Artur-Aron Weber

Platelet CD40 ligand (CD40L) – subcellular localization, regulation of expression, and inhibition by clopidogrel

This study compares the subcellular localization and the regulation of expression of the platelet activation markers CD62P and CD63 with CD40 ligand (CD40L) on the surface of washed human platelets. CD40L was expressed upon stimulation with a wide range of platelet activators. However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L expression was low. Upon stimulation with thrombin receptor-activating peptide (TRAP-6), all activation markers were expressed. In contrast, upon stimulation with low concentrations of collagen (1-3 w g/ml), CD40L, but not the granule proteins (CD62P, CD63), were expressed. Using immunofluorescence microscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic localization of CD40L was verified by Western blotting of subcellular platelet fractions. The staining of CD40L was different from that of filamentous actin and only little association of CD40L with platelet cytoskeleton was found. Surface expression of CD40L was dependent on internal Ca 2+ stores and protein kinase C, while the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 w M)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level of CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in the promegakaryocytic cell line MEG-01.

Comparative Pharmacology of GP IIb/IIIa Antagonists

GP IIb/IIIa antagonists are qualitatively different from classical antiplatelet agents, such as aspirin or clopidogrel. They do not inhibit platelet activation, i.e. intraplatelet signal generation or conduction but primarily act outside the platelet by competing with ligand (e.g. fibrinogen) binding that is essential for platelet bridging and aggregate formation. Three compounds are in clinical use: abciximab, an antibody fragment and two low-molecular weight compounds, tirofiban and eptifibatide. In comparison to the low-molecular weight compounds, abciximab has a substantially longer platelet half-life (4 h), i.e. slow off-rate and a short plasma half-life (20–30 min) without significant distribution into the extravascular space. The plasma half-life of tirofiban and eptifibatide is about 2 h and parallels the antiplatelet effect. The off-rate from the platelet GP IIb/IIIa receptor is much faster and there is a significant distribution into the extravascular space. These pharmacokinetic variables might influence the competition between the antagonists and fibrinogen for GP IIb/IIIa binding. Other pharmacological variables are a partial agonistic activity, facilitation of thrombolysis, modification of other integrin-related actions, including inflammatory responses, effects on vascular cells and apoptosis. Importantly, GP IIb/IIIa antagonists might also interfere with prothrombin binding to the platelet surface and, thus, might influence the coagulation pathway. There is no clear evidence that the biological activity of the agents is modified by gene polymorphism (HPA-1). All three compounds may cause thrombocytopenia, possibly related to drug-induced antibodies.There is no clear data suggesting that these pharmacological differences transfer into significant differences in clinical outcome, for example in patients with acute coronary syndromes (ACS) subjected to acute percutaneous coronary interventions (PCI). The only head-to-head comparison of all three clinically used parenteral compounds did not demonstrate differences in major adverse cardiac effects (MACE) at 30 days although those have been described in particular with long-term use of oral antagonists. The inherent problems with all GP IIb/IIIa antagonists are the narrow therapeutic range because the same mechanisms are involved in hemostasis and thrombosis and their inability to inhibit platelet activation.

Regulation of Thrombomodulin Expression in Human Vascular Smooth Muscle Cells by COX-2–Derived Prostaglandins

There is concern that cyclooxygenase (COX)-2 inhibitors may promote atherothrombosis by inhibiting vascular formation of prostacyclin (PGI2) and an increased thrombotic risk of COX-2 inhibitors has been reported. It is widely accepted that the prothrombotic effects of COX-2 inhibitors can be explained by the removal of platelet-inhibitory PGI2. Using microarray chip technology, we have previously demonstrated that thrombomodulin (TM) mRNA is upregulated in cultured human coronary artery smooth muscle cells by the stable prostacyclin mimetic iloprost. This study is the first to demonstrate a stimulation of the expression of functionally active thrombomodulin in human smooth muscle cells by prostaglandins, endogenously formed via the COX-2 pathway. Because TM is an important inhibitor of blood coagulation, these findings provide a novel platelet-independent mechanism to explain the prothrombotic effects of COX-2 inhibitors. The full text of this article is available online at http://circres.ahajournals.org.

