Contribution of arachidonic acid cycle enzymes to platelet activation by low-density lipoproteins

The mechanism of platelet activation by low-density lipoproteins (LDL) was studied using inhibitors of arachidonic acid cycle enzymes. Lipoxygenase and cyclooxygenase inhibitors (indomethacin, acetylsalicylic acid, NDGA, and BW755C) inhibited LDL-induced platelet aggregation to a small extent, as was indicated by mere 20% to 30% decreases in the maximal rate of change in light transmission. 4-Bromophenacyl bromide inhibited LDL-induced platelet aggregation in a dose-dependent, manner, almost complete inhibition occurring at concentrations in excess of 20 μM. The results support the conclusion that enzymes to the arachidonic acid cycle do not contribute substantially to platelet activation by LDL. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 10, pp. 376–379, October, 1995 Presented by Yu. M. Lopukhin, Member of the Russian Academy of Medical Sciences     

Inhibition of platelet aggregation by monoclonal antibodies against glycoprotein IIb–IIIa complex

Monoclonal antibodies CRC64 are obtained against Ca²⁺-dependent glycoprotein IIb–IIIa complex of the platelet membrane which possess the ability to inhibit completely fibrinogen-dependent platelet aggregation. CRC64 is directed against the epitope formed by the glycoprotein IIb–IIIa complex and does not interact with proteins isolated after platelets are treated with ethylenediamine tetraacetate. Complete, reproducible blockade of platelet aggregation caused by 5 μM adenosine diphosphate is noted in an MCA concentration of 3 μg/ml, while in the case of a stronger inductor, namely 1 U/ml thrombin, platelet aggregation is inhibited in a concentration of 5 μg/ml. F(ab′)₂ fragments are also able to inhibit platelet aggregation completely and are usually effective in concentrations lower than native monoclonal antibodies. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 10, pp. 402–405, October, 1994 Presented by V. N. Smirnov, Member of the Russian Academy of Medical Sciences     

Effect of interleukin-1β on platelet aggregation in August, Wistar, and WAG rats in acute emotional stress

Acute emotional stress inhibits platelet aggregation in August and WAG rats and reduces it in Wistar rats. Functional parameters of platelets are altered predominantly in passive rats. Interleukin-1β reduces the rate of platelet aggregation in nonstressed August and WAG rats and elevates it in Wistar rats. Preliminary injection of interleukin-1β reduces the stress-induced changes in platelet aggregation in August and WAG rats; while in Wistar rats this effect is not observed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 144–147, August, 1997     

Effect of recombinant forms of urokinase plasminogen activator on platelet aggregation and intracellular calcium accumulation

Urokinase caused plasmin-dependent inhibition of platelet aggregation in platelet-rich plasma, while its proteolytically inactive form had no such effect. Both forms potentiated the increase in platelet calcium concentration induced by aggregation inductors and facilitated aggregation of washed platelets. In contrast to full-length urokinase molecule, its aminoterminal fragment inhibited platelet aggregation and the corresponding elevation of intracellular calcium. These data suggest that urokinase exerts a plasmin-independent effect on platelet activity. This effect depends on urokinase structure. Translated formByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 339–343, September 1999     

Inhibition of platelet aggregation under the action of sodium hypochlorite. Effect of blood plasma components

The proteins fibrinogen and serum albumin and the amino acid alanine modified by sodium hypochlorite are shown to inhibit thrombin-induced aggregation of isolated platelets. The hypochlorite sodium-treated proteins and amino acids acquire the capacity to counter platelet aggregation as a result of the formation of chloramine derivatives. The aggregating capacity of hypochlorite sodium-inactivated platelets can be restored by native plasma and fibrinogen. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 5, pp. 488–490, May, 1995 Presented by Yu. M. Lopukhin, Member of the Russian Academy of Medical Sciences     

Hydrocortisone inhibition of adenosine diphosphate (ADP)-induced platelet aggregation in horse

