to be located in the head region of GP 4. Aukrust P, Muller F, Ueland T, et al. Enhanced levels of soluble IIb/IIIa (38). However, recent studies by Takagi et al. (39) and membrane-bound CD40 ligand in patients with unstable angina: possible reflection of T lymphocyte and platelet involve- suggest that the molecule may assume variably bent confor- ment in the pathogenesis of acute coronary syndromes. Circulation mations. Because the orientation of the transmembrane 1999;100:614–20. domain and extracellular “legs” are uncertain, it is impossi- 5. Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA. The inflammatory action of CD40 ligand (CD154) expressed on activated ble to assign a height to the ligand binding region. In any human platelets is temporally limited by coexpressed CD40. Blood event, this three-dimensional arrangement would not pre- 2001;98:1047–54. JACC Vol. 43, No. 12, 2004 Furman et al. 2325 June 16, 2004:2319–25 Regulation of CD40L Release From Platelets

6. Jin Y, Nonoyama S, Morio T, Imai K, Ochs HD, Mizutani S. 24. Cairns JA, Theroux P, Lewis HD Jr., Ezekowitz M, Meade TW. Characterization of soluble CD40 ligand released from human acti- Antithrombotic agents in coronary artery disease. Chest 2001;119: vated platelets. J Med Dent Sci 2001;48:23–7. 228S–52S. 7. Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet- 25. Popma JJ, Ohman EM, Weitz J, Lincoff AM, Harrington RA, Berger derived CD40L: the switch-hitting player of cardiovascular disease. P. Antithrombotic therapy in patients undergoing percutaneous cor- Circulation 2002;106:896–9. onary intervention. Chest 2001;119:321S–36S. 8. Andre P, Prasad KS, Denis CV, et al. CD40L stabilizes arterial 26. Heeschen C, Dimmeler S, Hamm CW, et al. Soluble CD40 ligand in thrombi by a beta3 integrin-dependent mechanism. Nature Med acute coronary syndromes. N Engl J Med 2003;348:1104–11. 2002;8:247–52. 27. Nannizzi-Alaimo L, Alves VL, Phillips DR. Inhibitory effects of 9. Urbich C, Dernbach E, Aicher A, Zeiher AM, Dimmeler S. CD40 glycoprotein IIb/IIIa antagonists and aspirin on the release of soluble ligand inhibits endothelial cell migration by increasing production of CD40 ligand during platelet stimulation. Circulation 2003;107: endothelial reactive oxygen species. Circulation 2002;106:981–6. 1123–8. 10. Mitchell WB, Li J, Singh F, et al. Two novel mutations in the ␣IIb 28. Simoons ML, GUSTO IV-ACS Investigators. Effect of glycoprotein calcium-binding domains identify hydrophobic regions essential for IIb/IIIa receptor blocker abciximab on outcome in patients with acute ␣IIb␤3 biogenesis. Blood 2003;101:2268–76. coronary syndromes without early coronary revascularisation: the 11. Solorzano CC, Ksontini R, Pruitt JH, et al. A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor alpha GUSTO IV-ACS randomised trial. Lancet 2001;357:1915–24. (TNF alpha) and abrogates endotoxin-induced lethality. Shock 29. Agah R, Plow EF, Topol EJ. GP IIb/IIIa antagonists. In: Michelson 1997;7:427–31. AD, editor. Platelets. New York, NY: Academic Press/Elsevier Sci- 12. Fox JEB, Phillips DR. Inhibition of actin polymerization in blood ence, 2002:769–85. platelets by cytochalasins. Nature 1981;292:650–2. 30. Michelson AD, Krueger LA, Barnard MR, Fox ML, Frelinger AL, 13. Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the Furman MI. GP IIb/IIIa antagonists reduce thromboinflammatory platelet membrane glycoprotein IIb/IIIa complex during platelet acti- processes in patients with acute coronary syndromes undergoing vation. J Biol Chem 1985;260:11107–14. percutaneous coronary intervention (abstr). J Thromb Haemost 2003; 14. Amour A, Knight CG, Webster A, et al. The in vitro activity of 1:P1767. ADAM-10 is inhibited by TIMP-1 and TIMP-3. FEBS Letts 31. Zondlo SC, Wang X, Feuerstein GZ. Effects of orally active glycop- 2000;473:275–9. rotein IIb/IIIa antagonists on platelet CD40 ligand (CD154) expres- 15. Kiener PA, Moran-Davis P, Rankin BM, Wahl AF, Aruffo A, sion and platelet-heteroaggregate formation. Thromb Res 2002;106: Hollenbaugh D. Stimulation of CD40 with purified soluble gp39 35–40. induces proinflammatory responses in human monocytes. J Immunol 32. Chew DP, Bhatt DL, Sapp S, Topol EJ. Increased mortality with oral 1995;155:4917–25. platelet glycoprotein IIb/IIIa antagonists: a meta-analysis of phase III 16. Hollenbaugh D, Mischel-Petty N, Edwards CP, et al. Expression of multicenter randomized trials. Circulation 2001;103:201–6. functional CD40 by vascular endothelial cells. J Exp Med 1995;182: 33. Hermann A, Rauch BH, Braun M, Schror K, Weber AA. Platelet 33–40. CD40 ligand (CD40L)—subcellular localization, regulation of expres- 17. Karmann K, Hughes CC, Schechner J, Fanslow WC, Pober JS. CD40 sion, and inhibition by clopidogrel. Platelets 2001;12:74–82. on human endothelial cells: inducibility by cytokines and functional 34. Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek RA. regulation of adhesion molecule expression. Proc Natl Acad Sci USA A soluble form of TRAP (CD40 ligand) is rapidly released after T cell 1995;92:4342–6. activation. Eur J Immunol 1995;25:1749–54. 18. Wiley JA, Geha R, Harmsen AG. Exogenous CD40 ligand induces a 35. Weber AA, Hermann A, Rauch BH, Schror K. Molecular identity of pulmonary inflammation response. J Immunol 1997;158:2932–8. platelet CD40 ligand (CD40L) (abstr). Thromb Haemost 2001;86: 19. Nannizzi-Alaimo L, Rubenstein MH, Alves VL, Leong GY, Phillips 718. DR, Gold HK. Cardiopulmonary bypass induces release of soluble 36. Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF, Kestin CD40 ligand. Circulation 2002;105:2849–54. 20. Tsakiris DA, Tschopl M, Wolf F, Labs KH, Jager KA, Marbet GA. AS. Downregulation of the platelet surface glycoprotein Ib-IX com- Platelets and cytokines in concert with endothelial activation in plex in whole blood stimulated by thrombin, ADP or an in vivo patients with peripheral arterial occlusive disease. Blood Coagul wound. Blood 1991;77:770–9. Fibrinol 2000;11:165–73. 37. Hato T, Ginsberg MH, Shattil SJ. Integrin alphaIIb-beta3. In: 21. Viallard JF, Solanilla A, Gauthier B, et al. Increased soluble and Michelson AD, editor. Platelets. New York, NY: Academic Press/ platelet-associated CD40 ligand in essential thrombocythemia and Elsevier Science, 2002:105–16. reactive thrombocytosis. Blood 2002;99:2612–4. 38. Adair BD, Yeager M. Three-dimensional model of the human platelet 22. Kato K, Santana-Sahagun E, Rassenti LZ, et al. The soluble CD40 integrin ␣IIb␤3 based on electron microscopy and x-ray crystallogra- ligand sCD154 in systemic lupus erythematosus. J Clin Invest 1999; phy. Proc Natl Acad Sci USA 2002;99:14059–64. 104:947–55. 39. Takagi J, Petre BM, Walz T, Springer TA. Global conformational 23. Schonbeck U, Varo N, Libby P, Buring J, Ridker PM. Soluble CD40L rearrangements in integrin extracellular domains in outside-in and and cardiovascular risk in women. Circulation 2001;104:2266–8. inside-out signaling. Cell 2002;110:599–611.