Endothelial progenitor cells bind and inhibit platelet function and thrombus formation.

BACKGROUND: Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. METHODS AND RESULTS: Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. CONCLUSIONS: Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

Health research and innovation: Can we optimize the interface between startups/pharmaceutical companies and academic health care institutions or not?

Although France has numerous assets in the realm of health care, such as the excellence of its research teams, the reputation of its healthcare system, and the presence of many startups, all of which are necessary to become a leader in innovation, it also has combined cultural and regulatory barriers that limit the flexibility and efficiency of interactions between companies/startups and public health institutions. Therefore, the aim of the roundtable discussion was to optimize the interface between those businesses and institutions. Several institutions have successfully implemented teams and procedures which aim to facilitate this interface, with regard to assessments of technology, services provided, the transfer of biological material, R&D collaboration, and licensing agreements. However, there is still a notable absence of entrepreneurial culture among hospital and academic research practitioners; their training regarding innovation remains insufficient and business-related value-creation is non-existent in their career evolution. Pharmaceutical companies, and particularly startups, often lack knowledge about hospital environments and their constraints. As a result, the recommendations of the roundtable participants are as follows: (1) promote reciprocal acculturation between public health institutions and startups through multidisciplinary training in innovation, promoting project development and staff recognition within the institution, and improving pharmaceutical companies' understanding regarding the health care system; (2) provide those involved with means and resources dedicated to innovation by reserving time for innovation at work, securing the status of the staff involved, and aiding in the search for funding; (3) develop and use standard methodologies and tools; and (4) co-design and co-construct innovative health solutions, encouraging the emergence of participatory and interdisciplinary creative spaces. All of these recommendations should help to make the interface between startups/companies and public health institutions more fluid and attractive for those in the health sector.

Matching the evaluation of the clinical efficacy of clopidogrel to platelet function tests relevant to the biological properties of the drug.

OBJECTIVES: This study aimed to explore platelet function tests relevant to the biological effects of clopidogrel that could help the clinical monitoring of drug efficacy. BACKGROUND: Clopidogrel selectively inhibits the P2Y12 receptor, the major role of which is stabilization of aggregation, whereas initiation of aggregation depends on activity of both P2Y1 and P2Y12 receptors. METHODS: Tests used were peak aggregation (Agg(max)) and late aggregation (Agg(6min)), and disaggregation, relating to P2Y1 and P2Y12 activity, respectively; and monoclonal antibody binding activated glycoprotein (GP) IIb/IIIa receptors (PAC-1) and P-selectin, measuring activation and secretion. A first study compared hirudin/PPACK (r-hirudin and D-phenylalanyl-prolyl-arginine chloromethyl ketone) with citrate as blood anticoagulant (16 patients), and a second control study compared the effects of clopidogrel, aspirin, or both (20 normal controls). RESULTS: Clopidogrel similarly inhibited adenosine 5'-diphosphate (ADP)-induced Agg(max) with either anticoagulant, but significantly more Agg(6min) (75% vs. 31%), P-selectin (72% vs. 53%), and PAC-1 (62% vs. 24%) in hirudin/PPACK. In the control study, it inhibited Agg(max) by 22%, and Agg(6min), P-selectin, and PAC-1, by 69%, 66%, and 55%, respectively (all p < 0.05). Disaggregation at six min reached 62% with clopidogrel, but was virtually absent with placebo and aspirin. Non-responsiveness as evaluated by inhibition of Agg(max) in citrate was diagnosed in 35% of patients; in half this rate by Agg(6min), P-selectin, and PAC-1; and in 6% to 12% with the latter tests performed in hirudin/PPACK. CONCLUSIONS: The evaluation of clopidogrel responsiveness by platelet function tests is largely influenced by the choice of blood preservative and functional tests. Measures of aggregation stabilization, and of consequent secretion and activation, identified most patients as responders, contrasting with measures of peak aggregation, by likely reflecting better the interactions clopidogrel and the P2Y12 receptor.

Variations of dopamine, serotonin, and amino acid concentrations in Noda epileptic rat (NER) retina.

