Dehydration of blood platelets by zeodration: in vitro characterization and hemostatic properties in vivo.

BACKGROUND: Platelets (PLTs) are currently stored at room temperature (RT) for 5 to 7 days. So far, there exists no validated method for the preparation and long-term storage of dehydrated PLTs suitable for transfusion after rehydration. In this study, a desiccation process, zeodration, was applied to PLTs. STUDY DESIGN AND METHODS: A complete procedure of dehydration at RT by zeodration was employed. Zeodrated human and mouse PLTs were characterized in vitro. Zeodrated mouse PLTs were transfused into clopidogrel-treated mice to evaluate their hemostatic properties. RESULTS: The optimal conditions for dehydration of PLTs at RT in a laboratory scale zeodrator were defined as 145 mbar and 20.2 ± 1.5 °C. The recovery rate was 85 ± 2% and the dryness of zeodrated PLTs (Z_PLTs) indicated that they were sufficiently stable for long-term storage. Rehydrated Z_PLTs were round, were not aggregated, and expressed the glycoproteins required for PLT function. Z_PLTs agglutinated in the presence of von Willebrand factor (VWF) and aggregated in response to thrombin or collagen independently of an active metabolism. In a flow system, Z_PLTs could adhere to VWF and form aggregates on a collagen matrix. Thrombin was generated at the surface of Z_PLTs as efficiently as on fresh PLTs. In clopidogrel-treated mice, which exhibited a severely prolonged bleeding time, continuous perfusion of Z_PLTs restored normal hemostasis. CONCLUSION: Zeodration represents a new strategy to prepare PLTs with partly preserved aggregative properties after storage and rehydration. Z_PLTs have potential hemostatic properties provided it is possible to improve their transfusion efficacy.

Smoking related diseases: the central role of monoamine oxidase.

Smoking is a major risk factor of morbidity and mortality. It is well established that monoamine oxidase (MAO) activity is decreased in smokers. Serotonin (5-HT), a major substrate for MAO that circulates as a reserve pool stored in platelets, is a marker of platelet activation. We recently reported that smoking durably modifies the platelet 5-HT/MAO system by inducing a demethylation of the MAO gene promoter resulting in high MAO protein concentration persisting more than ten years after quitting smoking. The present data enlarges the results to another MAO substrate, norepinephrine (NE), further confirming the central role of MAO in tobacco use-induced diseases. Thus, MAO could be a readily accessible and helpful marker in the risk evaluation of smoking-related diseases, from cardiovascular and pulmonary diseases to depression, anxiety and cancer. The present review implements the new finding of epigenetic regulation of MAO and suggests that smoking-induced MAO demethylation can be considered as a hallmark of smoking-related cancers similarly to other aberrant DNA methylations.

Smoking induces long-lasting effects through a monoamine-oxidase epigenetic regulation.

BACKGROUND: Postulating that serotonin (5-HT), released from smoking-activated platelets could be involved in smoking-induced vascular modifications, we studied its catabolism in a series of 115 men distributed as current smokers (S), never smokers (NS) and former smokers (FS) who had stopped smoking for a mean of 13 years. METHODOLOGY/PRINCIPAL FINDINGS: 5-HT, monoamine oxidase (MAO-B) activities and amounts were measured in platelets, and 5-hydroxyindolacetic acid (5-HIAA)--the 5-HT/MAO catabolite--in plasma samples. Both platelet 5-HT and plasma 5-HIAA levels were correlated with the 10-year cardiovascular Framingham relative risk (P<0.01), but these correlations became non-significant after adjustment for smoking status, underlining that the determining risk factor among those taken into account in the Framingham risk calculation was smoking. Surprisingly, the platelet 5-HT content was similar in S and NS but lower in FS with a parallel higher plasma level of 5-HIAA in FS. This was unforeseen since MAO-B activity was inhibited during smoking (P<0.00001). It was, however, consistent with a higher enzyme protein concentration found in S and FS than in NS (P<0.001). It thus appears that MAO inhibition during smoking was compensated by a higher synthesis. To investigate the persistent increase in MAO-B protein concentration, a study of the methylation of its gene promoter was undertaken in a small supplementary cohort of similar subjects. We found that the methylation frequency of the MAOB gene promoter was markedly lower (P<0.0001) for S and FS vs. NS due to cigarette smoke-induced increase of nucleic acid demethylase activity. CONCLUSIONS/SIGNIFICANCE: This is one of the first reports that smoking induces an epigenetic modification. A better understanding of the epigenome may help to further elucidate the physiopathology and the development of new therapeutic approaches to tobacco addiction. The results could have a larger impact than cardiovascular damage, considering that MAO-dependent 5-HT catabolism is also involved in addiction, predisposition to cancer, behaviour and mental health.

