Endothelin does not affect aggregation of human platelets

Endothelin (ET-1) is a recently discovered endothelial-derived peptide with pronounced vasoconstrictor activity. The present study addressed whether ET-1, in analogy with several other vasoactive agents, can induce or modulate aggregation of human platelets in vitro. Venous blood from healthy donors was collected in citrate or heparin and platelet-rich plasma (PRP) was prepared. Portions of the PRP were added to drugs, and platelet aggregation was recorded according to Born & Cross (1963). ET-1 added to the PRP (final concentrations 1-100 nM) did not induce aggregation of platelets, either in citrate- or heparin-containing plasma. Adenosine-diphosphate (0.5-2 microM) or thrombin (0.1-0.4 NIH units ml-1) induced dose-dependent aggregation of platelets in citrate- or heparin-containing PRP; such aggregation was, however, not affected by ET-1 (1-100 microM) either. We conclude that ET-1, in contrast to other endothelial-derived vasoactive agents, lacks direct effect on platelet aggregation in vitro.     

Alpha fetoprotein inhibits aggregation of human platelets

The effect of human alpha-fetoprotein (AFP) on platelet aggregation induced by physiological (ADP, PAF, collagen, arachidonic acid) and aphysiological (ionophore A 23,187) activators was investigated. It was found that AFP at the concentrations of 60-750 micrograms/ml, i.e. far below that observed in human blood, inhibits platelet aggregation induced by physiological activators. Platelet aggregation induced by arachidonic acid was two-four times more sensitive to inhibition by AFP than that induced by other physiological agonists, whereas aggregation induced by A 23,187 was not affected by AFP even at concentration of 750 micrograms/ml.     

Eosinophils infiltrating interleukin-5 gene-transfected tumors do not suppress tumor growth

Tumor-associated eosinophils have been observed in human tumors and in experimental tumor models, but their function is poorly understood. To study the role of eosinophils during tumor growth, the plasmacytoma J558L and the mammary adenocarcinoma TS/A were transfected with an expression vector encoding the murine gene for interleukin-5 (IL-5), a cytokine inducing proliferation and activation of eosinophils. Injection of parental cells, mock-transfectants and IL-5-producing cells into syngeneic mice showed that local IL-5 secretion induced rapid tumor infiltration by eosinophils, as evidenced by immunohistochemical staining, but nevertheless did not alter the tumor growth kinetics of IL-5 transfectants. Therefore, the mere presence of IL-5 and eosinophils was not sufficient to induce a protective host immune response.     

Separate induction of human blood platelet aggregation or cytotoxicity by different concentrations of PAF-acether and thrombin

Using decreasing concentrations of PAF-acether or thrombin, it was possible to observe on human platelets, first, aggregation, classically associated to activation, then, below a threshold, cytotoxicity towardsSchistosoma mansoni larvae, proposed here as stimulation. These two activities appeared as distinct and antithetic. However, their induction might be the consequence of triggering of the same receptors with different intensity, since PAF-induced, but not thrombin-induced, cytotoxicity could be inhibited with specific PAF-antagonists BN 52021 and BN 52024 also known to inhibit PAF-induced aggregation. These results give credit to the hypothesis that haemostatic and cytotoxic properties of platelets are two distinct functions of these blood elements.     

Surface-activated bovine platelets do not spread, they unfold.

The present study has examined the response of bovine platelets to surface activation and compared it to the reaction of human cells. Human platelets react to surfaces by losing their discoid shape, extending pseudopods, converting to dendritic forms, and finally, spreading into thin films resembling pancakes. Bovine platelets do not spread, they unfold. Surface activation causes them to transform from discs to irregular, flattened shapes resembling dendritic platelets, but they are unable to fill in spaces between pseudopods, a step required for spreading. Bovine platelets lack the surface-connected open canalicular system (OCS), which serves as a reservoir of membrane for human platelet spreading. Its absence may be the major factor in the failure of bovine platelet spreading, but there are other possible factors. Circumferential microtubules are more resistant to disassembly in surface-activated bovine than human cells, and their stability as rings or fractured bundles may limit spreading. Actin filament assembly is similar in human and bovine platelets, but the organization is different. Human platelets form a peripheral weave of actin that expands the membrane between pseudopods. A peripheral weave does not form in surface-activated bovine platelets. The absence of the OCS and differences in cytoskeletal organization in bovine platelets may also affect spreading of the surface membrane. Fibrinogen-gold (Fgn-Au) probes added to spread human platelet move from pseudopods and the cell margin toward the center and concentrate in the OCS. Fgn-Au particles bind to surface-activated bovine cells, but move very little, or not at all. All of these factors may contribute to the inability of bovine platelets to react to surfaces by spreading like human cells, but absence of the OCS appears to be the major cause.     

