EDTA induced changes in platelet structure and function: influence on particle uptake

Ethylenediaminetetracetic acid (EDTA) causes structural, biochemical and functional damage to blood platelets. The alterations induced are considered irreversible. However, the degree of irreversibility, and whether all functions are similarly compromised by EDTA have not been fully evaluated. The present study has examined platelets treated with EDTA to produce the structural changes in channels of the open canalicular system (OCS) associated with irreversible dissociation of the fibrinogen receptor, GPIIb-IIIa ( alpha IIb beta 3 ), for their ability to interact with particulates in suspension. Despite severe narrowing and near occlusion of peripherally oriented OCS channels by EDTA, treated cells were able to bind, translocate and take up fibrinogen-coated gold particles (Fgn/Au), colloidal gold and latex spheres. Thus exposure to EDTA may compromise some aspects of platelet function, but not others which may be important for participation in hemostatic events.     

Neuroadaptations in adenosine receptor signaling following long-term ethanol exposure and withdrawal

Ethanol affects the function of neurotransmitter systems, resulting in neuroadaptations that alter neural excitability. Adenosine is one such receptor system that is changed by ethanol exposure. The current review is focused on the A(1) and the A(2A) receptor subtypes in the context of ethanol-related neuroadaptations and ethanol withdrawal because these subtypes (i) are activated by basal levels of adenosine, (ii) have been most well-studied for their role in neuroprotection and ethanol-related phenomena, and (iii) are the primary site of action for caffeine in the brain, a substance commonly ingested with ethanol. It is clear that alterations in adenosinergic signaling mediate many of the effects of acute ethanol administration, particularly with regard to motor function and sedation. Further, prolonged ethanol exposure has been shown to produce adaptations in the cell surface expression or function of both A(1) and the A(2A) receptor subtypes, effects that likely promote neuronal excitability during ethanol withdrawal. As a whole, these findings demonstrate a significant role for ethanol-induced adaptations in adenosine receptor signaling that likely influence neuronal function, viability, and relapse to ethanol intake following abstinence.     

Effect of garlic on human platelet aggregation in vitro arun bordia

In 6 healthy adults the effect of essential oil of garlic on platelet aggregation was studied in vitro with an aggreganometer. The blood was collected in a siliconized centrifuge tube containing sodium citrate. The aggregating agents used were ADP, epinephrine and collagen. In each subject aggregation was studied 3 times: (i) initial fasting control; (ii) immediately after (i) but with essential oil of garlic drawn into the syringe together with the sodium citrate; (iii) 5 days after feeding 0.5 mg of essential oil of garlic daily.

Addition of essential oil of garlic inhibited in-vitro platelet aggregation induced by ADP, epinephrine or collagen; the effect was dose-related. Oral administration of garlic also decreased platelet aggregation. Thus, garlic seems to inhibit some aspects of thrombus formation.     

Inhibition of NMDA receptor function with an anti-GluN1-S2 antibody impairs human platelet function and thrombosis

GluN1 is a mandatory component of N-methyl-D-aspartate receptors (NMDARs) best known for their roles in the brain, but with increasing evidence for relevance in peripheral tissues, including platelets. Certain anti-GluN1 antibodies reduce brain infarcts in rodent models of ischaemic stroke. There is also evidence that human anti-GluN1 autoantibodies reduce neuronal damage in stroke patients, but the underlying mechanism is unclear. This study investigated whether anti-GluN1-mediated neuroprotection involves inhibition of platelet function. Four commercial anti-GluN1 antibodies were screened for their abilities to inhibit human platelet aggregation. Haematological parameters were examined in rats vaccinated with GluN1. Platelet effects of a mouse monoclonal antibody targeting the glycine-binding region of GluN1 (GluN1-S2) were tested in assays of platelet activation, aggregation and thrombus formation. The epitope of anti-GluN1-S2 was mapped and the mechanism of antibody action modelled using crystal structures of GluN1. Our work found that rats vaccinated with GluN1 had a mildly prolonged bleeding time and carried antibodies targeting mostly GluN1-S2. The monoclonal anti-GluN1-S2 antibody (from BD Biosciences) inhibited activation and aggregation of human platelets in the presence of adrenaline, adenosine diphosphate, collagen, thrombin and a protease-activated receptor 1-activating peptide. When human blood was flowed over collagen-coated surfaces, anti-GluN1-S2 impaired thrombus growth and stability. The epitope of anti-GluN1-S2 was mapped to α-helix H located within the glycine-binding clamshell of GluN1, where the antibody binding was computationally predicted to impair opening of the NMDAR channel. Our results indicate that anti-GluN1-S2 inhibits function of human platelets, including dense granule release and thrombus growth. Findings add to the evidence that platelet NMDARs regulate thrombus formation and suggest a novel mechanism by which anti-GluN1 autoantibodies limit stroke-induced neuronal damage.     

