Inhibition of platelet aggregation by primary amines. Evidence for a possible role of membrane-associated calcium

The aggregation of human blood platelets by thrombin, adenosine diphosphate, wheat germ agglutinin or ristocetin was inhibited by primary amines. In general, thrombin-induced platelet aggregation was strongly affected by the amines while the effect was weak on cell aggregation by ristocetin. Usually, the diamines were stronger inhibitors of aggregation than the monoamines with cadaverine as the strongest and ethylamine as the weakest inhibitor. At concentration where platelet aggregation was inhibited, the amines neither displaced serotonin from serotinin-loaded platelets nor caused lysis of human red cells. The lectin activity of wheat germ agglutinin on human red cells was not affected by the amines indicating that the amines probably acted on platelets and not on the agglutinin. The clotting activity of thrombin on fibrinogen was partially inhibited by the amines while its esterolytic activity remained unaltered. The inhibitory action of the amines on platelet aggregation could be overcome with small amounts of calcium while other divalent cations tested had little effect. It is suggested that the amines affect platelet aggregation by interfering with the actions of membrane-associated calcium.     

重组炭疽水肿因子的表达与生物活性分析

炭疽毒素包括3种蛋白因子,即保护性抗原（PA）、致死因子（LF）和水肿因子（EF）。EF是钙调蛋白依耐的腺苷酸环化酶,可使细胞cAMP浓度升高,导致宿主防御能力下降。为深入研究炭疽毒素的作用机理,构建了原核表达质粒,在大肠杆菌中表达出重组EF（rEF）。经鉴定,rEF以可溶形式表达于细菌胞质中。经过金属螯和层析、阳离子交换层析和凝胶层析,每升诱导培养物可获得约5mg 重组蛋白。用重组蛋白免疫家兔获得了兔多抗,能够在细胞试验中中和rEF,体外细胞试验显示rEF具有很好的生物活性,在J774A.1和CHO细胞试验中,能与LF共同竞争和PA的结合位点,相互抑制。上述工作为深入研究炭疽毒素的作用机理,开发针对EF的毒素抑制剂打下基础     

Inhibition of platelet aggregation by omega-3 polyunsaturated fatty acids is gender specific—Redefining platelet response to fish oils

Existence of gender differences in cardiovascular disease (CVD) following long-chain omega-3 polyunsaturated fatty acid (LCn-3 PUFA) supplementation have suggested that sex hormones play a role in cardio-protection. The objective of this study was to determine gender specific responses in the efficacy of LCn-3 PUFA to inhibit platelet aggregation in vitro. Blood was analyzed for collagen-induced platelet aggregation following pre-incubation with LCn-3 PUFA in healthy adults (n=42). Eicosapentaenoic acid (EPA) was significantly more effective in reducing platelet aggregation compared with docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). When grouped by gender, this differential pattern was followed in males only. In females, DHA, DPA and EPA were all equally effective. Between group analyses (LCn-3 PUFA vs. gender) showed that both DHA and DPA were significantly less effective in males compared with females. EPA was equally effective in reducing platelet aggregation in both groups. These findings show that significant gender differences exist in platelet aggregation in response to various LCn-3 PUFA treatments.     

Endogenous platelet serotonin release monitored during aggregation by means of a new electrochemical technique

Differential pulse voltammetry combined with electrochemically treated carbon fibre microelectrodes was used to monitor endogenous serotonin release occurring during platelet aggreagtion. After platelet stimulation by thrombin, an oxidation peak was recorded at +280 mV. HPLC analyses performed with fluorimetric detection have shwon that this released electroactive compound was essentially serotonin. Moreover, serotonin measurements in the same samples by the technique reported here and by fluorimetry were found to be very similar (1.15 ± 0.30 μM and 1.17 ± 0.15 μM (mean ± dS.D., n = 6), respectively). Extracellular serotonin concentrations could be estimated either directly during aggregation or in supernatants obtained from stimulated or lysed platelets. Maximal serotonin concentrations have been found to be 6.93 ± 0.37 and 3.28 ± 0.39 nmol/10⁹ platelets from rat and human, respectively. Using the reported procedure, we have observed that no serotonin was released from thrombin-stimulated platelets prepared from rats treated with reserpine. Our new technique represents a selective and performant tool for rapid determination of endogenous serotonin platelet secretion.     