The aim of this study was to evaluate the effect of hydrocortisone on adenosine diphosphate (ADP)-induced platelet aggregation in horse. The study was carried out on 35 healthy horses of various breeds and gender. On all horses, citrated blood samples were collected by jugular venipuncture to assess platelet aggregation. On all samples, platelet-rich and platelet-poor plasma were prepared by centrifugation and divided into two different aliquots. On the first aliquot, platelet aggregation was determined after platelet activating with 1 and 0.5 μM ADP (group A). On the last one, the effect of 20-min preincubation with hydrocortisone in ADP-induced aggregation was determined at final ADP concentrations of 1 and 0.5 μM (group B). By means of an aggregometer, the platelet response was quantitated as maximum degree of platelet aggregation and initial velocity of platelet aggregation. All data were analyzed using unpaired Student's t test. A P ≤ 0.05 was considered statistically significant. The obtained results showed a statistically significant effect of hydrocortisone on both the maximum degree of platelet aggregation and the slope of platelet aggregation at the two final concentrations of ADP used. These results are compatible with the possibility that the vessel wall releases smooth muscle-relaxing prostaglandins when injured and that the inhibition of prostaglandin formation by hydrocortisone enhances hemostasis by allowing vasoconstriction to be maintained. Further investigations could be useful utilizing different concentrations of hydrocortisone in order to apply the knowledge of the effect of glucocorticoids on platelet aggregation.     

Effect of allicor on platelet aggregationin vitro andex vivo

An extract of garlic powder in isotonic phosphate buffer and adjoen (bioactive compound isolated from garlic powder) suppress human blood platelet aggregation induced by ADP and arachidonic acidin vitro. Adjoen more effectively than aspirin inhibits ADP-induced platelet aggregation but is inferior to aspirin if platelet aggregation is induced by arachidonic acid.Ex vivo oral intake of one Allicor tablet significantly decreases rabbit platelet aggregation induced with ADP. It is suggested that long-acting garlic powder tablets prevent thromboembolic complications and are recommended for correcting hemostasis parameters in patients with atherosclerotic involvement of blood vessels. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 7, pp. 79–100, July, 1997     

Contractile effects of polycations in permeabilized smooth muscle

The polycations spermine, neomycin and polylysine potentiated Ca2+-activated force in β-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in α-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by β-escin, which eliminated the contractile effect of GTPγS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca2+-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca2+-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca2+-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40μm. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR to inhibit phosphatase activity was increased by the polycations, but only at [Ca2+]<0.3μm. The results suggest that polycations increase Ca2+-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

The metabolism of serotonin in neuronal cells in culture and platelets

The aim of this study is to find a relationship between serotonin (5-HT) and its metabolite 5-hydroxy indol acetic acid (5-HIAA) in hippocampus, frontal neocortex and platelets. Serotonin and 5-HIAA were measured in cultured neurons and compared with those produced by human platelets. The cortical neuronal 5-HIAA/serotonin ratio was 4.7 and for hippocampal neurons it was 3.2. In human platelets, this ratio was 1.35 suggesting that the highest serotonin metabolism occurs in the frontal neocortex followed by the hippocampus and platelets. In the presence of 0.3 μM of p-chlorophenylalanine both cultured neurons and platelets exhibited an approximately 50% decrease in serotonin and 5-HIAA concentration suggesting similarities in the metabolic profile in both preparations. In addition, we found that serotonin by itself does not play any role in platelet aggregation but potentiates this phenomenon in the presence of calcium ionophore A23187. This synergistic interaction between serotonin (2–5 μM) and A23187 (0.5–2 μM) was inhibited by serotonin receptor blockers [methysergide (IC₅₀ = 18 μM) and cyproheptadine (IC₅₀, 20 μM)] and calcium channel blockers (verapamil and diltiazem, IC₅₀ = 20 and 40 μM, respectively) that indicate both mechanisms are receptor mediated. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect of serotonin and ionophore at an IC₅₀ value of 9.2 μM. Wortmannin, a phosphoinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC₅₀ = 2.6 μM) by inhibiting respiratory burst. However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C (PKC) inhibitor, affected aggregation. Our results are strongly suggestive of a synergistic interaction between serotonin type-2 and Ca-ionophore via a PLC/Ca signalling pathway.     

Mechanism of action of aspirin on platelet function

Inhibition of platelet aggregation and of the reaction of liberation of platelet factor 3 under the influence of aspirin was shown to be due to the action of the drug not only on the platelets, but also on plasma cofactors: In experimentsin vitro the blood plasma of rats receiving aspirin reduced the aggregating power of the platelets of intact animals; blood plasma of intact rats increased the aggregating power and accessibility of factor 3 of the platelets of animals receiving aspirin.Department of Radiation Pathological Physiology, Scientific-Research Institute of Medical Radiology, Academy of Medical Sciences of the USSR, Obninsk. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 434–435, April, 1977.     