Inhibition of platelet function in thrombosis

Accumulating experimental and clinical evidence indicates that a time for reappraisal of therapeutic modalities designed to inhibit the eicosanoid pathway as it may affect vascular disease may be approaching. Pharmacologic agents originally used were chosen because they were capable of suppressing platelet functions such as aggregation, release, and adhesion. The goals of clinical trials were to evaluate medications that would prevent or reduce platelet accumulation in critically located blood vessels of the heart, brain, and extremities and on vascular prostheses. Evaluation of results of therapeutic trials has been difficult and this is superimposed on less-than-complete knowledge of the basic pharmacology of the drugs that have been used. Participation of neutrophils and possibly macrophages in the thrombotic process is now well recognized on morphologic grounds. Because different cell types such as platelets, neutrophils, and endothelial cells have been shown to interact biochemically by sharing precursors and intermediates of the eicosanoid pathway, the pharmacologic approach to inhibition of vascular disease may require reevaluation. Neutrophils appear to lack a cyclooxygenase pathway but serve as a source of the lipoxygenase product leukotriene B4 (LTB4). Actions of LTB4 include neutrophil aggregation, adhesion of neutrophils to endothelial cells, chemotaxis, chemokinesis, and plasma exudation. We have demonstrated in vitro that released free arachidonic acid from aspirin-treated platelets can serve as a source of neutrophil LTB4. Leukotrienes C4, D4, and E4 are agonists for various functions of vascular endothelium and smooth muscle. Most pharmacologic agents used in the treatment of vascular diseases inhibit the cyclooxygenase pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of selective serotonin reuptake inhibitors on platelet function in whole blood and platelet concentrates

Several studies report that patients who are treated with selective serotonin reuptake inhibitors (SSRIs) for depression may have increased risk of bleeding, particularly from the gastrointestinal tract. This may be related to low intraplatelet serotonin concentrations. Several blood banks do not store platelets from donors using SSRIs for transfusion, although the possible effects of SSRIs on platelet storage are not documented. We conducted a case-control pilot study of apheresis platelet concentrates prepared from donors using SSRIs (n?=?8) and from donors without medication (n?=?10). The platelet concentrates were stored for 5 days. Light transmission aggregometry (LTA), thrombelastography (TEG), and flow cytometric analyses were preformed for in vitro measurements of platelet function. Platelet function and platelet serotonin content were investigated in whole blood and in platelet concentrates stored for up to 5 days. LTA, TEG, and flow cytometric analysis of glycoprotein expression did not reveal any significant differences between the two groups. All 18 platelet concentrates performed well according to the standards set for platelet quality in relation to transfusion. Blood donors using SSRIs had significantly lower platelet serotonin compared to blood donors without medication. The results from our pilot study indicate that platelets from donors using SSRIs may be suitable for transfusion after storage for 5 days, but further laboratory and clinical studies are necessary to confirm this.     

Targeting platelet receptor function in thrombus formation: The risk of bleeding

In this review, we presume that the process of thrombus formation, as assessed in whole blood flow studies and in experimental (murine) thrombosis studies, reflects the platelet responses in human haemostasis and thrombosis. Following this concept, we give an up-to-date overview of the main platelet receptors and signalling pathways that contribute to thrombus formation and are used as targets in (pre)clinical intervention studies to prevent cardiovascular disease. Discussed are receptors for thrombin, thromboxane, ADP, ATP, prostaglandins, von Willebrand factor, collagen, CLEC-2 ligand, fibrinogen and laminin. Sketched are the consequences of receptor deficiency or blockage for haemostasis and thrombosis in mouse and man. Recording of bleeding due to (congenital) platelet dysfunction or (acquired) antiplatelet treatment occurs according to different protocols, while common laboratory methods are used to determine platelet function.     

Physical inactivity and platelet function in humans and brown bears: A comparative study

Physical inactivity increases the risk of thromboembolism. However, good standardized human models on inactivity are in short supply and experimental models are few.