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Small molecule inhibitors of anthrax edema factor

Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5′-Fluorosulfonylbenzoyl 5′-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production.     

IL-17A increases ADP-induced platelet aggregation

The increased risk of thromboembolism and higher incidence of cardiovascular disorders are among the most common causes of morbidity in patients suffering from autoimmune diseases. In this study we tested the hypothesis that IL-17A, a key pro-inflammatory cytokine involved in the development of autoimmune diseases, exerts pro-aggregant effects on both human and mouse platelets. Human or murine platelets were incubated with IL-17A for 2 min at 37 °C prior the addition of the stimuli. Aggregation was monitored in a light transmission aggregometer measuring changes in turbidity with continuous observation over a 5-min interval after the addition of the stimuli. IL-17RA, CD42b and CD62P expression as well as fibrinogen bindings were measured by FACS while Erk-2 phosphorylation was analyzed by western blot using phospho-specific antibodies. Pre-incubation with IL-17A increased ADP-, but not collagen-induced platelet aggregation and accelerated CD62P expression and exposure of fibrinogen binding sites. These effects were associated with a faster kinetic of ADP-induced Erk-2 phosphorylation and were lost in platelets deficient in the IL-17 receptor. Together these results unveil a novel aspect of the inflammatory nature of IL-17A suggesting, at the same time, that therapeutic strategies targeting this cytokine might provide further benefit for the treatment of autoimmune diseases by reducing the risk of cardiovascular-related pathologies.     

Effects of trace element levels on platelet aggregation

Platelet aggregation was measured by an optical method in 32 patients with iron-deficiency anemia at the time of diagnosis and after a period of supplementation with iron. Epinephrine- and adenosine diphosphate-induced platelet aggregation were lower in anemic patients than in the controls (p<0.05). After iron-supplementation therapy, these values showed no significant differences. If induced by collagen or ristocetin, platelet aggregation was the same for patients and controls, but increased after treatment of patients (p<0.05). The plasma zinc values did not show significant differences among the subjects included in this study. These results show that iron is involved in the enzymatic systems that regulate platelet aggregation. The exact nature of this interaction is still to be determined.     

Inhibition of platelet activating factor (PAF)-induced aggregation of human thrombocytes by ginkgolides: considerations on possible bleeding complications after oral intake of Ginkgo biloba extracts

The special Ginkgo biloba leaf extract EGb 761 is marketed for more then two decades. During this time its therapeutic efficacy and favorable safety profile have been proven in numerous clinical trails as well as by postmarketing surveillance in accordance with German drug regulations. During recent years, however, several cases of hemorrhage have been reported to occur in coincidence with the use of Ginkgo products. Although a clear causality between Ginkgo intake and bleeding could not be established, these observations have generally been explained by the platelet-activating factor (PAF)-antagonistic action of ginkgolides, which represent characteristic constituents of Ginkgo extracts. PAF was originally characterized by inducing aggregation and secretion of serotonin and histamine from rabbit platelets. We now confirmed that induction of aggregation of human platelets by PAF requires at least 200 times higher concentration when compared to rabbit cells. Under the chosen experimental conditions, PAF-mediated aggregation of human platelets was half-maximally inhibited by ginkgolide B, A, C and J at concentrations of 2.5, 15.8, 29.8 and 43.5 microg/ml, respectively. These concentrations are generally more than 100 times higher as the peak plasma values measured after oral intake of EGb 761 at recommended doses between 120 and 240 mg. As PAF is a 'weak' platelet activator, which does not appear to be of importance for primary hemostasis, our results rise serious doubts that the PAF antagonistic effect of ginkgolides could be responsible for hemorrhage in patients taking EGb 761.     

A novel surface-active peptide derivative exhibits in vitro inhibition of platelet aggregation

A tetrapeptide corresponding to a region of the N-terminal portion of lactotransferrin with hydrophobic alkyl groups at the terminal ends was synthesized and its physicochemical properties as well as its effect on thrombin-stimulated platelet aggregation were examined. The tetrapeptide derivative, in the aggregated state, produced inhibitory effect on platelet aggregation. The concentration dependent activity of the peptide was analyzed in the light of micelle formation, with the micellar aggregate comprising four tetrapeptide units. The unique action of this peptide derivative on the inhibition of platelet aggregation might be useful in the development of potent antithrombotic drugs.     