Allium compounds, dipropyl and dimethyl thiosulfinates as antiproliferative and differentiating agents of human acute myeloid leukemia cell lines.

Epidemiologic studies support the premise that Allium vegetables may lower the risk of cancers. The beneficial effects appear related to the organosulfur products generated upon processing of Allium. Leukemia cells from patients with acute myeloid leukemia (AML) display high proliferative capacity and have a reduced capacity of undergoing apoptosis and maturation. Whether the sulfur-containing molecules thiosulfinates (TS), diallyl TS (All(2)TS), dipropyl TS (Pr(2)TS) and dimethyl TS (Me(2)TS), are able to exert chemopreventative activity against AML is presently unknown. The present study was an evaluation of proliferation, cytotoxicity, differentiation and secretion of AML cell lines (U937, NB4, HL-60, MonoMac-6) in response to treatment with these TS and their related sulfides (diallylsulfide, diallyl disulfide, dipropyl disulfide, dimethyl disulfide). As assessed by flow cytometry, ELISA, gelatin zymogaphy and RT-PCR, we showed that Pr(2)TS and Me(2)TS, but not All(2)TS and sulfides, 1) inhibited cell proliferation in dose- and time-dependent manner and this process was neither due to cytotoxicity nor apoptosis, 2) induced macrophage maturation, and 3) inhibited the levels of secreted MMP-9 (protein and activity) and TNF-alpha protein, without altering mRNA levels. By establishing for the first time that Pr(2)TS and Me(2)TS affect proliferation, differentiation and secretion of leukemic cell lines, this study provides the opportunity to explore the potential efficiency of these molecules in AML.

Thiosulfinates modulate platelet activation by reaction with surface free sulfhydryls and internal thiol-containing proteins.

Thiosulfinates are characteristic flavors of Allium vegetables, with a highly reactive S-S=O group, that we previously showed to inhibit platelet aggregation through calpain-dependent mechanisms. With the aim to clarify the mode of action of these redox phytochemicals, we studied their effect on extracellular free sulfhydryls in relation to their effect on platelet responses (Ca2+ signals, release reaction, and aIIb3 integrin activation state). At the platelet surface, thiosulfinate dose-dependently increased the basal level of free sulfhydryls, independently of protein disulfide isomerase activity. This generation of new free sulfhydryls was associated with: (i) a three fold increase in labeling of resting platelets with an anti ligand-induced binding site antibody and (ii) marked inhibition of subsequent aIIb3 activation by agonists. Thiosulfinates increased the basal intracellular Ca2+ level of platelets. In activated platelets, they markedly inhibited the Ca2+ mobilization independently of the external Ca2+, the calpain-induced SNAP-23 cleavage and the granule release. In platelet free systems, thiosulfinates inhibited the activity of purified calpain and the free sulfhydryl of glutathione without any reducing properties on disulfides. The results demonstrate for the first time that thiosulfinates rapidly interact with sulfhydryls both at the platelet surface and inside the cell on intracellular cysteine-proteins, especially calpain. Inhibition of free cysteine and glutathione in whole blood may also contribute to their anti-aggregant properties. Such sulfur compounds are of interest for the development of a new class of antithrombotic agents.

Decreased number of circulating CD34+KDR+ cells in asymptomatic subjects with preclinical atherosclerosis.