Our objective was to investigate how standardized bed rest affects platelet aggregation in humans and to investigate if aggregation is altered in a translational model system – the hibernating brown bear (Ursus arctos). We collected blood from (1) healthy male volunteers participating in a 21-day bed rest study in head-down tilt position (?6°) 24 h a day; (2) free-ranging brown bears captured during winter hibernation and again during active state in summer. We analyzed platelet function using multiple electrode platelet aggregometry. In total, 9 healthy male volunteers (age 31.0 ± 6.4 years) and 13 brown bears (7 females and 6 males, age 2.8 ± 0.6 years) were included. In hibernating bears adenosine diphosphate, arachidonic acid, thrombin receptor activating peptide, and collagen impedance aggregometry tests were all halved compared to summer active state. In human volunteers no statistically significant changes were found between baseline and the end of bed rest. In human male volunteers 3 weeks of bed rest did not affect platelet function. In hibernating brown bears platelet aggregation was halved compared to summer and we hypothesize that this is a protective measure to avoid formation of thrombi under periods of low blood flow.     

A comparison of von Willebrand Factor antigen with platelet activity in vitro in normal and venous occlusion blood

Von Willebrand Factor (vWF) is essential for normal haemostasis involving platelet aggregation induced by high shear forces. In vitro a functional test of platelet aggregation using the filterometer is abnormal in von Willebrand's disease. However in normal people there is no significant correlation between the antigenic assay of vWF and the filter results. To study this discrepancy normal blood before and during venous occlusion, and blood before and after infusion of 1 deamino-(8-D-arginine) vasopressin was studied. During venous occlusion (VO) the increase in vWF due to the release of large multimers correlated precisely with the increase in the filterometer results. That this was due to the plasma vWF and not to any change induced in the platelets was shown as follows: The methodology was altered so that a small amount of the donor's platelet-poor plasma (PPP) was added to homologous normal substrate blood. The effect of the added donor's PPP was then shown to be closely correlated to the increase in the antigenic assay. Analysis of vWF multimer size showed during VO an increase in large multimers. We conclude that the effect of vWF on normal blood may be obscured by variation in platelet aggregability. In the filterometer system as elsewhere the large active multimers probably play a major part in causing platelet adhesion, aggregation and filter blocking. The filterometer test is influenced by the amount of vWF antigen, by the molecular size and activity of the vWF and by platelet sensitivity. Clinically this is a useful global test.     

Evaluation of platelet function and lack of response to epinephrine in pregnant women

Previous studies in healthy subjects have demonstrated a lack of response of platelets to epinephrine at a rate of 16-40% on an aggregometer. An association between the increased procoagulant factors during pregnancy and venous thromboembolism is known, and it has also been shown that prolactin levels increase platelet aggregation. We evaluated whether platelet functions in pregnant women and also assessed the lack of response to epinephrine during this period. We compared 27 healthy and volunteering pregnant women with 26 similar control subjects. Platelet functions were assessed with an aggregometer and a Platelet Function Analyzer (PFA-100). Less than 40% response to epinephrine on the aggregometer was defined as an impaired epinephrine response. The aggregation response of epinephrine was normal in 25 of the 27 pregnant women, while two of them showed a late-rising response. Eight of the 26 subject control group (30.8%) showed an impaired response to epinephrine. When we compared the 25 pregnant and 18 control subjects with normal aggregation responses, the maximum aggregation responses to ADP and epinephrine, and the Col/Epi and Col/ADP cartridge closure time values were significantly lower in pregnant women. There were no difference between second and third trimesters as regards platelet function parameters. The fact that no impaired response to epinephrine was detected in pregnant women while a 30% rate was observed in non-pregnant women indicates that the platelet malfunction caused by a disorder in the Gi protein and intracellular mechanisms is bypassed during pregnancy thanks to some physiological changes.     

Involvement of Protein Kinase C in Ethanol-Induced Inhibition of NMDA Receptor Function in Cerebellar Granule Cells

Ethanol inhibits N -methyl- d -aspartate (NMDA)-stimulated increases in intracellular Ca²⁺ in cerebellar granule cells apparently by reducing the potency of glycine to act as a co-agonist at the NMDA receptor. The inhibitory effect of ethanol on the NMDA response in these cells can be reversed not only by a high concentration of glycine, but also by the protein kinase inhibitors, staurosporine and calphostin C. We previously showed that activation of protein kinase C in cerebellar granule cells also resulted in inhibition of the NMDA response, and in decreased potency of glycine at the NMDA receptor. Furthermore, the inhibitory effects of ethanol and protein kinase C activation are not additive. These results suggest a role for protein kinase C in ethanol inhibition of NMDA responses in cerebellar granule cells. In contrast, although ethanol can inhibit the response to kainate in these cells in a "competitive" manner, this response is not affected by activation of protein kinase C.     

