Antagonism of the antithrombotic and anti-atherosclerotic actions of aspirin by rofecoxib in the cholesterol-fed rabbit.

BACKGROUND AND PURPOSE: Most patients at elevated cardiovascular risk receive long-term aspirin (ASA) anti-platelet treatment. The present study specifically addresses the pharmacological interactions between selective COX-2 inhibitors and ASA and the possible consequences for the thrombotic risk during long-term treatment. EXPERIMENTAL APPROACH: New Zealand white rabbits were fed a standard laboratory diet supplemented with 1% cholesterol (CON) for 12 weeks. Age-matched control rabbits were fed the same standard diet without addition of cholesterol (SD). Rabbits were randomly assigned to one of the following groups: rofecoxib (ROFE, 25 mg·kg⁻¹, bid), acetylsalicylic acid (ASA, 5 mg·kg⁻¹, bid) or a combination of both (ASA + ROFE). At the end of the feeding period, the severity of atherosclerotic plaque formation was assessed in the aorta. Thrombus formation was assessed in the left carotid artery using a modified Folts procedure. KEY RESULTS: Treatment of cholesterol-fed rabbits with ASA significantly reduced plaque formation. This reduction in lesion size was not observed in animals treated with the combination of rofecoxib and ASA. In the modified Folts model, treatment with either rofecoxib or ASA increased the total blood flow above that of untreated animals. This increase was statistically significant in the case of ASA, while cotreatment with rofecoxib abolished this ASA effect completely and reduced the total flow rate to the levels seen in untreated hypercholesterolaemic controls. CONCLUSIONS: COX-2 inhibition by rofecoxib attenuates the antithrombotic and anti-atherosclerotic effects of ASA during long-term treatment in cholesterol-fed rabbits.

Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladder cancer cells.

BACKGROUND AND PURPOSE: Sorafenib is an inhibitor of several intracellular signalling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in tumour cells. Sorafenib is used in the therapy of advanced renal cell carcinoma, and several phase II clinical trials are being carried out in patients with urothelial carcinomas. EXPERIMENTAL APPROACH: Using a panel of human bladder cancer cell lines (RT4, T24, J82), we characterized systematically the effects of sorafenib on intracellular signalling, migration, proliferation and apoptosis. KEY RESULTS: We demonstrated that at low concentrations (<1 microM), sorafenib is capable of significantly stimulating migration and proliferation of the bladder cancer cells. We hypothesize that these stimulatory effects on tumour cell functions might be explained by an activation of the Ras/ERK-1/2 signal transduction pathway. In addition, the comparison of different bladder cancer cell lines not only revealed a different biology (e.g. cell migration), but also a differential susceptibility to the anti-apoptotic effects of sorafenib. Finally, we confirmed in different bladder cancer cell lines the known inhibitory actions of sorafenib in pharmacological concentrations (> or =3 microM) on ERK-1/2 phosphorylation, migration and proliferation, as well as the pro-apoptotic effects of the compound. CONCLUSIONS AND IMPLICATIONS: Taken together, these findings suggest that although sorafenib has the potential to be used in the treatment of urothelial carcinoma, this compound might also activate bladder cancer cells at low concentrations. This should be relevant for dosing regiments to optimize the treatment with this promising anti-tumour drug.

Epac inhibits apoptosis of human leukocytes.

cAMP is known to participate in the regulation of apoptosis in leukocytes. Depending on the cell type, pro- and antiapoptotic effects of cAMP have been described. Thus far, most of the cAMP-dependent effects have been attributed to the activation of PKA. However, Epac proteins (direct cAMP targets and guanine nucleotide exchange factors for Ras-like GTPases) have been shown recently to contribute to cAMP-dependent regulation of apoptosis. Therefore, we investigated the effects of the selective Epac activators 8-pCPT and Sp on apoptosis in human leukocytic cells (U937, HL-60, primary human mononuclear cells). We report here that Epac activation inhibits leukocyte apoptosis significantly.

Effects of fibroblast extracellular matrix calcification on platelet adhesion in vitro.

Calcified atherosclerotic lesions are more prone to rupture during angioplasty than non-calcified lesions and are associated with an increased risk of thrombotic complications following angioplasty. This study investigates the possible role of extracellular matrix (ECM) calcification for platelet adhesion. Human cultured fibroblasts (CRL-1635) were subjected to beta-glycerophosphate (10 mM) for 10 to 16 days. Calcification was visualized by von Kossa staining and quantified by the O-cresolphthalein complexone method. Adhesion of calcein-labelled platelets was measured by fluorescence microscopy at static conditions and in a parallel-flow chamber at a shear rate of 1000 s(-1). beta-glycerophosphate treatment resulted in a marked calcification of the ECM. In parallel, a small, albeit significant increase in platelet adhesion under static conditions was observed. In contrast, at flow conditions, the area covered by thrombi was significantly lower when calcified ECM was used. The number of thrombi was not significantly different which is compatible with a smaller thrombus size. Taken together, it appears unlikely that calcification of atherosclerotic lesions contributes to thrombotic complications by an increased platelet adhesion.

