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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^* and Alan D. Michelson, MD^*,*

^* Center for Platelet Function Studies, University of Massachusetts Medical School, Worcester, Massachusetts, USA
Division of Cardiovascular Medicine, Departments of Pediatrics and Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

View larger version (20K): [in a new window]   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 FcRIIa blocking antibody, and then incubated (22°C, 20 min) with either buffer alone, abciximab (6.4 µg/ml), eptifibatide (1.0 µg/ml), or tirofiban (50 ng/ml). Samples were then activated (37°C, 1 h) with 50 µM 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 ± SEM, n = 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.

View larger version (19K): [in a new window]   Figure 2 The release of sCD40L from the surface of activated platelets is inhibited by glycoprotein (GP) IIb/IIIa antagonists. The platelet-rich plasm (with platelet count adjusted to 300,000/µl) was incubated (22°C, 20 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 µM isoTRAP, fluorescein isothiocyanate-conjugated activated GP IIb/IIIa-specific monoclonal antibody PAC1, and PerCP-conjugated GP IIIa-specific monoclonal antibody RUU-7F12, then fixed with 1% formalin and analyzed by flow cytometry. The remaining aliquot was then activated (37°C, 1 h) with 50 µM isoTRAP without stirring, centrifuged twice, and the supernatants were tested in duplicate for sCD40L by enzyme-linked immunosorbent assay. Values for "Released sCD40L" were determined by subtracting sCD40L values obtained from samples that were not isoTRAP-activated from values obtained from samples that were iso-TRAP activated. Data are mean ± SEM, n = 3. Asterisks indicate significance as compared with samples with no GP IIb/IIIa antagonist.

View larger version (16K): [in a new window]   Figure 3 The activation-dependent translocation of CD40L to the platelet surface is not reduced in Glanzmann platelets (A), but the release of sCD40L from the surface of activated platelets is markedly reduced in Glanzmann platelets (B). (A) Platelet surface CD40L, in the presence of 50 µM isoTRAP, was measured as in Figure 1. (B) Released sCD40L in platelet rich plasma was measured as in Figure 2. Data are mean ± SEM, n = 3 for normal control samples and mean of n = 2 for Glanzmann thrombasthenia. Asterisks indicate significance as compared with normal control samples.

View larger version (17K): [in a new window]   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 µM isoTRAP and either no inhibitor, EDTA 5 mM, cytochalasin D 60 µM, or GM6001 30 µM, was measured as in Figure 1. Data are mean ± SEM, n = 4. The asterisk indicates significance as compared with no inhibitor.

View larger version (27K): [in a new window]   Figure 5 The ethylenediaminetetraacetic acid (EDTA), cytochalasin D, and GM6001 inhibit the release of sCD40L from the surface of activated platelets. Released sCD40L in platelet rich plasma, in the presence of 50 µM isoTRAP and either no inhibitor, EDTA 5 mM, cytochalasin D 60 µM, or GM6001 30 µM, was measured as in Figure 2. (A) Inhibitors added before isoTRAP. (B) Inhibitors added 3 min after isoTRAP. Data are mean ± SEM, n = 3. Asterisks indicate significance as compared with no inhibitor.