OBJECTIVES: To assess whether circulating endothelial progenitor cells (CEPCs) can be considered as a cardiovascular risk marker before event has occurred, that is less firmly established than in clinically overt atherosclerosis. METHODS: Number of CD34+KDR+ cell number per ml blood was measured by flow cytometry in 84 untreated subjects without cardiovascular disease. Atherosclerotic plaque was detected by ultrasound in carotid, abdominal aortic and femoral sites and the number of sites affected by plaque among these three sites was counted as 0, 1, 2 or 3. Additionally, intima-media thickness (IMT) was measured by computerized ultrasound imaging of both common carotid segments. RESULTS: CD34+KDR+ cell number decreased by 48, 29 or 30% in the presence of carotid, aortic or femoral plaque (p<0.001, 0.05, 0.05, respectively) as compared to the absence of plaque and by 70% in the presence of three sites affected with plaque as compared with 0 site with plaque (p<0.01) but did not change with increasing IMT tertiles. Adjustment for Framingham risk score, that was also associated with decreased CD34+KDR+ cell number (p<0.001), made CD34+KDR+ cell number associations with plaque insignificant, except at the carotid site (p<0.01). CONCLUSIONS: Reduced CEPC number may participate to preclinical stage of atherosclerosis and provide additional information to traditional risk factors as regards global risk assessment.

Differential regulation of platelet aggregation and aminophospholipid exposure by calpain.

Aggregation, exposure of procoagulant phospholipids and shedding of microparticles are platelet responses that depend on activating conditions. To determine how these different responses are interconnected, we simultaneously measured fibrinogen (Fg) binding and aminophospholipid exposure on activated platelets by means of flow cytometry. Low calcium ionophore (A23187) concentrations induced Fg binding but not annexin V binding. In contrast, high A23187 concentrations induced annexin V binding but not Fg binding. Collagen, both alone and in the presence of thrombin, induced both Fg and annexin V binding. Dual labelling was found on 38 +/- 9% of platelets stimulated by thrombin plus collagen. The regulatory role of calpain in these platelet functions was investigated. When calpain was partially inhibited by 2 microg/ml calpeptin, Fg binding still occurred but aminophospholipid exposure was limited. By contrast, complete inhibition of calpain by 100 microg/ml calpeptin or E64d decreased Fg binding but enhanced aminophospholipid exposure. In these latter conditions, cytosolic calcium-extruding systems were inhibited. The results suggest that (i) conditions that favour aminophospholipid exposure tend to decrease the aggregation process and (ii) calpain determines the switch to either aggregation or aminophospholipid exposure by controlling intracellular calcium.

Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II.

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

Integrin alpha(IIb)beta3 signals lead cofilin to accelerate platelet actin dynamics.

Cofilin, in its Ser3 dephosphorylated form, accelerates actin filament turnover in cells. We report here the role of cofilin in platelet actin assembly. Cofilin is primarily phosphorylated in the resting platelet as evidenced by a specific antibody directed against its Ser3 phosphorylated form. After stimulation with thrombin under nonstirring conditions, cofilin is reversibly dephosphorylated and transiently incorporates into the actin cytoskeleton. Its dephosphorylation is maximal 1-2 min after platelet stimulation, shortly after the peak of actin assembly occurs. Cofilin rephosphorylation begins 2 min after activation and exceeds resting levels by 5-10 min. Cofilin is dephosphorylated with identical kinetics but fails to become rephosphorylated when platelets are stimulated under stirring conditions. Cofilin is normally rephosphorylated when platelets are stimulated in the presence of Arg-Gly-Asp-Ser (RGDS) peptide or wortmannin to block alpha(IIb)beta3 cross-linking and signaling or in platelets isolated from a patient with Glanzmann thrombasthenia, which express only 2-3% of normal alpha(IIb)beta3 levels. Furthermore, actin assembly and Arp2/3 complex incorporation in the platelet actin cytoskeleton are decreased when alpha(IIb)beta3 is engaged. Our results suggest that cofilin is essential for actin dynamics mediated by outside-in signals in activated platelets.