Mechanisms of the inhibitory effects of epigallocatechin-3 gallate on platelet-derived growth factor-BB-induced cell signaling and mitogenesis

An enhanced activity of receptor tyrosine kinases (RTKs), such as the platelet‐derived growth factor (PDGF) α‐receptor (PDGF‐Rα) or the PDGF β‐receptor (PDGF‐Rβ), is involved in the development of proliferative diseases. We have previously demonstrated that green tea catechins containing a galloyl group in the third position of the catechin structure interfere with PDGF‐BB‐ induced mitogenic signaling pathways by inhibiting tyrosine phosphorylation of the PDGF‐Rβ. However, the underlying cellular and molecular mechanisms are unknown. Using human vascular smooth muscle cells (VSMC) and porcine endothelial cells (AEC) stably transfected with PDGF‐Rα and ‐β, respectively, we demonstrate that EGCG preferably inhibited PDGF‐BB isoform‐mediated signal transduction pathways and cell proliferation. To elucidate cellular and molecular mechanisms of the inhibitory effects of EGCG, we studied the distribution of incorporated EGCG into cellular compartments after subcellular fractionation. Interestingly, most (85%) of the EGCG was found in the cytoplasmic fraction, whereas only ∼2% was found within the cell plasma membranes. However, no alteration of membrane fluidity has been observed after treatment of VSMC with 50 µM EGCG. Binding studies with [125I]‐PDGF‐BB on EGCG‐treated VSMC demonstrated that the specific binding of PDGF‐BB was completely abolished. Moreover, when [125I]‐PDGF‐BB was incubated with VSMC in the presence of EGCG, a 50% reduction of cellular [125I]‐PDGF‐BB binding was observed. Our findings suggest that plasma membrane incorporated EGCG or soluble EGCG directly interacts with PDGF‐BB, thereby preventing specific receptor binding.

Platelet-Derived Microparticles Stimulate Coronary Artery Smooth Muscle Cell Mitogenesis by a PDGF-Independent Mechanism

This study investigates the role of platelet-derived microparticles for vascular smooth muscle cell (SMC) proliferation. Microparticles concentration dependently stimulated p42/p44 MAP kinase phosphorylation, c-fos induction, DNA synthesis, and proliferation of cultured bovine coronary artery SMC. The maximum mitogenic effects of microparticles were significantly higher than those of platelet-derived growth factor (PDGF)-BB. Microparticle-induced SMC mitogenesis was heat sensitive, whereas the effects of PDGF were not. In addition, neutralizing anti-PDGF antibodies prevented PDGF-induced DNA synthesis but did not inhibit the effects of microparticles. In contrast to PDGF, which potently stimulated SMC migration, microparticles had only minor migratory activity. These results demonstrate a novel mechanism of SMC mitogenesis by platelet-derived microparticles that is probably independent of PDGF.

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

The thienopyridine clopidogrel is a specific inhibitor of ADP‐induced platelet aggregation ex vivo. No direct effects of clopidogrel (100 μM) on platelet aggregation in vitro have been described so far. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet‐rich plasma and in washed platelets. Incubation of platelet‐rich plasma with clopidogrel (100 μM) for up to 8 h did not result in any inhibition of ADP (6 μM)‐induced platelet aggregation. Incubation of washed platelets with clopidogrel resulted in a time‐ (maximum effects after 30 min) and concentration‐dependent (IC50 1.9±0.3 μM) inhibition of ADP (6 μM)‐induced platelet aggregation. Clopidogrel (30 μM) did not inhibit collagen (2.5 μg ml−1)‐, U46619 (1 μM)‐ or thrombin (0.1 u ml−1)‐induced platelet aggregation. The inhibition of ADP‐induced aggregation by clopidogrel (30 μM) was insurmountable indicating a non‐equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 μM) was significantly less active than clopidogrel (30 μM) in inhibiting platelet aggregation (32±5 % vs 70±1 % inhibition, P<0.05, n=5). In washed platelets, clopidogrel (30 μM) did not significantly reverse the inhibition of prostaglandin E1 (1 μM)‐induced platelet cyclic AMP formation by ADP (6 μM). The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml−1) was present in the test buffer. It is concluded that clopidogrel specifically inhibits ADP‐induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP‐induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1‐stimulated cyclic AMP by ADP.

Tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E1 in coronary artery smooth muscle cells

This study compares the antimitogenic effects of iloprost and prostaglandin E1 on platelet-derived growth factor-BB stimulated DNA synthesis ([3H]thymidine incorporation) in bovine coronary artery smooth muscle cells. When added 20-24 h after stimulation with platelet-derived growth factor-BB (20 ng/ml), both iloprost and prostaglandin E1, concentration-dependently (IC50 3-5 nM) inhibited DNA synthesis. However, when added together with the growth factor (0-24 h), the inhibition of DNA synthesis by iloprost was markedly attenuated, indicating tolerance development. In contrast, no tolerance to antimitogenic effects of prostaglandin E1 or forskolin were observed. When added to iloprost-tolerant cells, both prostaglandin E1 and forskolin, still inhibited DNA synthesis. There was no evidence for transcriptional down-regulation of prostacyclin receptor gene by iloprost. The data demonstrate a tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E1 and suggest that the receptors, mediating the antiproliferative actions of these prostaglandins, may be different.

Direct inhibition of platelet function by organic nitrates via nitric oxide formation

This study investigates the mechanisms of platelet inhibition by the nitrate esters isosorbide dinitrate, isoidide dinitrate, isomannide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate as compared to the spontaneous nitric oxide (NO)-donor linsidomine, the active metabolite of molsidomine. Nitrates and linsidomine dose-dependently inhibited aggregation, ATP secretion and thromboxane formation of washed human platelets at a rank order of potency, identical with that for stimulation of cyclic GMP in cultured rat lung fibroblasts. While linsidomine (0.1 mM) caused a 3-fold platelet cGMP elevation, there was a weak (< or = 30%) but significant cGMP stimulation by organic nitroesters, which was tightly correlated with inhibition of platelet aggregation (r = 0.926, P = 0.008). Zaprinast (2 microM) potentiated, while methylene blue (1 microM) and oxyhemoglobin (10 microM) reversed the antiaggregatory effects. Linsidomine (0.5 microM-0.1 mM) dose-dependently released NO in a cell-free system. No spontaneous NO release was detected with organic nitroesters (0.1 mM). These data suggest that, to some extent, bioactivation of organic nitroesters occurs in platelets, resulting in platelet inhibition via the NO/cGMP system.

Flow cytometry analysis of platelet cyclooxygenase‐2 expression: induction of platelet cyclooxygenase‐2 in patients undergoing coronary artery bypass grafting

Summary. There are conflicting reports about the expression of cyclooxygenase (COX)‐2 in human platelets. The present study describes a flow cytometric method for the measurement of platelet COX. Both COX‐1 and COX‐2 were shown to be expressed in platelets from patients undergoing a coronary artery bypass graft. There was a significant increase in COX‐2 expression at day 5 as compared with pre‐surgery values (mean fluorescence 12·31 ± 0·88 versus 9·15 ± 0·88; means ± SEM, n = 7, P < 0·05), whereas COX‐1 levels did not change (13·45 ± 1·11 versus 12·38 ± 1·41; n = 7, P > 0·05).

Alternatively spliced human tissue factor (asHTF) is not pro-coagulant

It has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells. As expected, a marked pro-coagulant activity (FX activation, thrombin generation) was observed on the surface, in lysates, and on microparticles from HTF transfected cells. In contrast, no pro-coagulant activity of asHTF was observed.