Noda epileptic rats (NER) exhibit frequent spontaneous tonic-clonic convulsions which represent a valuable model of human epilepsy. If implication of brain neurotransmitters was largely reported, little is known about retina. However, it has been reported that human epilepsy syndrome varies not only with the location of seizure foci but also according to rhythmic patterns, for which retina has a major role in the transmission of external light-dark cycle information. The purpose of this work was to evaluate dopamine (DA), DA metabolites, serotonin (5-HT), and amino acid [glutamate, aspartate, glycine, gamma aminobutyric acid (GABA), and taurine] level variations in retina from NER, at two different nycthemeral periods (11 a.m. and 11 p.m.) and at different ages (2, 6, and 12 months). In NER, retinal dopaminergic function was decreased as soon as 2 months, whereas GABA levels were increased, even if no differences among the different ages could be distinguished. These variations were associated to a slight increase in 5-HT. Other amino acids tested were not affected by epilepsy, whereas taurine decreased with aging in NER as well as in control rats. Retinal 5-HT occurs principally as a precursor of melatonin (MEL). A triangular interaction may be hypothesized: MEL could decrease DA synthesis or release by enhancing GABA activity. Taken together, these results suggest that the retinal physiology is affected by the epileptic status and that information transmitted from retina to the brain should be affected by epilepsy in NER.

In vivo comparative antithrombotic effects of ioxaglate and iohexol and interaction with the platelet antiaggregant clopidogrel.

RATIONALE AND OBJECTIVES: Experiments were designed to (1) compare the effects of iodinated contrast media (CM) on a rat model of arterial thrombosis, (2) evaluate which element of the ioxaglate solution supports its antithrombotic activity, and (3) investigate the interaction of ionic and non-ionic CM with the antiplatelet agent clopidogrel. MATERIALS AND METHODS: Carotid thrombosis was induced in rats by extravascular application of a filter paper soaked in FeCl3 (35% vol/wt), proximal to an ultrasonic flow probe. (1) The antithrombotic potential of low-osmolar ionic (ioxaglate Na/meglumine) or nonionic contrast media (iohexol and iodixanol) (all 1600 mg iodine/kg, IV) was assessed by measuring the time to occlusion (TTO) of the carotid artery and the thrombus weight (TW). (2) Isotonic saline and iso-osmolar (280 mOsm/kg) and hyperosmolar (560 mOsm/kg) solutions of meglumine hydrochloride, meglumine ioxaglate (560 mOsm/kg), sodium ioxaglate (600 mOsm/kg) and sodium and meglumine ioxaglate (commercial solution) were tested under similar conditions. (3) Interaction with clopidogrel was tested by injecting lower dose of CM (960 mg iodine/kg) 2 hours after clopidogrel (2 mg/kg per os). RESULTS: (1) Ioxaglate prolonged TTO when compared with saline (30.0 +/- 1.1 minute vs. 19.6 +/- 2.4 minutes, P< 0.001), whereas iohexol had no effect (21.3 +/- 1.3 minutes). Ioxaglate's effect was associated with a reduction in TW with ioxaglate versus saline (2.6 +/- 0.4 mg and 4.7 +/- 0.7 mg, respectively, P< 0.05) whereas TW remained unchanged in the iohexol group (4.2 +/- 0.4 mg). The nonionic dimer iodixanol induced a direct vasoconstrictor effect on the carotid artery and was consequently excluded from the study. (2) Neither iso-osmolar nor hyperosmolar solutions of meglumine had any effect on TTO whereas both sodium and meglumine salts of ioxaglic acid prolonged TTO, suggesting that the antithrombotic effect of ioxaglate is mediated by the ioxaglic acid moiety alone as neither meglumine, osmolality or sodium played a significant role. (3) A synergistic effect on TTO was found when ioxaglate was associated with clopidogrel whereas no such effect was observed with iohexol. CONCLUSIONS: These data show a greater in vivo antithrombotic potential for the ionic contrast medium ioxaglate than for the non-ionic contrast medium iohexol and, for the first time, a synergistic effect between a contrast medium and a platelet antiaggregant drug in vivo.

Clopidogrel 600-mg double loading dose achieves stronger platelet inhibition than conventional regimens: results from the PREPAIR randomized study.