Effect of storage of platelet concentrate and products of myeloperoxidase reaction on initial platelet aggregation

An original nephelometric method allows one to measure initial platelet aggregation after a long-term storage of platelet concentrate. This attests to a high level of platelet activity despite the fact that final platelet aggregation cannot be detected by usual turbidimetric method. N-chloroalanine more slowly inhibits initial platelet aggregation in comparison with sodium hypochlorite, which implies different mechanisms of their membrane-modifying effects. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 523–526, November, 1997     

Early stages in the mechanism of action of glucocorticoids on human platelets. Effect of hydrocortisone on intracellular cAMP and Ca2+ content

Changes in basal and stimulated levels of cAMP and calcium induced by hydrocortisone in a wide range of concentration (0.1–25 μM) are studies in a suspension of washed human platelets. The effects of hydrocortisone on the activity of preparations modulating various stages of the adenylate cyclase system (forskolin, adenosine, adrenaline, and 3-isobutyl-1-methylxanthine) are compared. Platelets are stimulated with collagen, platelet activating factor, and thapsigargin. Hydrocortisone in different concentrations acts as both activator and inhibitor of calcium metabolism in platelets. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 6, pp. 663–665, June, 1997     

Serotonin-induced platelet aggregation and release of3H-serotonin in patients with migraine

Serotonin-induced platelet aggregation in patients with migraine without aoura is less pronounced than in healthy individuals. In 50% of the patients the platelet serotonin transport system is characterized by increased sensitivity to serotonin: serotonin induced a more potent (by 26%) release of³H-serotonin from platelets, while the serotonin transport inhibitor imipramine blocked this effect. Thus, the serotonin transport system in patients with migraine differs from that of healthy indiduals. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 156–159, August, 1998     

A novel role of andrographolide,an NF-kappa B inhibitor,on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP

Andrographolide is a novel NF-κB inhibitor from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of thrombotic diseases. However, no data are available concerning the effects of andrographolide in platelet activation. The aim of this study was to examine the mechanisms of andrographolide in preventing platelet activation. Andrographolide (25–75 μΜ) exhibited a more potent activity of inhibiting platelet aggregation stimulated by collagen. Andrographolide inhibited collagen-stimulated platelet activation accompanied by relative Ca²⁺ mobilization; thromboxane A₂ formation; and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Andrographolide markedly increased cyclic GMP, but not cyclic AMP levels. Andrographolide also stimulated endothelial nitric oxide synthase (eNOS) expression, NO release, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. ODQ, an inhibitor of guanylate cyclase, markedly reversed the andrographolide-mediated inhibitory effects on platelet aggregation, p38 MAPK and Akt phosphorylation, and the andrographolide-mediated stimulatory effect on VASP phosphorylation. Furthermore, a PI3 kinase inhibitor (LY294002) but not a PKC inhibitor (Ro318220) significantly diminished p38 MAPK phosphorylation; nevertheless, a p38 MAPK inhibitor (SB203580) and LY294002 diminished PKC activity stimulated by collagen. Andrographolide also reduced collagen-triggered hydroxyl radical (OH^⋅) formation. In vivo studies revealed that andrographolide (22 and 55 μg/kg) is effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism and significantly prolonged platelet plug formation in mice. This study demonstrates for the first time that andrographolide possesses a novel role of antiplatelet activity, which may involve the activation of the eNOS-NO/cyclic GMP pathway, resulting in the inhibition of the PI3 kinase/Akt-p38 MAPK and PLCγ2–PKC cascades, thereby leading to inhibition of platelet aggregation.     

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion,secretion and aggregation

Adrenaline (1 to 10M) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca²⁺ ionophore A23187. Adrenaline (0.5 M) potentiates the aggregation and secretion induced by all the previous agonists in citrated plateletrich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: i) nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC₅₀ 0.22 M and 0.28 M respectively); ii) nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC₅₀ ranging from 0.1 to 2.5 M) or in washed platelets (IC₅₀ ranging from 0.1 to 0.8 M); iii) nicergoline inhibits the biding of³H-yohimbine to washed human platelets (IC₅₀ 0.26 M); iv) the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly theex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC₅₀ range 108 to 670 M) and in washed platelets (IC₅₀ range 27 to 140 M) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activationin vivo could justify the use of nicergoline (Sermion^®), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.     