Analysis of platelet surface conformation in thrombin-induced aggregation

We used flow cytometry to investigate the change of platelet membrane glycoproteins (GPIb and GP IIb/IIIa) and the distributions of fibrinogen (Fbg), thrombospondin (TSP) and fibronectin (Fn) on the surface of thrombin-stimulated platelets. The binding of a monoclonal antibody directed at the von Willebrand factor binding site on GPIb decreased in thrombin-stimulated platelets. This antibody caused a reactive delay in thrombin-induced aggregation, but had little influence on aggregability. Slight thrombin-induced aggregation was observed even after blocking the binding of Fbg to GP II b/IIIa. The new expression of GP II b/IIIa was detected on the surface of thrombin-stimulated platelets, whereas there was little increase of Fbg dependent on this GP II b/IIIa. An increase of TSP after thrombin stimulation was observed on the surface of platelets of healthy controls and patients with Glanzmann's thrombasthenia (Type I). The level of on platelet surface was slightly increased by thrombin stimulation. The mechanism involved in thrombin-induced aggregation appears to differ from that in ADP-induced aggregation.     

Inhibitors of receptor tyrosine kinases do not suppress progesterone-induced [Ca2+]i signalling in human spermatozoa

Previous studies have implicated receptor tyrosine kinases in progesterone-induced [Ca2+]i signalling, and consequent induction of the acrosome reaction, in human spermatozoa. We have investigated the effects of tyrosine kinase inhibition on [Ca2+]i responses in large numbers of individual human spermatozoa. Genistein (5, 50 and 250 micromol/l), an inhibitor of receptor-linked tyrosine kinases, significantly inhibited the progesterone-induced acrosome reaction (P < 0.05). However, we could detect no effect of genistein on progesterone-induced [Ca2+]i signalling. In control experiments, application of progesterone induced a significant transient [Ca2+]i response in approximately 77% of cells and a sustained [Ca2+]i ramp/plateau in approximately 48% of cells (n = 26; 5411 cells). In preparations pretreated with genistein (50 micromol/l), significant transient and sustained responses were detected in 69.5 and 39.1% of cells respectively (n = 5; 1109 cells). The amplitudes of both transient and sustained [Ca2+]i responses were similar in control and genistein-pretreated preparations. Tyrphostin A47 (100 micromol/l), another receptor tyrosine kinase inhibitor, also failed to inhibit either the transient or sustained [Ca2+]i response (n = 3; 468 cells). Assessment of tyrosine phosphorylation of two sperm proteins (p105/81) showed greatly increased levels of phosphotyrosine in response to capacitation but a negligible increase in response to progesterone stimulation. Pretreatment with genistein (50 and 250 micromol/l) decreased capacitation-induced tyrosine phosphorylation and resulted in a loss of phosphorylation in response to progesterone treatment. We conclude that neither the transient nor sustained phases of the progesterone-induced [Ca2+]i response require receptor tyrosine kinase signalling. Previous reports of modulation of the progesterone-induced [Ca2+]i signal by tyrosine kinase inhibition probably reflect inhibition of the acrosome reaction.     

