Thomas Hohlfeld

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.

Drug/drug interaction of common NSAIDs with antiplatelet effect of aspirin in human platelets

Nonsteroidal anti-inflammatory drugs (NSAIDs) may interfere with the anti-platelet activity of aspirin at the level of the platelet cyclooxygenase-1 (COX-1) enzyme. In order to examine the interference of common NSAIDs with the anti-platelet activity of aspirin the human platelet rich plasma from voluntary donors was used for arachidonic acid-induced aggregation and determination of thromboxane synthesis. Further, docking studies were used to explain the molecular basis of the NSAID/aspirin interaction. The experimental results showed that celecoxib, dipyrone (active metabolite), ibuprofen, flufenamic acid, naproxen, nimesulide, oxaprozin, and piroxicam significantly interfere with the anti-platelet activity of aspirin, while diclofenac, ketorolac and acetaminophen do not. Docking studies suggested that NSAIDs forming hydrogen bonds with Ser530, Arg120, Tyr385 and other amino acids of the COX-1 hydrophobic channel interfere with antiplatelet activity of aspirin while non interfering NSAIDs do not form relevant hydrogen bond interactions within the aspirin binding site. In conclusion, docking analysis of NSAID interactions at the COX-1 active site appears useful to predict their interference with the anti-platelet activity of aspirin. The results, demonstrate that some NSAIDs do not interfere with the antiplatelet action of aspirin while many others do and provide a basis for understanding the observed differences among individual non-aspirin NSAIDs.

Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis.

Summary.  Background: Anti‐inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug–drug interaction at the level of platelet cyclooxygenase‐1 (COX‐1).

Cardiospecific Overexpression of the Prostaglandin EP3 Receptor Attenuates Ischemia-Induced Myocardial Injury

Background—The generation of prostaglandin E2 (PGE2) is significantly increased in acute myocardial ischemia and reperfusion. PGE2, in addition to other prostaglandins, protects the reperfused ischemic myocardium. It has been hypothesized that this cardioprotection is mediated by E-type prostaglandin receptors of the Gi-coupled EP3 subtype. Methods and Results—We tested this hypothesis by generating transgenic (tg) mice with cardiospecific overexpression of the EP3 receptor. According to ligand binding, a 40-fold overexpression of the EP3 receptor was achieved in membranes prepared from tg hearts compared with wild-type (wt) littermates. In isolated cardiomyocytes from tg mice, the forskolin-induced rise in cAMP was markedly attenuated, indicating coupling of the overexpressed EP3 receptor to inhibitory G proteins (Gi) with constitutive receptor activity. There was no evidence for EP3 receptor coupling to Gq-mediated protein kinase C signaling. Isolated hearts from tg and wt mice were subjected to 60 minutes of no-flow ischemia and 45 minutes of reperfusion. In tg hearts, ischemic contracture was markedly delayed compared with wt hearts, and the ischemia-induced increase in left ventricular end-diastolic pressure was reduced by 55%. Creatine kinase and lactate dehydrogenase release was significantly decreased by 85% and 73%, respectively, compared with wt hearts. Conclusions—Constitutive prostaglandin EP3 receptor signaling exerts a protective effect on cardiomyocytes, which is probably Gi mediated and results in a remarkable attenuation of myocardial injury during ischemia and reperfusion. Cardioprotective actions of E-type prostaglandins may be mediated by this receptor subtype.

Activation of IP and EP3 receptors alters cAMP-dependent cell migration

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G(s)-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP(3) receptors with opposite (i.e., G(i)) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP(3) receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP(3) receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP(3)). Chemotaxis of CHO cells transfected with G(s)-coupled IP receptors was concentration-dependently inhibited by iloprost (2-100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP(3) receptors showed a significant, concentration dependent (1-100 nM) increase of cell migration in presence of the selective EP(3) agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G(s)-mediated increase of intracellular cAMP. EP(3) receptors conversely stimulate CHO migration.

Expression, Function, and Regulation of E-Type Prostaglandin Receptors (EP3) in the Nonischemic and Ischemic Pig Heart

The action of prostacyclin, prostaglandin E1 (PGE1), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [3H]PGE1, with a Kd of 3.7 nmol/L and a Bmax of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype-selective agonists sulprostone (EP1 and EP3) and M&B 28.767 (EP3) were active, suggesting the presence of an EP3 receptor subtype. PGE1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP3 receptor coupling to an inhibitory G protein (Gi). Additional in vivo experiments showed that intracoronary infusion of PGE1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of beta-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg i.v.), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-gamma-S-induced decrease of [3H]PGE1 binding. These data demonstrate for the first time that myocardial receptors for PGE1 belong to the EP3 subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.

Detection and duration of aspirin resistance after coronary artery bypass grafting

A spirin resistance is associated with a significant increase in major adverse cardiovascular events. We have previously reported an insufficient antiplatelet effect of aspirin in most patients after coronary artery bypass grafting (CABG). Nevertheless, the duration of aspirin resistance after CABG remains unknown, and the suitability of available tools to discriminate those with reduced response to aspirin is controversial. This work determined the antiplatelet effect of aspirin at the time of CABG and 6 months later and examined the reliability of different methods for the diagnosis of aspirin resistance.