Differences in Ethanol Sensitivity of Brain NMDA Receptors of Long-Sleep and Short-Sleep Mice

Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred mice that differ in the duration of anesthesia produced by an acute dose of ethanol, were used to determine the possible association of differing ethanol sensitivity of brain NMDA receptors with differing sensitivity to the anesthetic effects of ethanol in vivo. NMDA receptor-mediated responses were determined by measurement of l -glutamate-stimulated increases in free intracellular calcium concentration (Ca_l) using the fluorescent indicator for Ca₁, Indo 1, in microsacs (a cell-free brain membrane vesicle preparation) isolated from hippocampi or cerebral cortices of the two mouse lines. In the absence of added drugs, NMDA responses did not differ between the two lines in hippocampal or cerebrocortical microsacs. However, a high concentration of ethanol (200 m_m) inhibited NMDA responses in hippocampal microsacs from LS mice. In contrast, a moderate concentration of ethanol (50 m_m) stimulated NMDA responses in hippocampal microsacs isolated from SS mice. In cerebrocortical microsacs, ethanol inhibited NMDA responses in the two lines to an equivalent degree. MK-801, a noncompetitive blocker of NMDA receptors, blocked NMDA responses at lower concentrations in hippocampal microsacs from LS mice than in SS mice, but produced a similar degree of inhibition of NMDA responses in cerebrocortical microsacs from the two lines. A high concentration of ethanol (200 mm ) increased resting Ca₁ in hippocampal microsacs from LS mice but not in hippocampal microsacs from SS mice, and increased resting Ca_l in cerebrocortical microsacs isolated from both lines of mice equally. The small change in resting Ca, produced by MK-801 in cerebrocortical microsacs did not differ between the two lines. These results show that hippocampal NMDA receptors of LS and SS mice differ in their sensitivity to ethanol, possibly because of differences in allosteric modulation at the MK-801 site or some other site that interacts with the MK-801 site of the NMDA receptor.     

脂质体携载前列腺素E_1抑制血小板功能降低心肌梗死面积

目的　在家兔心肌缺血 再灌注模型上观察脂质体携载前列腺素E1(Lipo PGE1)对血小板功能的抑制作用和对缺血心肌的保护作用。方法　 2 0只家兔随机分成Lipo PGE1治疗组及对照组 ,每组 10只。两组家兔左冠状动脉前降支结扎 4 0min后松开 ,再灌注 2h。于再灌注前 10min分别自耳缘静脉注射Lipo PGE1( 2 μg/kgPGE1)及等容量的脂肪乳剂 (Lipo PGE1的溶剂 )。整个实验过程中连续记录血压及心电变化。家兔处死后以Evans蓝及氯化三苯基四氮唑 (TTC)双重染色确定缺血及梗死心肌范围 ,心肌梗死范围以梗死心肌占危险区心肌重量百分比 (MI/RISK)表示。通过测定血浆血栓素B2 (TXB2 )及α颗粒膜蛋白 14 0 (GMP 14 0 )含量反映周围血小板活性状态。结果　再灌注前静脉应用 2 μg/kgLipo PGE1能减少家兔再灌注期心律失常的发生 ,不产生明显的血流动力学障碍。Lipo PGE1治疗组心肌梗死范围 ( 3 3 .6± 5 .5 ) %较对照组 ( 4 3 .8± 5 .1) %显著缩小 (P <0 .0 1)。Lipo PGE1治疗组再灌注期血浆TXB2 及GMP 14 0含量较对照组明显降低 (P <0 .0 5及P <0 .0 1) ,血小板最大聚集率较对照组也明显降低 (P <0 .0 1)。结论　Lipo PGE1能显著抑制血小板聚集、活化 ,挽救缺血心肌 ,缩小心肌梗死范围。     

Ethanol-Induced Inhibition of Cell Proliferation Is Modulated by Insulin-Like Growth Factor-I Receptor Levels