Influence of fibrinolysis system on ADP- and serotonin-induced aggregation of human platelets

Urokinase, a component of the fibrinolytic system, induces a time-dependent decrease in platelet aggregation activated by ADP and serotonin. Significant inhibition of ADP-induced aggregation was observed on the 30th–60th min and serotonin-induced on the 3th–10th min of preincubation with urokinase and depended on urokinase concentration. The plasmin inhibitor aprotinin partially abolished urokinase-induced reduction of the amplitude and rate of ADP-induced aggregation and had no effect on serotonin-induced aggregation. Our results favor multiple mechanisms of urokinase influence on platelet activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 158–161, February, 1998     

Anticonvulsants do not suppress long-term potentiation (LTP) in the rat hippocampus

Long-term potentiation (LTP) of population spikes in the CA1 area of rat hippocampus was induced by tetanic stimulation of stratum radiatum in slices kept submerged in a perfusion chamber. Addition of the two antiepileptic drugs phenytoin or the diazepine midazolam to the medium did not significantly alter this phenomenon within 22 min after the tetanus. The early enhancement (post-tetanic potentiation, PTP) was reduced only by phenytoin. Therefore an interaction of these drugs with N-methyl-D-aspartate (NMDA) receptors and LTP induction is unlikely.     

Characterization of human platelets separated from blood by ADP-induced aggregation.

Separation of platelets from plasma is achieved by adding ADP (final concentration 10-5 M) to platelet-rich plasma and allowing aggregates to form. Aggregates are removed quickly by brief, gentle centrifugation, washed two to three times with 0.9% NaCl (saline), and then incubated for 10 minutes in the presence of apyrase, albumin and calcium. Platelet aggregates deaggregate completely during this incubation period. The platelet suspension is then subjected to 1100g for 12 minutes, gently resuspended in a small volume of saline, and finally diluted with an appropriate medium to the desired concentration. The entire separation procedure requires approximately 30 minutes. Platelets obtained by this procedure are a) comparable in aggregability to the platelet preparations obtained by gel filtration, b) have normal intracellular amounts of ATP and ADP, and c) except for slight dilatation of the surface-connected canalicular system, have normal ultrastructural appearance. When suspended in an appropriate medium, these separated platelets take up serotonin 14-C and subsequently release it in nearly normal quantities when exposed to thrombin, collagen or ADP.     

Changes in guanylate cyclase activity of human platelets in adp-induced aggregation

Laboratory of Biochemistry and Pathochemistry of the Guanylate Cyclase System, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 2, pp. 152–154, February, 1991.     

Exercise- and allergen-induced asthma do not change the production of Paf-acether by neutrophils and platelets

Paf-acether, whose role has been suggested in asthma, is a mediator released by stimulated neutrophils, platelets and other cells. Neutrophils and platelets are activated in vivo during exercise or allergen-induced asthma. Upon in vitro stimulation, macrophages from mice treated with an inflammatory stimulus, such as thioglycoccollate, release less paf-acether than macrophages from non-treated mice. We hypothesized that upon in vitro activation platelets and neutrophils should produce less paf-acether after exercise- or allergen-induced asthma. To test this hypothesis, we measured the production of paf-acether by neutrophils and platelets obtained before, 15 and 75 min after exercise in seven normal subjects and five asthmatic subjects with exercise-induced asthma, and in five other asthmatic subjects after specific challenge with Dermatophagoides Pteronyssinus. Purified neutrophils and washed platelets were incubated independently for 10 min at 37 degrees C with no specific activator, with a platelet activator (thrombin, 1 IU.ml-1), a neutrophil activator (opsonized zymosan, 1 mg.ml-1), and both together. We found no significant difference between asthmatic and normal subjects in the amount of paf-acether synthesized by platelets or neutrophils and no fall in the production of paf-acether after exercise- or allergen-induced asthma. However, our method may lack sensitivity in detecting partial activation of these cells and is based on the assumption that changes in peripheral blood cells are representative of changes of these cells in lungs.     

Effect of toxic substances of diphtheria corynebacteria on aggregation of human platelets

The effect of the toxic substances of diphtheria corynebacteria (diphtheria toxin, diphtheria anatoxin, and codivac) on the aggregation of human plateletsin vitro was demonstrated using platelet-rich plasma prepared from citrated blood and the standard platelet activator ADP (2×10⁻⁵ M). These substances induce platelet aggregation in a dose-dependent manner. Incubation of diphtheria toxin and anatoxin with platelets reduces ADP-induced and total platelet aggregation, the effect being dependent on the inducer dose and incubation time. By contrast, codivac stimulates ADP-induced and total platelet aggregation in all experimental series. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp 623–625, December, 1994 Presented by V. I. Pokrovskii, Member of the Russian Academy of Medical Sciences     