[Methods to evaluate aspirin and clopidogrel resistance]. / Methoden zur Messung der Azetylsalizylsäure- bzw. Clopidogrelresistenz.

Based on the concept that the so-called resistance to anti-platelet drugs is meant to describe a phenomenon where the drug does not hit its direct pharmacodynamic target, assays, used to evaluated the effects of anti-platelet drugs, should as closely as possible measure the direct pharmacodynamic effect of a particular drug. Thus, for the detection of aspirin effects, thromboxane concentrations or arachidonic acid-induced responses (light aggregometry, whole-blood aggregometry) should be measured. For the detection of clopidogrel actions, VASP phosphorylation (flow cytometry) or ADP-induced responses (light aggregometry, whole blood aggregometry) should be analysed.

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

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). OBJECTIVE: We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. METHODS: Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. RESULTS: 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. CONCLUSION: This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

Aspirin resistance - does it clinically matter?

A variable responsiveness to acetylsalicylic acid(ASA) is a clinical reality that does not principally differ from variable responses to other kinds of drug treatment in other therapeutic fields. Two questions arise: (i) is any resulting "treatment failure"due to a pharmacological failure of the drug to act and (ii) is any reduced antiplatelet activity to ASA related to the clinical outcome oft he patient?Two major laboratory techniques are available to quantify platelet variability to ASA ex vivo: Measurement of platelet function and measurement of thromboxane formation. Both methods have limitations and did not yet result in a generally accepted definition of a pharmacological ASA "resistance".A "true" pharmacological resistance to ASA exists in selected groups of patients. However, unless more information is available,results from in vitro assays of platelet function should not be over-interpreted. More data from prospective trials are required,predominantly by measuring serum thromboxane formation which is a platelet-specific, ASA sensitive reaction. At this time,there is no reason to change there commended daily maintenance dose of about 100 mg ASA without particular requirements in patients who need coronary protection.

Differential effects of vasodilatory prostaglandins on focal adhesions, cytoskeletal architecture, and migration in human aortic smooth muscle cells.

OBJECTIVE: Cyclooxygenases 1 and 2 are expressed in atherosclerotic arteries, and local generation of prostacyclin and prostaglandin E2 (PGE2) occurs. However, the role of cyclooxygenases and individual prostaglandins during plaque progression is currently uncertain. The present study characterizes the effect of vasodilatory prostaglandins on morphology, focal adhesion (FA) function, and migration in human aortic smooth muscle cells (SMCs). METHODS AND RESULTS: The stable prostacyclin analog iloprost transiently induced: (1) disassembly of FA and stress fibers, (2) partial retraction and rounding of SMCs, (3) hypophosphorylation of FA kinase (FAK) and paxillin, and (4) inhibition of platelet-derived growth factor-BB-induced migration. Inhibition of FAK phosphorylation and morphological changes were mimicked by forskolin, inhibited by H89, and prevented by the protein tyrosine phosphatase inhibitor vanadate and by calpeptin. PGE2 was by far less efficient with respect to all parameters investigated. This difference correlated with the respective cAMP induction in response to iloprost and PGE2. CONCLUSIONS: Inhibition of FAK phosphorylation and FA function is a new target of vasodilatory prostaglandins, which might be causally involved in the antimigratory effects of prostaglandins. Importantly, prostacyclin analogs and PGE2 differ dramatically with respect to dephosphorylation of FAK and inhibition of migration, which might be of relevance for their respective functions in atherosclerosis.

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 alpha-granule secretion were effectively inhibited by 30 or 100 micromol/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.

[Combination therapy with anticoagulants and platelet-active drugs]. / Kombination von Antikoagulanzien und Plättchenfunktionshemmern.

Platelets and the coagulation system cannot be regarded as completely separate systems. Both systems become activated upon vessel injury and multiple functional interactions exist. Thus with a combination therapy with anticoagulants and platelet-active drugs, drug interactions relevant for hemostasis must be expected. This includes a synergistic inhibition of the hemostatic system but also possible synergistic effects on bleeding complications. This concise review discusses the clinical efficacy of a combination therapy with anticoagulants and platelet-active drugs in patients with acute coronary syndromes (unstable angina, myocardial infarction).