Polymorphonuclear leukocyte and monocyte activation induced by plasma from patients with heparin-induced thrombocytopenia in whole blood.

Heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy, results from platelet activation by heparin-dependent antibodies. Previously, we have shown that plasma from patients with HIT (HIT plasma) induces leukocyteplatelet aggregation in blood. In this report, we examined leukocyte activation by HIT plasma and the contribution of heparin and platelets to this activation, in whole blood. Degranulation of leukocytes from HIT patients was evaluated as a leukocyte activation marker. We showed that polymorphonuclear leukocytes (PMN) and monocytes were the leukocyte subpopulations involved in platelet-leukocyte aggregation induced by HIT plasma in healthy donor blood. PMN and monocyte activation, reflected by increased surface expression of the CD11b adhesion molecule, was induced by HIT plasma in a heparin-dependent manner. The CD11b increase induced by HIT plasma was observed on PMN only when they were associated with platelets. Moreover, the increased CD11b expression on monocytes and PMN correlated strongly with the degree of platelet adhesion to these cells. Degranulation of leukocytes from HIT patients and control subjects (non-HIT heparin-treated patients and healthy subjects) was evaluated in vivo by measuring the plasma myeloperoxidase concentration. HIT plasma contained higher myeloperoxidase concentrations than control plasma, suggesting leukocyte degranulation during HIT. In conclusion, this study provides the first evidence that PMN activation is induced by HIT plasma. HIT plasma induced PMN and monocyte activation in a heparin-dependent manner. In whole blood, platelet association with monocytes and PMN, and the activation of these leukocytes by HIT plasma were interrelated. Finally, leukocyte degranulation could be involved in HIT physiopathology.

Plasma cGMP and large artery remodeling in asymptomatic men.

cGMP regulates vascular smooth muscle tone and arterial wall response to proliferative signals. We determined plasma cGMP and carotid artery intima-media thickness (IMT) and diameter in 84 asymptomatic men submitted to investigation of their cardiovascular risk profiles. Plasma cGMP was positively associated with IMT (P<0.01) and diameter (P<0.05), independently of coexisting risk factors. These associations were reinforced in the subgroup of subjects with high-sensitivity C-reactive protein level or multiple atherosclerotic plaques. A positive relationship existed between diameter and IMT (P<0.01) and disappeared after cGMP adjustment. This suggests a link between cGMP pathway and arterial wall geometry that is revealed by vascular injury conditions and may participate in early large artery remodeling.

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin.

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.

Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2.

The oxidation of plasma LDLs (low-density lipoproteins) is a key event in the pathogenesis of atherosclerosis. LPC (lysophosphatidylcholine) and oxysterols are major lipid constitutents of oxidized LDLs. In particular, 7-oxocholesterol has been found in plasma from cardiac patients and atherosclerotic plaque. In the present study, we investigated the ability of 7-oxocholesterol and LPC to regulate the activation of eNOS (endothelial nitric oxide synthase) and cPLA2 (cytosolic phospholipase A2) that synthesize two essential factors for vascular wall integrity, NO (nitric oxide) and arachidonic acid. In endothelial cells from human umbilical vein cords, both 7-oxocholesterol (150 microM) and LPC (20 microM) decreased histamine-induced NO release, but not the release activated by thapsigargin. The two lipids decreased NO release through a PI3K (phosphoinositide 3-kinase)-dependent pathway, and decreased eNOS phosphorylation. Their mechanisms of action were, however, different. The NO release reduction was dependent on superoxide anions in LPC-treated cells and not in 7-oxocholesterol-treated ones. The Ca2+ signals induced by histamine were abolished by LPC, but not by 7-oxocholesterol. The oxysterol also inhibited (i) the histamine- and thapsigargin-induced arachidonic acid release, and (ii) the phosphorylation of both cPLA2 and ERK1/2 (extracellular-signal-regulated kinases 1/2). The results show that 7-oxocholesterol inhibits eNOS and cPLA2 activation by altering a Ca2+-independent upstream step of PI3K and ERK1/2 cascades, whereas LPC desensitizes eNOS by interfering with receptor-activated signalling pathways. This suggests that 7-oxocholesterol and LPC generate signals which cross-talk with heterologous receptors, effects which could appear at early stage of atherosclerosis.