OBJECTIVES: The objective of this study was to compare the level of platelet inhibition achieved by 3 different clopidogrel loading regimens in patients undergoing elective angiography and percutaneous coronary intervention when appropriate. BACKGROUND: Optimal platelet inhibition is a key therapeutic goal for patients undergoing percutaneous coronary intervention. Although 600 mg has been described as the maximum absorbed dose when given as a single bolus, the effects of 2 boluses given 24 h apart have not been described. METHODS: Patients (n = 148) were randomly assigned to one of 3 regimens: Group A, clopidogrel 300 mg the day before (>or=15 h) + 75 mg the morning of the procedure; Group B, clopidogrel 600 mg the morning of the procedure (>or=2 h); and Group C, clopidogrel 600 mg the day before (>or=15 h) and 600 mg the morning of the procedure (>or=2 h). Blood samples were obtained at baseline and immediately before angiography. Peak and late platelet aggregation were measured in platelet rich plasma, with researchers blinded to treatment allocation. RESULTS: There was a consistent difference favoring Group C in all aggregation parameters. Percent inhibition in Groups A, B, and C was 31.4%, 29.0%, and 49.5%, respectively, for peak aggregation (5 micromol/l adenosine diphosphate; p < 0.0001) and 54.1%, 57.7%, and 81.1%, respectively, for late aggregation (p < 0.0001). Similar striking reductions were observed when 20 micromol/l adenosine diphosphate was used. All comparisons between Group C and the other 2 groups were statistically significant, and those between Groups A and B were not. CONCLUSIONS: Clopidogrel 600-mg double bolus achieves greater platelet inhibition than conventional single loading doses.

Coping with new challenges in acute coronary syndromes.

The acute coronary syndromes have become well-established clinical entities and are used as working diagnoses tied in with elaborate investigation and treatment practice guidelines. However, these syndromes remain challenging because their prevalence, morbidity and mortality remain high. Furthermore, these syndromes are demanding on the health care system because an early invasive management strategy is recommended for most patients with these conditions. Coping with these challenges requires the following initiatives: stepping beyond current diagnostic and management algorithms to a better performing risk stratification scheme that considers specific patient risk factors in addition to the disease risk, and a reintegration of acute coronary syndromes into the more global perspective of coronary artery disease; more effective antithrombotic therapy that does not further compromise bleeding risk, or drugs that control thrombogenic stimuli; and medical and revascularization therapies targeted to more specific individual pathophysiologies identified by novel blood markers and imaging techniques.

Inhibition and reversal of platelet aggregation by alphaIIbbeta3 antagonists depends on the anticoagulant and flow conditions: differential effects of Abciximab and Lamifiban.

Shear influences platelet aggregate formation and stability, as well as the inhibitory capacities of antithrombotic drugs. We compared the inhibitory and disaggregating properties of two distinct alphaIIbbeta3 antagonists, Abciximab and Lamifiban, on platelet aggregation induced by adenosine diphosphate (ADP) (5 micromol/l) in platelet-rich plasma (PRP), in an aggregometer (poorly defined low shear, <100/s) and in a microcouette at arterial shear rate (1,000/s). Platelet aggregation was detected by changes in light transmission in the aggregometer (TA), and by particle counting with a flow cytometer (PA). Lamifiban (1 mumol/l) completely inhibited TA or PA induced by ADP in citrated PRP in the aggregometer or microcouette. In contrast, Abciximab (2 micromol/l) only partially inhibited PA in the microcouette while blocking both TA and PA in the aggregometer. Moreover, Abciximab did not reverse platelet aggregates formed either in the microcouette or in the aggregometer, whereas Lamifiban caused complete reversal. On the contrary, Abciximab completely inhibited platelet aggregation induced by ADP in hirudin/d-Phe-Pro-Arg-chloromethylketone PRP in the microcouette. Our results demonstrate a marked dependence of inhibitory capacity of Abciximab on shear conditions, with citrate anticoagulant responsible for the residual aggregation, in contrast to Lamifiban, another alphaIIbbeta3 antagonist interacting with a distinct site on beta3.

Serotonin synthesis and its light-dark variation in the rat retina.

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.