Karsten Schrör

Functional and Biochemical Evaluation of Platelet Aspirin Resistance After Coronary Artery Bypass Surgery

Background—Aspirin inhibits platelet activation and reduces atherothrombotic complications in patients at risk of myocardial infarction and stroke. However, a sufficient inhibition of platelet function by aspirin is not always achieved. The causes of this aspirin resistance are unknown. Methods and Results—Patients undergoing coronary artery bypass grafting (CABG) have a high incidence of aspirin resistance. To evaluate functional and biochemical responses to aspirin, platelet-rich plasma was obtained before and at days 1, 5, and 10 after CABG. Thromboxane formation, aggregation, and &agr;-granule secretion were effectively inhibited by 30 or 100 &mgr;mol/L aspirin in vitro before CABG, but this inhibition was prevented or attenuated after CABG. Whereas the inhibition of thromboxane formation and aggregation by aspirin in vitro partly recovered at day 10 after CABG, oral aspirin (100 mg/d) remained ineffective. The inducible isoform of cyclooxygenase in platelets, COX-2, has been suggested to confer aspirin resistance. In fact, immunoreactive COX-2 was increased 16-fold in platelets at day 5 after CABG, but the COX-2 selective inhibitor celecoxib did not alter aspirin-resistant thromboxane formation. By contrast, the combined inhibitor of thromboxane synthase and thromboxane receptor antagonist terbogrel equally prevented thromboxane formation of platelets obtained before (control) and after CABG. Conclusions—Platelet aspirin resistance involves an impairment of both in vivo and in vitro inhibition of platelet functions and is probably due to a disturbed inhibition of platelet COX-1 by aspirin.

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.

Aspirin in the Chemoprevention of Colorectal Neoplasia: An Overview

Considerable evidence supports the effectiveness of aspirin for chemoprevention of colorectal cancer (CRC) in addition to its well-established benefits in the prevention of vascular disease. Epidemiologic studies have consistently observed an inverse association between aspirin use and risk of CRC. A recent pooled analysis of a long-term posttrial follow-up of nearly 14,000 patients from four randomized, cardiovascular disease prevention trials showed that daily aspirin treatment for about five years was associated with a 34% reduction in 20-year CRC mortality. A separate metaanalysis of nearly 3,000 patients with a history of colorectal adenoma or cancer in four randomized adenoma prevention trials showed that aspirin reduced the occurrence of advanced adenomas by 28% and any adenoma by 17%. Aspirin has also been shown to be beneficial in a clinical trial of patients with Lynch syndrome, a hereditary CRC syndrome; in those treated with aspirin for at least two years, there was a 50% or more reduction in the risk of CRC commencing five years after randomization and after aspirin had been discontinued. A few observational studies have shown an increase in survival among patients with CRC who use aspirin. Taken together, these findings strengthen the case for consideration of long-term aspirin use in CRC prevention. Despite these compelling data, there is a lack of consensus about the balance of risks and benefits associated with long-term aspirin use, particularly in low-risk populations. The optimal dose to use for cancer prevention and the precise mechanism underlying aspirins anticancer effect require further investigation. Cancer Prev Res; 5(2); 164–78. ©2011 AACR.

The pharmacology of cilostazol

Cilostazol (6‐[4‐(1‐cyclohexyl‐1H‐tetrazol‐5‐yl)butoxy]‐3,4‐dihydro‐2(1H)‐quinolinone; OPC‐13013) is a 2‐oxo‐quinoline derivative with antithrombotic, vasodilator, antimitogenic and cardiotonic properties. The compound is a potent inhibitor of phosphodiesterase (PDE) 3A, the isoform of PDE 3 in the cardiovascular system (IC50: 0.2 µm). In addition, there is inhibition of adenosine uptake, eventually resulting in changes in cAMP levels, dependent on the type of adenosine receptors (A1 or A2). Cilostazol inhibits platelet aggregation and has considerable antithrombotic effects in vivo. The compound relaxes vascular smooth muscle and inhibits mitogenesis and migration of vascular smooth muscle cells. In the heart, cilostazol causes positive inotropic and chronotropic effects. Most, if not all, of these actions are cAMP‐mediated, including the modification of cAMP‐controlled gene expression. Cilostazol decreases levels of serum triglycerides and causes some increase in HDL‐cholesterol levels. The compound has a number of additional effects which might contribute to its overall clinical efficacy. Cilostazol undergoes intensive and finally complete hepatic metabolism via the cytochrome P450 systems. This might result in some drug interaction, i.e. with erythromycin and omeprazole. The half‐life is approximately 10 h, resulting in about 2‐fold accumulation of the drug during repeated administration.

Cellular adhesion molecules on vascular smooth muscle cells

Several studies during the last years have shown that, in addition to endothelial cells, vascular smooth muscle cells also express the cellular adhesion molecules ICAM-1 and VCAM-1 in atherosclerosis, restenosis and transplant vasculopathy. In vitro studies have characterized stimulatory and inhibitory factors that regulate the expression of ICAM-1 and VCAM-1 on cultured smooth muscle cells. There is evidence for a role of adhesion molecules on smooth muscle cells for leukocyte accumulation and activation of mononuclear cells. Some recent data suggest that the expression of adhesion molecules on smooth muscle cells are cell cycle-dependent and influence smooth muscle cell proliferation and differentiation. Therefore, ICAM-1 and VCAM-1 on smooth muscle cells may contribute to the inflammatory reaction in the vascular wall and may actively be involved in the progression and stability of atherosclerotic plaques.

