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 pro- thrombotic 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 stan- dard 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.