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

A novel disintegrin-like domain of a high molecular weight metalloprotease inhibits platelet aggregation

Disintegrin is one of the functionally distinct domains in high molecular weight metalloproteases from various snake venoms and generally has an Arg-Gly-Asp (RGD) sequence that is recognized by specific cell surface integrins. A cDNA encoding the disintegrin-like domain of a snake venom metalloprotease was cloned, expressed in Pichia pastoris, and molecular function of the recombinant protein was characterized. The cDNA sequence indicated that the disintegrin-like domain contains an Asp-Glu-Cys-Asp (DECD) sequence in place of the RGD motif. The expressed disintegrin-like protein was designated as halydin and it was able to inhibit human platelet aggregation in a dose-dependent manner. Unlike other typical RGD-disintegrins, the recombinant non-RGD disintegrin, halydin, inhibited platelet aggregation by suppressing platelet adhesion to collagen rather than by blocking fibrinogen binding to glycoprotein (GP) IIb-IIIa on the platelet surface. Experimental evidence suggests that halydin binds to integrin alpha2beta1 on the platelet surface.     

Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin

The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U. The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N-->I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation.     

Antithrombotic effects of peroxynitrite: Inhibition and reversal of aggregation in human platelets

The inhibition of platelet aggregation by peroxynitrite, a reactive oxygen species derived from the interaction of nitric oxide (NO) and superoxide, was examined in platelet-rich plasma. In this report, we have used a preparation of peroxynitrite that was free of H₂0₂ and MnO₂. As such, peroxynitrite dose-dependently (50–200 μA) inhibited aggregation of human platelets stimulated by ADP (5 μM), collagen (0.5 μg), thrombin (0.5 UlmL) and U46619 (1 PM). In addition, peroxynitrite reversed platelet aggregation induced by collagen, ADP, and thrombin. Peroxynitrite, preincubated with platelet-poor plasma or albumin (7%) for 30 min, did not alter the inhibition of platelet aggregation. This suggested that the inhibitory action of peroxynitrite may be due to nitrosylation of proteins, which by themselves possess activity, rather than conversion to NO or NO donors. Furthermore, we show that peroxynitrite increased the cGMP level only at 200 μM concentrations, further suggesting that the action of peroxynitrite was not completely due to its conversion to NO or NO donors.     

Synthesis and screening of small molecule inhibitors of anthrax edema factor

The synthesis and development of a novel class of molecules that inhibit anthrax edema factor, an adenylyl cyclase, is reported. These molecules are derived from the initial discovery that histidine and imidazole adducts of the prostaglandin PGE₂ reduce the net secretory response of cholera toxin-challenged mice and act directly on the action of anthrax edema factor, a calmodulin-dependent adenylyl cyclase. The simple enones examined in this letter were prepared by palladium-catalyzed Suzuki reaction.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate,and its mechanism

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Anti-thrombosis effect of diosgenyl saponins in vitro and in vivo

Thrombosis in coronary or cerebral arteries is the major cause of morbidity and mortality worldwide. Diosgenin and total steroidal saponins extracted from the rhizome of Dioscorea zingiberensis C.H. Wright are demonstrated to have anti-thrombotic activity. However, few studies describe the anti-thrombotic activity of the diosgenyl saponin monomer. In the present study, a simple and convenient method for the preparation of a new disaccharide saponin, diosgenyl β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranoside (3), is described. We evaluated the anti-thrombotic effects of diosgenin and four diosgenyl saponins by measuring the bleeding time; the results showed that compound 3 exhibits outstanding efficiency in prolonging the bleeding time. Furthermore, we assessed whether compound 3 could alter platelet aggregation in vitro and in vivo. In addition, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), coagulation factors and protection rate in mice were measured to evaluate the anti-thrombotic effect of compound 3. The results show that compound 3 inhibited platelet aggregation, prolonged APTT, inhibited factor VIII activities in rats, and increased the protection rate in mice in a dose-dependent manner. Taken together, these findings suggested that diosgenyl saponins, especially compound 3, had anti-thrombotic activity. It may execute anti-thrombotic activity through inhibiting factor VIII activities and platelet aggregation.