Effect of proton pump inhibitors on clinical outcome in patients treated with clopidogrel: a systematic review and meta-analysis

Summary.  To investigate whether proton pump inhibitors (PPIs) negatively affect clinical outcome in patients treated with clopidogrel. Systematic review and meta‐analysis. Outcomes evaluated were combined major adverse cardiac events (MACE), myocardial infarction (MI), stent thrombosis, death and gastrointestinal bleeding. Studies included were randomized trials or post‐hoc analyzes of randomized trials and observational studies reporting adjusted effect estimates. Twenty five studies met the selection criteria and included 159 138 patients. Administration of PPIs together with clopidogrel corresponded to a 29% increased risk of combined major cardiovascular events [risk ratio (RR) = 1.29, 95% confidence intervals (CI) = 1.15–1.45] and a 31% increased risk of MI (RR = 1.31, 95%CI = 1.12–1.53). In contrast, PPI use did not negatively influence the mortality (RR = 1.04, 95%CI = 0.93–1.16), whereas the risk of developing a gastrointestinal bleed under PPI treatment decreased by 50% (RR = 0.50, 95% CI = 0.37–0.69). The presence of significant heterogeneity might indicate that the evidence is biased, confounded or inconsistent. The sensitivity analysis, however, yielded that the direction of the effect remained unchanged irrespective of the publication type, study quality, study size or risk of developing an event. Two studies indicate that PPIs have a negative effect irrespective of clopidogrel exposure. In conclusion, concomitant PPI use might be associated with an increased risk of cardiovascular events but does not influence the risk of death. Prospective randomized trials are required to investigate whether a cause‐and‐effect relationship truly exists and to explore whether different PPIs worsen clinical outcome in clopidogrel treated patients as the PPI‐clopidogrel drug–drug interaction does not seem to be a class effect.

The Basic Pharmacology of Ticlopidine and Clopidogrel

Ticlopidine and clopidogrel are two thienopyridines with potent and apparently irreversible platelet inhibitory properties. The antiplatelet effects are mainly directed against ADP-induced stimulation of platelet function, in particular ADP-induced inhibition of adenylyl cyclase stimulation. There is evidence for additional effects, including inhibition of agonist-induced intracellular Ca(++) mobilization, interference with GpIIb/IIIa receptor/agonist interaction and inhibition of α-granule secretion. However, these actions are probably secondary to the ADP-antagonistic action. Thienopyridines do not directly interfere with arachidonic acid metabolism. The substances are inactive in vitro and have to undergo some form of bioactivation in vivo which requires 3 to 5 days of treatment for a maximum effect. The nature of the postulated active metabolite(s) is still unknown. From a pharmacological point of view, thienopyridines may be considered interesting alternatives to acetylsalicylic acid with particular value in shear-stress-mediated platelet activation, for example in prevention of acute thrombembolic risk in injury-related vessel stenosis.

Transformation of arachidonic acid and prostaglandin endoperoxides by the guinea pig heart. Formation of RCS and prostacyclin

The metabolism of arachidonic acid (AA) was studied in perfused isolated hearts from guinea pigs. The coronary effluent was continuously bioassayed for prostaglandin-like substances (PLS) using the cascade technique of Vane. Injections of AA in doses between 1--50 microgram into the perfusion fluid prior to the heart produced vasodilatation of the coronary vascular bed followed by a contraction of the rat stomach strip (RSS), chick rectum (CR) and rat colon (RC) as well as relaxation of the bovine coronary artery (BCA). At the higher doses of AA there was also contraction of the rabbit aorta (RbA). The same pattern of effects on the bioassay tissues was seen when prostaglandin endoperoxide (PGH2) was perfused through the heart. The response of the bank of superfused tissues provided evidence for the formation of prostacyclin (PGX or PGI2), PGE2 and PGF2alpha. Chromatographic studies showed that 6-oxo-PGF1alpha together with other prostaglandins was present in the perfusate after acidification, which suggested that the bovine coronary relaxing substance consists mainly of PGI2. Moreover, the rabbit aorta contracting substance (RCS) released in the perfusate was due to prostaglandin endoperoxides and not to thromboxane (TXA2). The formation of PLS from AA was completely blocked after treatment of the heart with the cyclo-oxygenase inhibitors, indomethacin or meclofenamic acid. Pretreatment of the heart with 15-hydroperoxyarachidonic acid (15-HPAA), a selective inhibitor of prostacyclin synthetase, inhibited the effect of AA on the coronary vasculature and diverted the metabolic transformation of AA towards PGE2 and PGF2alpha.

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.

Release of sphingosine-1-phosphate from human platelets is dependent on thromboxane formation.

Summary.  Background: Platelets release the immune‐modulating lipid sphingosine‐1‐phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. Objectives: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods: S1P was detected using thin‐layer chromatography in [3H]sphingosine‐labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results: Release of S1P from platelets was stimulated with protease‐activated receptor‐1‐activating peptide (PAR‐1‐AP, 100 μm). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500‐mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U‐46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC‐transport. Furthermore, thrombin‐induced release of S1P was attenuated in platelets from TP‐deficient mice. Supernatants from PAR‐1‐AP‐stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P‐dependent manner via S1P receptors‐1 and ‐3. These effects were inhibited by ASA‐pretreatment of platelets. Conclusions: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti‐inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.