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     

Effect of diquertin on platelet content of cyclic nucleotides

The effects of diquertin on the content of cyclic nucleotides in human platelets was studied. Diquertin in a concentration of 5 mM increased the content of cAMP and cGMP in native and thrombin-activated platelets probably due to inhibition of phosphodiesterase. Increasing the concentration of diquertin above 5 mM did not potentiate this effect. The antiaggregation effect of diquertin was probably associated with the increase in platelet levels of cyclic nucleotides. Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny,Vol. 128, No. 9, pp. 267–269, September, 1999     

Human platelets as the object of investigation of the molecular mechanisms underlying nongenomic effects of glucocorticoid hormones

The effect of hydrocortisone (100 nM–10 μM) on the major biochemical parameters of platelet activity (intracellular free calcium concentration, thromboxane B₂ content, and baseline and stimulated levels of cAMP and cGMP) is examined. The results obtained indicate that the inhibitory effect of glucocorticoids on platelet aggregation is mediated by activation of the adenylate cyclase system and suppression of the calcium response. Presumably, neither guanylate cyclase nor phospholipase A₂-dependent systems are the targets of nongenomic actions of glucocorticoid hormones. Platelets can serve as a convenient tool for the investigation of nongenomic effects of glucocorticoid hormones. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 285–287, September, 1996     

Contraction of tubulointerstitial fibrosis tissue in diabetic nephropathy,as demonstrated in an in vitro fibrosis model

Tubulointerstitial fibrosis in diabetic nephropathy (DN) was investigated using an in vitro tissue model of remodeling, to determine the pathogenic mechanism of fibrosis that leads to renal atrophy, i.e., renal failure. The remodeling model consisted of a renal fibroblast-populated collagen lattice (FPCL). The overexpression of transforming growth factor (TGF)-β1 in the diabetic kidney gave rise to FPCL contraction. FPCL relaxation was induced by the subsequent addition of cytochalasin D. The FPCL failed to contract when exposed to TGF-β1 plus Y27632, a Rho kinase inhibitor. TGF-β1 induced the phosphorylation of myosin light chains, and Y27632 blocked this activity. TGF-β1-induced FPCL contraction was suppressed by the addition of 2,3-butanedione monoxime, a myosin ATPase inhibitor. As shown in the video, the contraction rate of the projections of the cells in the FPCL was significantly greater in the TGF-β1 group than in the control group. Collectively, these results indicate that TGF-β1-induced FPCL contraction is attributable to actin–myosin interactions in the fibroblasts through the activation of Rho kinase, the phosphorylation of myosin light chains, and the subsequent activation of myosin ATPase. We propose that via these mechanisms, tubulointerstitial fibrosis generates tissue contraction that leads to renal atrophy and renal failure in DN.     

Glucosamine,a naturally occurring amino monosaccharide,suppresses the ADP-mediated platelet activation in humans

Objective: To evaluate the anti-thrombotic action of glucosamine, a naturally occurring amino monosaccharide, platelets were stimulated with ADP in the presence of glucosamine, and its effects on platelet functions were examined.Materials and Methods: Human platelet-rich plasma was stimulated with 2.5 M ADP in the presence of glucosamine (0.01 ~ 1 mM) or other aminosugars (N-acetyl-glucosamine, galactosamine or N-acetyl-galactosamine, 1 mM), and platelet aggregation was monitored. Furthermore, the effects of glucosamine on the thromboxane A2 production, release of granule contents, intracellular calcium mobilization and phosphorylation of Syk (a 72 kD protein tyrosine kinase) were evaluated following ADP-stimulation. In addition, the binding of [³H] ADP to its receptors was examined.Results: Glucosamine (>0.01 mM) dose-dependently suppressed platelet aggregation in response to ADP (p < 0.05), whereas N-acetyl-glucosamine, galactosamine or N-acetyl-galactosamine (1 mM) did not affect the ADP-induced platelet aggregation. Furthermore, glucosamine (>0.1 mM) inhibited the extracellular release of granule contents (ATP and platelet factor 4) and production of thromboxane A₂ from ADP-stimulated platelets (p < 0.05). Moreover, glucosamine significantly repressed the intracellular calcium mobilization at >0.1 mM and phosphorylation of Syk at >0.01 mM upon ADP-stimulation (p < 0.05). In addition, glucosamine (>0.1 mM) inhibited the binding of ADP to its receptors (p < 0.05).Conclusion: Glucosamine is able to suppress platelet aggregation, release of granule constituents, thromboxane A₂ production, calcium mobilization and phosphorylation of Syk possibly via the inhibition of ADP-binding to the receptors. Glucosamine could be expected as a novel anti-platelet agent for thrombotic disorders due to its suppressive actions on platelets.Received 18 June 2004; returned for revision 11 July 2004; accepted by M. Katori 9 August 2004