Adolescent ischemic stroke associated with anabolic steroid and cannabis abuse.

We report on a 16-year-old body builder who suffered from an acute ischemic stroke. In the urine, cannabis metabolites as well as metabolites of the oral androgenic-anabolic steroid methandrostenolone were detected, both known to be associated with stroke events. This report highlights the role of cannabis and steroid abuse that induce strokes in the absence of arteriopathy, cardioembolism or thrombophilia. Owing to new upcoming socio-behavioral aspects of late childhood and early adolescent life, this formally rare abuse of cannabis and/or anabolic steroids as well as their associations with strokes becomes more current than ever.

Expression and translation of the COX-1b gene in human cells--no evidence of generation of COX-1b protein.

Cyclooxygenase 1b (COX-1b) is a splice variant of COX-1, containing a retained intron 1 within the signal peptide sequence. COX-1b mRNA is found in many species, but the existence of a functionally active protein, which is possibly related to different species-dependent lengths of intron 1, is controversially discussed. The human intron 1 comprises 94 bp, and the resulting frameshift at the intron 1-exon 2 junction creates a premature stop codon. Nevertheless, full-length human COX-1b protein expression, including translated intron 1 and the signal peptide, has been reported and was explained by a frameshift repair. In this study, the fate of COX-1b mRNA in a human overexpression system is analyzed. Independent of the hypothetical frameshift repair mechanism, the splicing of the COX-1b intron 1, resulting in COX-1 mRNA and removal of the signal peptide during protein maturation, with subsequent generation of a COX-1 protein is demonstrated.

Crossed cerebellar diaschisis after status epilepticus in a young child.

We report on a 3.8-year-old girl who was born preterm. Due to a posthemorrhagic hydrocephalus she had a ventriculoperitoneal shunt. Magnetic resonance imaging (MRI) showed mild atrophy of the left cerebellum. She was found unresponsive in a febrile state. After the application of midazolam she regained consciousness. There were no epileptic discharges on electroencephalogram. MRI with diffusion-weighted sequences showed areas of hyperintensity in the right cerebrum. After the patient deteriorated again, MRI showed signs of increased intracranial pressure and high signal intensity throughout the right cerebral and left cerebellar hemispheres, suggesting crossed cerebellar diaschisis (CCD) most likely resulting from a nonconvulsive status epilepticus (SE). A follow-up MRI showed progressive brain atrophy. CCD after SE might be caused by cortical excitatory input through the cortico-pontine-cerebellar pathway. Alternatively, the cerebral edema in SE may decrease neuronal cell activity in the contralateral cerebellar hemisphere. The unilateral cerebellar atrophy before the onset of CCD might be attributed to impaired neuronal connections after peripartal cerebral injury. This case presents a young child with a combination of two CCDs, at first due to perinatal brain injury, and at second to SE. MRI with diffusion-weighted sequences can detect CCD at an early stage.

Determination of thromboxane formation, soluble CD40L release and thrombopoietin clearance in apheresis platelet concentrates.

All deleterious changes in platelet morphology, structure and function that occur in platelet concentrates (PC) during storage are titled as the 'platelet storage lesion'. No single in vitro test currently available is sufficient in assessing these changes of platelet quality. The release of soluble CD40 Ligand (sCD40L), the formation of thromboxane (TXB2) and the thrombopoietin (TPO) clearance reflect different aspects of platelet metabolism and activitiy, and were used to examine platelet quality in apheresis platelet products. At days 1, 3 and 5, in single-donor apheresis platelet products (n = 10) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). TPO (measured by ELISA) was determined after an incubation time of 5 h at 37°C with platelet-rich plasma (adjusted initial TPO concentration of about 500 pg/mL). Results were related to a therapeutic unit (TU = 2 × 10(11) platelets). Immediately after platelet preparation, sCD40L release was 41 ± 7.6 ng/TU, TXB2 formation 1688 ± 374 ng/TU and TPO clearance 1.22 ± 0.32 ng/h/TU. At days 1, 3 and 5, sCD40L was reduced to 89 ± 7%, 71 ± 12% and 57 ± 9%, TXB2 release to 91 ± 6%, 74 ± 12% and 58 ± 9% and TPO clearance to 90 ± 15%, 84 ± 5% and 79 ± 10% of the respective control values. In conclusion, in single-donor apheresis PC, sCD40L release and TXB2 formation as well as TPO clearance by the platelets were dependent on storage duration and reduced to about 60% to 80% of the respective control values after a storage period for 5 days. These findings are in line with literature data, indicating that a loss of platelet functionality of about 30% will occur after 5 days of storage.