Influence of synaptic serotonin level on [18F]altanserin binding to 5HT2 receptors in man

The feasibility of in vivo serotonin 5HT(2) receptor binding measurement using [18F]altanserin as a radioligand has been well established. In this study, the postsynaptic receptor binding potential of this ligand was examined as a possible indicator of synaptic serotonin content after pharmacological challenge. Studies were performed in 11 subjects with a history of recurrent major depression. Six of them received serotonergic antidepressive treatment at the time of the experiment, the other five patients were untreated. Two PET measurements were carried out in each subject within 2 or 3 days. Before one of the measurements, 25 mg of the serotonin re-uptake inhibitor clomipramine were given intravenously, the other measurement was done without pharmacological challenge. The data were analyzed using non-linear least-square regression and Logans graphical method. In the whole group of subjects, binding potential and distribution volume of altanserin decreased following clomipramine challenge. The decrease was between 14 (P=0.03) and 23% (P=0.004). This effect was mainly seen in subjects not on antidepressive medication. Clomipramine challenge probably increased the synaptic serotonin level, which competed with altanserin leading to the lowered binding potential. The paradigm might, thus, be useful to estimate serotonin release in vivo. Pretreatment with serotonergic antidepressants reduces the effect of clomipramine.

Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity

OBJECTIVE The cardioprotective effects of trapidil on ischemic reperfused (I/R) rabbit hearts were studied. Recently, we had shown that trapidil might activate protein kinase A (PKA). In this study, we examined the exact mode of PKA stimulating activity of trapidil. Finally, we investigated the effect of trapidil on the phosphorylation state of phospholamban (PLB), a major PKA target in the heart and key regulator of Ca(2+) sequestration via the sarcoplasmic reticulum Ca(2+)-ATPase. METHODS Langendorff-hearts of New Zealand White rabbits were perfused at constant volume and subjected to global low-flow ischemia for 2 h, followed by 1 h of reperfusion. Subsequently, hearts were used for Western blot analysis of PLB phosphorylation. Furthermore, three different regulatory subunits and one catalytic subunit of PKA were overexpressed in E. coli. These PKA subunits were purified and used in an in vitro assay system to test the impact of trapidil on PKA activities in the absence and presence of cAMP. RESULTS I/R resulted in a significant increase in left ventricular end-diastolic pressure and creatine kinase efflux in the hearts. Trapidil (10 microM) prevented these alterations. Using recombinant cAMP-free PKA isoforms, it was found that trapidil specifically stimulated PKAII but only did so in the presence of small amounts of added cAMP. Furthermore, the PKA-dependent 16Ser phosphorylation of PLB was markedly reduced in I/R. Trapidil largely normalized the 16Ser phosphorylation of PLB. CONCLUSIONS The data demonstrate cardioprotective actions of trapidil in I/R and show a PKAII-dependent cAMP sensitizing effect of the compound. They also indicate PKA-dependent PLB phosphorylation as a target, suggesting an improved Ca(2+) uptake by the sarcoplasmic reticulum. This action might be involved in the cardioprotective effects of trapidil.

Overexpression of prostaglandin EP3 receptors activates calcineurin and promotes hypertrophy in the murine heart

AIMS Prostaglandin E(2) (PGE(2)) has been shown to mediate anti-ischaemic effects and cardiomyocyte hypertrophy and there is evidence for an involvement of the prostaglandin EP(3)-receptor subtype. This study focuses on the EP(3)-mediated hypertrophic action and investigates intracellular signalling pathways of the EP(3)-receptor subtype in the murine heart. METHODS AND RESULTS Cardiac function was analyzed in vivo by magnetic resonance imaging (MRI) in transgenic (tg) mice with cardio-specific overexpression of the EP(3) receptor in comparison with wild-type (wt) mice. Left ventricular (LV) function was determined in isolated perfused hearts subjected to 60 min of zero-flow ischaemia and 45 min of reperfusion. Calcineurin activity and nuclear activity of nuclear factor of activated T-cells (NFAT) were determined by a modified malachite green assay and ELISA, respectively. Extracellular matrix compounds were analyzed by RT-PCR and histology. MRI indicated a significant increase in end-diastolic and end-systolic volume in tg hearts. LV ejection fraction was severely decreased in tg hearts while the relative LV mass was significantly increased. In Langendorff perfused hearts, EP(3)-receptor overexpression resulted in a marked blunting of the ischaemia-induced increase in LV end-diastolic pressure and creatine kinase release. Analysis of EP(3)-receptor-mediated signalling revealed significantly increased calcineurin activity and nuclear activity of NFAT in tg hearts. Moreover, elevated mRNA levels of collagen types I and III as well as the collagen-binding proteoglycans biglycan and decorin were detected in tg hearts. CONCLUSION EP(3)-receptor-mediated signalling results in a significant anti-ischaemic action and activation of the pro-hypertrophic calcineurin signalling pathway, suggesting the involvement of the EP(3) subtype in both PGE(2)-mediated cardioprotection as well as cardiac hypertrophy.