Ethanol inhibits the tyrosine autophosphorylation of the insulin-like growth factor (IGF)-1 receptor, an action that correlates with the inhibition of IGF-I-stimulated cell proliferation [J. Biol. Chem . 268:21777–21782 (1993)l. In the current study, the IGF-I-dependent proliferation of mouse BALB/c3T3 cells was completely inhibited by ethanol, but the growth of BALB/c3T3 cells that overexpress the IGF-l receptor (p6 cells) was only partially inhibited by ethanol. BALB/ c3T3 cells that simultaneously overexpress both the IGF-I receptor and IGF-I were insensitive to growth inhibition by ethanol. In p6 cells, increasing concentrations of IGF-l overcame the inhibition of IGF-l receptor tyrosine autophosphorylation in the presence of ethanol. The importance of the IGF-I receptor as a specific target for ethanol was further investigated in C6 rat glioblastoma cells that respond mitogenically to both epidermal growth factor (EGF) and IGF-I. The mitogenic response of C6 cells to EGF was abrogated In cells expressing antisense mRNA to the IGF-l receptor. Thus, EGF action in these cells is dependent on activation of an IGF-I/lGF-I receptor au-tocrine pathway. Indeed, EGF stimulated an increase in IGF-l receptor levels by more than 100%. Ethanol completely inhibited the prollferation of C6 cells in response to either EGF or IGF-I. However, ethanol did not directly interfere with EGF receptor function, because EGF-induced cell proliferation was unaffected by ethanol when added exclusively during a 1-hr exposure to EGF. Ethanol did not interfere with the EGF-induced increase in IGF-I receptor expression. The addition of both EGF and IGF-I overcame the inhibitory action of ethanol. In conclusion, the potency of ethanol as an inhibitor of IGF-I-mediated cell proliferation correlates with the level of IGF-I receptors. In contrast to its effect on the IGF-I receptor, ethanol has no direct effect on EGF receptor activation.     

软脂酸对酒精体外诱导的脂肪变性肝细胞的作用及机制

目的:探讨不同浓度的软脂酸对酒精体外诱导的脂肪变性肝细胞的作用及机制.方法:体外培养人正常肝细胞株L-02,设立空白对照组、酒精诱导组及软脂酸干预组.软脂酸干预组设6个浓度梯度(2.5、5、10、20、30、40μmol/L),空白对照组用正常培基培养96h,酒精诱导组和软脂酸干预组在细胞培养24h后加入终浓度为60mL/L的无水乙醇,继续培养24h后,软脂酸干预组加入各浓度软脂酸.采用MTT法测细胞增殖,油红O染色观察细胞内脂滴形成情况,试剂盒检测细胞内三酰甘油的含量,Westernblot法测定细胞核内成熟的固醇调节元件结合蛋白-1c(nSREBP-1c)的含量.结果:60mL/L的无水乙醇培养L-02细胞72h成功建立了脂肪变性模型,软脂酸干预后,软脂酸浓度≤10μmol/L时,软脂酸明显地促进酒精诱导的脂肪变性肝细胞的增殖(P<0.05),并且明显地减轻细胞内脂滴的形成,减少细胞内三酰甘油的含量和细胞核内nSREBP-1c的含量,并呈现出剂量效应关系;而软脂酸浓度≥20μmol/L时,明显地抑制酒精诱导的脂肪变性肝细胞的增殖(P<0.05),并且加重细胞内脂滴的形成,增加细胞内三酰甘油和细胞核内nSR...     

Ethanol Inhibition of Insulin Signaling in Hepatocellular Carcinoma Cells

Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyi phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such ceils were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyi phosphorylation of the insulin receptor β-subunit, tyrosyi phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyi phosphorylation of She. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on She phosphorylation, which activates p21^ras through an IRS-1 independent pathway.     

Ethanol Sensitivity of Brain NMDA Receptors in Mice Selectively Bred for Differences in Response to the Low-Dose Locomotor Stimulant Effects of Ethanol

Brain NMDA receptor responses and their sensitivity to ethanol in vitro were determined in replicate lines of FAST and SLOW mice, selectively bred for differences in sensitivity to the locomotor stimulant effects of a low dose of ethanol. L-Glutamate-stimulated increases in the intracellular free calcium concentration (Ca₁) were determined in microsacs, a cell-free brain membrane preparation, isolated from hippocampus or cerebral cortex. Previous work showed that l -glutamate-stimulated increases in Ca₁ in microsacs are mediated by activation of NMDA receptors. The concentration response for l -glutamate-stimulated increases in Ca, did not differ between the lines in either hippocampal or cerebrocortical microsacs. Ethanol produced a concentration-dependent decrease in l- glutamate-stimulated increase in Ca₁ in hippocampal and cerebrocortical microsacs from SLOW mice, but this effect of ethanol was reduced or absent in microsacs isolated from FAST mice. Resting Ca₁ and the ability of a high ethanol concentration to increase resting Ca₁ did not differ between the lines. These results suggest that differences in the sensitivity of brain NMDA receptors to the effects of ethanol determine, at least in part, differences in the locomotor stimulant effects of low doses of ethanol in FAST and SLOW mice. These differences are not due to ethanol effects on resting Ca₁.