8-pCPT-conjugated cyclic AMP analogs exert thromboxane receptor antagonistic properties.

Membrane-permeable 8-(4-chlorophenylthio)-2'-O-methyl cyclic AMP (8-pCPT-2'-O-Me-cAMP) has been shown to specifically activate cAMP-regulated Epac proteins, without direct effects on protein kinase A and protein kinase G. During isometric tension measurements in thoracic aortic rings from Wistar rats, we observed that 8-pCPT-2'-O-Me-cAMP selectively induced a rightward shift of the concentration response curve for the thromboxane mimetic U46619, without altering the contractile response to noradrenaline. We hypothesised that 8-pCPT-2'-O-Me-cAMP and similar compounds may function as direct thromboxane receptor antagonists. Indeed, in addition to 8-pCPT-2'-O- Me-cAMP, also 8-pCPT-cAMP, 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS) and 8-CPT-adenosine, but not 8-Bromo-2'-O-Me-cAMP, induced rightward shifts of the contractile response to U46619. Likewise, 8-pCPT-2'-O- Me-cAMP and Rp-8-CPT-cAMPS, but not 8-Bromo-2'-O-Me-cAMP, specifically reduced U46619-induced aggregation of human platelets. In addition, 8-pCPT-2'-O-Me-cAMP and Rp-8-CPT-cAMPS completely reversed U46619-induced reduction of intercellular adhesion molecule-1 expression and migration of human coronary artery endothelial cells. Most important, the cAMP analogs that reduced the contractile response to U46619 also concentration-dependently inhibited binding of the thromboxane receptor radioligand [5,6-3H]SQ29548 to human platelets. We conclude that 8-pCPT-conjugated cAMP analogs exert competitive thromboxane receptor antagonistic properties.

Thrombin receptors in vascular smooth muscle cells - function and regulation by vasodilatory prostaglandins.

The vast majority of thrombin (>95%) is generated after clotting is completed, suggesting that thrombin formation serves purposes beyond coagulation, such as tissue repair after vessel injury. Two types of vascular thrombin binding sites exist: protease-activated receptors (PARs) and thrombomodulin (TM). Their expression is low in contractile vascular smooth muscle cells (SMC), the dominating subendothelial cell population, but becomes markedly up-regulated upon injury. In human SMC, PAR-1, PAR-3, and PAR-4 mediate thrombin-induced proliferation, migration and matrix biosynthesis as well as generation of inflammatory and growth-promoting mediators. Thrombin-responsive PARs are transcriptionally down-regulated in human vascular SMC by vasodilatory prostaglandins (PGI2/PGE2). For PAR-1 and PAR-3 this mechanism involves cAMP-dependent inactivation of the transcription factor NFAT. The human PAR-4 promoter does not possess NFAT recognition motifs suggesting involvement of other cAMP-regulated effectors. Unlike PARs, TM is induced in SMC exposed to vasodilatory prostaglandins. Enhanced thrombin binding to TM might ameliorate PAR-mediated SMC stimulation. Also expressed in human SMC is the endothelial protein C receptor (EPCR), which serves as an anchor to facilitate generation of activated protein C (aPC) by TM-bound thrombin. Whether prostaglandins affect aPC-generation is not known. In SMC, thrombin and aPC act synergistically via PAR-1 to modify tissue remodelling, in contrast to their antagonistic interaction in the coagulation pathways. Overall, this will contribute to plaque stability and wound healing. The processes outlined here are likely to become clinically relevant after up-regulation of vascular cyclooxygenase2, the rate limiting step in vascular PGE2/PGI2 biosynthesis, such as in advanced atherosclerosis and acute coronary syndromes.

Preliminary evidence for a matrix metalloproteinase-2 (MMP-2)-dependent shedding of soluble CD40 ligand (sCD40L) from activated platelets.

Platelets are the major source of soluble CD40 ligand (sCD40L) in the blood. It has been demonstrated that CD40L is cleaved from the surface of activated platelets to release sCD40L. However, the enzyme involved in sCD40L shedding has not been identified yet. Using a panel of pharmacological inhibitors of serine, cysteine, aspartate, or metalloproteinases, preliminary evidence is presented for the hypothesis that matrix metalloproteinase-2 (MMP-2) might be the protease, primarily responsible for CD40L cleavage from platelet surface.