Therapeutic immunoglobulin reduces Ca2+ mobilization and von Willebrand factor secretion, and increases nitric oxide release in human endothelial cells.

Intravenous gamma-immunoglobulin (i.v.Ig) is commonly used in the treatment of autoimmune and inflammatory vascular disorders to prevent thrombotic complications. The mechanism of action of i.v.Ig is, however, not yet elucidated. In view of this, we investigated the ability of i.v.Ig to modulate i) Ca(2+) signals of fura-2 loaded endothelial cells, and ii) the associated release of nitric oxide (NO) and von Willebrand factor (vWf). NO was measured either indirectly by radioimmunoassay of cGMP in unstimulated cells or directly by electrochemistry at the surface of stimulated endothelial cells from human umbilical cord veins (HUVEC). Short-term treatment of unstimulated HUVEC with intact i.v.Ig decreased the basal cytosolic Ca(2+) concentration by 20% while it activated the NO/cGMP synthesis. Following i.v.Ig treatment of HUVEC, the Ca(2+) liberation from internal stores and the vWf secretion induced by ATP, thrombin or histamine were significantly reduced by 38 and 60%, respectively. The effects on Ca(2+) signals were observed with intact i.v.Ig as well as with the F(ab')2 or the Fc fragments indicating that both portions are involved in the mechanism of action. The i.v.Ig treatment of HUVECs had no effect on the NO release induced by thrombin or histamine. By contrast, the i.v.Ig treatment increased the ATP-activated NO release by amplifying the Ser1177-eNOS phosphorylation. The i.v.Ig also activated the NO-dependent cGMP release in resting and collagen-stimulated platelets. Since NO is a potent inhibitor of platelet activation and vWF is a platelet adhesion cofactor, the beneficial effects of therapeutic i.v.Ig may lie in the inhibition of platelet adhesion to damaged endothelium.

Differential effect of the inhibition of Grb2-SH3 interactions in platelet activation induced by thrombin and by Fc receptor engagement.

The adaptor protein Grb2 (growth factor receptor-bound protein 2) is involved in cell proliferation via the Ras signalling pathway. In order to study the role of Grb2 in blood platelet responses, we used a peptide containing two proline-rich sequences derived from Sos (peptidimer), which binds to Grb2-Src homology 3 domain (SH3) with a high affinity, and hence inhibits Grb2-SH3-mediated protein interactions. Platelet aggregation and 5-hydroxytryptamine (serotonin) release measured in the presence of the peptidimer were: (i) significantly decreased when induced by thrombin; and (ii) potentiated when induced by the engagement of the Fc receptor. In thrombin-activated platelets, the Grb2-SH2 domain formed an association with the beta3 subunit of the alphaIIb-beta3 integrin (GPIIb-IIIa), Shc, Syk, Src and SHP1 (SH2-containing phosphotyrosine phosphatase 1), whereas these associations did not occur after the engagement of the receptor for the Fc domain of IgG (FcgammaRIIa) or in resting platelets. Grb2-SH3 domains formed an association with the proline-rich sequences of Sos and Cbl in both resting and activated platelets, since the peptidimer abolished these associations. Inhibition of both fibrinogen binding and platelet aggregation by the peptide RGDS (Arg-Gly-Asp-Ser) had no effect on thrombin-induced Grb2-SH2 domain association with the aforementioned signalling molecules, indicating that these associations occurred during thrombin-induced 'inside-out' signalling. Platelet aggregation induced by direct activation via alphaIIb-beta3 ('outside-in' signalling) was potentiated by the peptidimer. The results show that inhibition of Grb2-SH3 interactions with signal-transduction proteins down-regulates thrombin-induced platelet activation, but also potentiates Fc receptor- and alphaIIb-beta3-mediated platelet activation.