Dietary vitamin E supplementation inhibits thrombin-induced platelet aggregation, but not monocyte adhesiveness, in patients with hypercholesterolaemia

Several recent studies have indicated the possible beneficial effects of antioxidants, specifically vitamin E, in primary and secondary coronary prevention. These studies suggest that a diet enriched in vitamin E is insufficient to have a significant protective effect, whereas supplements, in excess of 200 international units (IU) per day, are efficacious in preventing coronary disease in both men and women. The mechanisms by which vitamin E may exert its protection are uncertain, but, vitamin E is lipophilic and has been shown to inhibit the oxidative modification of low density lipoprotein (LDL), a process thought to be of crucial importance in atherogenesis. We have also previously shown that α-tocopherol (the biologically most potent isomer of vitamin E) has important direct effects on vascular endothelial and smooth muscle cells. In the present study we have investigated the effects of oral supplements of vitamin E (400 IU per day) on platelet and mononuclear cell function in patients with hypercholesterolaemia. We found that although vitamin E supplementation had no significant effect on mononuclear cell adhesion ex vivo , it had a significant effect on the thrombin-induced platelet aggregation ( P  < 0.01; anova ): 6 weeks after starting the vitamin E supplements, the mean EC₅₀ for thrombin-induced aggregation increased 132% ( P  < 0.05; paired t -test) compared to treatment with placebo. The effects of vitamin E on platelet function may, in part, explain its anti-atherogenic properties.     

Release of PAF-acether from human blood monocytes

Stimulation of human blood monocytes with ionophore A 23187 induced the release of platelet-activating factor (PAF-acether). Phagocytosis of zymosan, coated or not with complement, bacteria or immune complexes, stimulated the release of PAF-acether whereas that of latex particles was without effect. Such release did not occur at 4°C or in the presence of EDTA. PAF-acether derived from monocytes shared the same characteristics as hog leucocyte PAF-acether or synthetic 1-O-alkyl-2-acetyl-glyceryl-3-phosphorylcholine. In lung physiology, the release of PAF-acether from monocytes and alveolar macrophages could lead, via the platelets, to bronchoconstriction. It could represent a cause for asthma other than the classical IgE-mastocyte interaction.     

Effects on aggregation of human platelets of two xanthines and their interactions with adenosine

Adenosine receptor antagonism has been suggested to be the cellular basis for many extrapulmonary actions of xanthine derivatives, such as theophylline. Enprofylline (3-propylxanthine) is a poor adenosine antagonist but is five times as potent as theophylline as a bronchodilator in man. Adenosine is a potent inhibitor of platelet aggregation, but also xanthines are considered to exert this action. In the present study, effects of theophylline and enprofylline on ADP-induced aggregation of human platelets were studied in vitro. Theophylline alone in concentrations exceeding 280 microM inhibited platelet aggregation concentration-dependently. Enprofylline alone mimicked this effect but was about five times more potent than theophylline. At the lowest concentrations used, corresponding to upper therapeutic levels, neither of the two xanthines affected platelet aggregation by ADP. The interactions between these low concentrations of the xanthines and adenosine were then evaluated. In the presence of theophylline 110 microM the inhibitory effect of adenosine 4 microM was attenuated, whereas the presence of enprofylline 21 microM enforced the inhibitory effect of adenosine. Thus, at low concentrations where neither theophylline nor enprofylline inhibits platelet aggregation theophylline antagonizes the antiaggregatory effect of adenosine, whereas enprofylline acts in synergy with this nucleoside.     

Platelet-activating factor (PAF-acether): Thromboxane-independent synergism with adrenaline on human platelets and recent insights into its mode of action

The mode of action of PAF-acether on human and rabbit plasma-free platelets is reviewed. PAF-acether and adrenaline synergize to trigger aggregation of human platelets, and this synergism is refractory to aspirin. When degranulated rabbit platelets are stimulated with PAF-acether, with thrombin or with the snake venom component convulxin, aggregation is obtained in the absence of detectable secretion. Collagen-induced aggregation is reduced, and is suppressed when aspirin is applied to the degranulated platelets. The formation of PAF-acether by platelets, and their stimulation by PAF-acether itself, should be added to the newly recognized pathways for platelet stimulation.