B cell receptor-induced growth arrest and apoptosis in WEHI-231 immature B lymphoma cells involve cyclic AMP and Epac proteins.

Signaling by the B cell antigen receptor (BCR) is essential for B lymphocyte homeostasis and immune function. In immature B cells, ligation of the BCR promotes growth arrest and apoptosis, and BCR-driven balancing between pro-apoptotic extracellular signal-regulated kinase 1 and 2 (ERK1/2) and anti-apoptotic phosphoinositide 3-kinase-dependent Akt seems to define the final cellular apoptotic response. Dysfunction of these late BCR signaling events can lead to the development of immunological diseases. Here we report on novel cyclic AMP-dependent mechanisms of BCR-induced growth arrest and apoptosis in the immature B lymphoma cell line WEHI-231. BCR signaling to ERK1/2 and Akt requires cyclic AMP-regulated Epac, the latter acting as a guanine nucleotide exchange factor for Rap1 and H-Ras independent of protein kinase A. Importantly, activation of endogenously expressed Epac by a specific cyclic AMP analog enhanced the induction of growth arrest (reduced DNA synthesis) and apoptosis (nuclear condensation, annexin V binding, caspase-3 cleavage and poly-ADP-ribose polymerase processing) by the BCR. Our data indicate that cyclic AMP-dependent Epac signals to ERK1/2 and Akt upon activation of Rap1 and H-Ras, and is involved in BCR-induced growth arrest and apoptosis in WEHI-231 cells.

Differential effects of factor IIa inhibitors on the endogenous thrombin potential.

Calibrated automated thrombin generation measurement in clotting plasma (endogenous thrombin potential, ETP) is being used increasingly to monitor the effects of anticoagulant drugs. Calibrated automated thrombography measures the concentration of thrombin in clotting plasma by monitoring the cleavage of a fluorogenic substrate (Z-Gly-Gly-Arg-7-amino-4-methylcoumarin) and comparing it with a constant known thrombin activity in a parallel nonclotting sample. This study compared the concentration-dependent effects of different factor IIa inhibitors on the ETP. In accordance with a theoretical prediction, the monovalent factor IIa inhibitors melagatran and argatroban reduced peak thrombin concentrations without a marked effect on the lag time of thrombin generation. However, both bivalent factor IIa inhibitors lepirudin and bivalirudin markedly prolonged the lag time of thrombin generation and thus behaved like "super" factor Xa inhibitors according to the theoretical model. These findings have important consequences for therapeutic drug monitoring using thrombin generation assays. For monitoring of hirudins, the lag time of thrombin generation seems to be a very sensitive parameter. In contrast, for monitoring of argatroban, the evaluation of the ETP seems to be useful.

Impaired interaction between platelets and endothelial progenitor cells in diabetic patients.

This editorial focuses on new data on the role of platelets of the recruitment of endothelial progenitor cells to sites of vascular lesion and their differentiation into endothelium. Questions of further research are raised.

Aspirin "resistance".

Recent clinical studies have shown that the expected antiplatelet effect of aspirin is not always achieved. From the laboratory point of view, resistance to aspirin is the inability to achieve the expected inhibition of platelet cyclooxygenase-(COX-)1 with prevention of platelet thromboxane (TX) A2 formation. The failure to prevent atherothrombotic events (treatment failure) must be distinguished from aspirin resistance. Nevertheless, different definitions of aspirin resistance complicate the assessment of published data, a problem aggravated by discordant results of the available diagnostic laboratory techniques.The pharmacological mechanisms of aspirin resistance are not completely understood. Potential causes include pharmacokinetic and pharmacodynamic issues, such as reduced bioavailability, increased platelet turnover, interactions with nonsteroidal anti-inflammatory drugs, comorbidities (hypercholesterolemia or diabetes mellitus), alternative pathways of platelet activation, and genetic polymorphisms. Clinical trials demonstrated a negative impact of aspirin resistance on the clinical outcome: an about fourfold increased risk of major atherothrombotic events has been found in aspirin nonresponders suffering from vascular disease.An individualized antiplatelet therapy with aspirin will have to consider the possibility of aspirin resistance. Thus, standardized and inexpensive diagnostic assays are needed. The identification of aspirin-resistant patients is essential to individually tailor antiplatelet treatment. For example, increasing the dosage of aspirin or alternative antiplatelet drugs are potential therapeutic concepts, but these require careful future investigation.

Methods to evaluate the pharmacology of oral antiplatelet drugs.

Principally, there are two reasons why the pharmacological response to antiplatelet drugs should be measured: on the one hand, an insufficient inhibition of platelet function may result in atherothrombotic complications; on the other hand, an excessive inhibition of platelet function may lead to bleeding complications. The clinical importance to measure the effects of antiplatelet drugs is demonstrated by increasingly growing evidence for an association of resistance to antiplatelet drugs with thromboembolic events. It is often claimed that there is no generally accepted definition of "resistance" and, instead, there is an ongoing semantic discussion about the correct term to be used to describe this phenomenon. From the pharmacological point of view, there is only one acceptable definition of "resistance" to antiplatelet drugs: the term "resistance" should be used when a drug is unable to hit its pharmacological target. Thus, laboratory methods used to evaluate the effects of antiplatelet drugs should be designed to measure the direct pharmacodynamic effect of a drug, rather than the consequences for global platelet function. Based on physiological/pathophysiological, pharmacological, and practical considerations, the authors propose the following assays to be used to measure the effects of oral antiplatelet drugs: for the detection of aspirin actions, thromboxane or arachidonic acid-induced responses (light aggregometry, whole-blood aggregometry) should be measured; for the detection of clopidogrel actions, VASP (vasodilator-stimulated phosphoprotein) phosphorylation (flow cytometry) or ADP-(adenosine diphosphate-)induced responses (light aggregometry, whole-blood aggregometry, possibly also flow cytometry) should be measured.

[Platelet function analysis with point-of-care methods]. / Messung der Thrombozytenfunktion mit Point-of-Care-Methoden.

BACKGROUND: More and more patients are treated with antiplatelet drugs today. In this context a sufficient inhibition of platelet aggregation, on the one hand, is of essential importance to the efficiency of prophylaxis of myocardial and cerebral infarction and to avoiding thrombosis of drug-eluting stents. On the other hand, this medication can result in an increased risk of perioperative bleeding. In both situations control of the efficiency of therapy or rather the assessment of the impairment of hemostasis is of vital importance. METHODS: Platelet function analyzer (PFA-100), multiple platelet function analyzer (Multiplate), and rotational thrombelastometry (ROTEM) are reliable and easy to use point-of-care (POC) devices. Since these three analyzers monitor different aspects of platelet function and have different limitations, the selection of the right test system depends on the right question. RESULTS: PFA-100 enables a sensitive detection of von Willebrand's syndrome. Multiplate is apt to control efficiency of platelet inhibiton with acetylsalicylic acid, clopidogrel and glycoprotein IIb/IIIa receptor antagonists. ROTEM analysis offers the opportunity to assess hemostasis as a holistic system. Thereby, ROTEM analysis particularly detects hyperfibrinolysis, heparin effects, and fibrinogen-platelet interaction. CONCLUSION: Due to their easy handling the described POC devices are applicable to perioperative coagulation management as well as during and after coronary intervention or to monitoring of platelet function in cardiologic practice. They enable a quick assessment of platelet function and an individually guided therapy.

Complete downmodulation of P-selectin glycoprotein ligand in monocytes undergoing apoptosis.

OBJECTIVE: Apoptotic monocytes release membrane microparticles which may play a major role in thrombogenicity through a P-selectin glycoprotein ligand (PGSL-1)-mediated mechanism. We have studied systematically the regulation of PSGL-1 expression and function in apoptotic monocytic cells. METHODS AND RESULTS: PSGL-1 expression (flow cytometry, immunofluorescence microscopy, immunoblot) was virtually abolished in apoptotic monocytes by proteolytic shedding. This was accompanied by a complete loss of PSGL-1-mediated platelet-leukocyte (flow cytometry) and leukocyte-endothelial cell (parallel plate flow chamber) interactions. Systematic screening of protease inhibitors combined with knock-out and siRNA experiments characterized the PSGL-1-cleaving enzyme as an N-ethylmaleimide-inhibitable metalloproteinase of the ADAM family. CONCLUSIONS: Downmodulation of PGSL-1 in apoptotic monocytes may prevent ectopic cell clearance in the peripheral vasculature to reduce local inflammatory and proliferative responses. Depletion of PSGL-1 expression on apoptotic microparticles may also act as a molecular switch to modulate their thrombogenic activity.

Membrane environment rather than tissue factor expression determines thrombin formation triggered by monocytic cells undergoing apoptosis.

Monocyte apoptosis is an important determinant of atherothrombosis. Two major mechanisms for apoptosis-associated thrombogenicity have been described: exposure of negatively charged membrane phospholipids and up-regulation of tissue factor (TF). However, the relative importance of these mechanisms is unclear. Thus, procoagulant functions (thrombin generation) of apoptotic (staurosporine, 2 muM, 24 h) U937 cells versus cell-derived microparticles (MPs) were studied. In apoptotic U937 cells, a significant increase in TF mRNA (real-time PCR), surface expression of TF (flow cytometry), and total cellular amount of TF (Western blotting) was observed. Control cells only minimally triggered thrombin generation (endogenous thrombin potential), and apoptotic cells were highly procoagulant. However, addition of negatively charged membranes completely restored the thrombin generation capacity of control U937 cells to the levels of apoptotic cells. MPs (defined as CD45(+) particles of subcellular size), derived from apoptotic U937 cells, were highly procoagulant but did not exhibit an increased TF expression or annexin V binding. Taken together, our data support the concept that the membrane environment, independent of TF expression, determines the extent of thrombin formation triggered by apoptosis of monocytic cells. Externalization of negatively charged phospholipids represents the most important mechanisms for whole cells. Additional yet unknown mechanisms appear to be involved in the procoagulant actions of MPs derived from apoptotic monocytes.

Delta6-desaturase (FADS2) deficiency unveils the role of omega3- and omega6-polyunsaturated fatty acids.

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and alpha-linolenic acid. EFAs are converted into omega3- and omega6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of delta6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2 -/- mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood-testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2 -/- mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.

Alternative splicing of platelet cyclooxygenase-2 mRNA in patients after coronary artery bypass grafting.

Recently, we cloned from platelet mRNA a novel cyclooxygenase (COX)-2 splice variant, designated COX-2a, which is characterized by a partial deletion of exon 5. Preliminary studies of mRNA distribution of COX-2 isoforms in platelets from coronary artery bypass grafting (CABG) patients showed a variable increase in COX-2a mRNA expression after cardiac surgery. Thus, we assessed whether this variant may play a functional role in these patients. We report a marked (about 200-fold) increase in the expression of COX-2a mRNA after CABG. Evidence is presented that ribosomal frame-shifting may correct the coding sequence resulting in the expression of a full-length COX-2a protein. In addition, a reading frame-corrected COX-2a mutant (COX-2a delta G) was generated by site-directed mutagenesis and expressed in COS-7 cells using an adenoviral expression system. However, COX-2a protein was not active in terms of prostaglandin formation. Thus, alternative mRNA splicing might represent an intriguing posttranscriptional mechanism to oppose a transcriptional activation of the COX-2 gene. Evolutionary, this mechanism may prevent COX-2-dependent thromboxane synthesis in the platelet, which would potentiate the likelihood of thrombosis; pharmacologically, this mechanism would prevent an aspirin-insensitive pathway of thromboxane formation.

Aspirin-induced platelet inhibition in patients undergoing cardiac surgery.

Platelet function and response to pharmacological inhibition are altered by cardiac surgery. For example, aggregation is increased early after aortic valve replacement (AVR) and platelet response to aspirin is often insufficient after coronary artery bypass grafting (CABG). We hypothesized that the effect of aspirin administration after cardiac surgery might be impaired due to platelet activation. Therefore, the antiplatelet effect of aspirin was compared in patients (n = 20 per group) after CABG and AVR surgery (bileaflet prosthesis). Arachidonic acid-induced aggregation (turbidimetry) and thromboxane formation (radioimmunoassay) were determined before and 1, 5, and 10 days after surgery. In CABG-patients, antiplatelet treatment had been discontinued 10 days before surgery. Oral aspirin was started on day 1 after CABG. AVR-patients did not receive oral aspirin. Before surgery, platelet aggregation and thromboxane formation were significantly higher in patients with aortic stenosis. After CABG, thromboxane formation was not significantly changed from control values before surgery (66 +/- 13% on day 10) despite oral aspirin treatment, whereas thromboxane formation in patients undergoing AVR significantly increased compared to values before surgery (216 +/- 29% on day 10). In both groups of patients, 100 micromol/l aspirin in vitro largely inhibited platelet function before surgery, with markedly attenuated effects after surgery. In conclusion, thromboxane formation increased after AVR but not after CABG. The antiplatelet effect of aspirin, therefore, may be impaired after CABG by increased platelet activity. An additional in vitro "resistance" of platelets was seen after both CABG and AVR.