PAF-Acether May Not Mediate the Third Pathway of Platelet Aggregation since Self-Desensitization Reduces the Effects of Low Thrombin but Enhances Those of Convulxin

SummaryPAF-acether (platelet-activating factor) was hypothesized as the mediator of the ADP and thromboxane-independent activation of platelets induced by thrombin (Thr) and by the snake venom glycoprotein convulxin (Cx). Aspirinized rabbit platelets self-desensitized to PAF-acether were less responsive to low amounts of Thr, as expected if PAF-acether would be formed, but were hyper-reactive to Cx, in contradiction with its hypothesized mediating role. Aggregation by higher concentrations of Thr overcame inhibition. Experiments with ADP-depleted platelets showed that secretion is neither involved with desensitization to PAF-acether nor with hyper-reactivity to Cx. Those effects required the presence of PAF-acether in the platelet suspension and persisted when transformation of PAF-acether into its recognized metabolite alkyl-acyl-glycerophosphorylcholine was inhibited. The ADP and thromboxane-independent activation of rabbit platelets by low and medium concentrations of Thr may be accounted for by platelet formation of PAF-acether, but overall the contrasting effects of platelet desensitization to PAF-acether on responsiveness to Thr and to Cx suggest that the third pathway of aggregation requires other explanations.

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Inhibition by Glaucocalyxin A of Aggregation of Rabbit Platelets Induced by ADP, Arachidonic Acid and Platelet-Activating Factor, and Inhibition of [3H]-PAF Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648470 ◽

1992 ◽

Vol 67 (04) ◽

pp. 458-460 ◽

Cited By ~ 12

Author(s):

Zhang Bin ◽

Long Kun

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Activating Factor ◽

Northeastern China ◽

Ethereal Extract ◽

Ic50 Value ◽

Rabbit Platelets ◽

Ic50 Values ◽

Induced Aggregation

SummaryGlaucocalyxin A is a new diterpenoid isolated from the ethereal extract of the leaves of Rabdosia japonica (Burm f) Hara var glaucocalyx (Maxim) Hara (Labiatae) collected in the northeastern China. When it was incubated with washed rabbit platelets, glaucocalyxin A inhibited ADP- or arachidonic acid-induced platelet aggregation with IC50 values of 4.4 μmol/1, 14.1 μmol/1 respectively. Glaucocalyxin A also inhibited PAF-induced aggregation of rabbit platelets which were refractory to ADP and arachidonic acid with an IC50 value of 13.7 μmol/1. Analysis of [3H]-PAF binding showed that glaucocalyxin A prevented [3H]-PAF binding to intact washed rabbit platelets with an IC50 value of 8.16 μmol/1, which was consistent with its inhibition of PAF-induced platelet aggregation.

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Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651597 ◽

1993 ◽

Vol 69 (03) ◽

pp. 286-292 ◽

Cited By ~ 13

Author(s):

Che-Ming Teng ◽

Feng-Nien Ko ◽

Inn-Ho Tsai ◽

Man-Ling Hung ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Inositol Phosphate ◽

Shape Change ◽

Platelet Activating Factor ◽

Atp Release ◽

Platelet Membrane ◽

Thromboxane B2 ◽

Dependent Manner ◽

Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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Pathways of Thrombin-Induced Aggregation and Release with Human and Rabbit Platelets

10.1055/s-0039-1682660 ◽

1977 ◽

Author(s):

R.L. Kinlough-Rathbone ◽

D.W. Perry ◽

M.A. Packham ◽

J.F. Mustard

Keyword(s):

Platelet Aggregation ◽

Direct Effect ◽

Thromboxane A2 ◽

Human Platelets ◽

The Third ◽

Rabbit Platelets ◽

Induced Aggregation

There are at least 3 mechanisms involved in thrombin-induced aggregation and release: (1) released ADP, (2) formation of thromboxane A2 and (3) a third mechanism(s). We have examined whether the third pathway is due to formation or release of a substance from platelets which affects other platelets. Washed human platelets were exposed to thrombin (2.5 u/ml) for 15 min at 37°C in the presence of indomethacin to block thromboxane A2 formation. Platelets were removed by centrifugation and the thrombin neutralized with hirudin or DFP. Addition of the superna te to washed human platelets prelabeled with 14C-serotonin caused platelet aggregation but release did not occur. Treatment of the supernate with apyrase, CP/CPK or dialysis abolished aggregation, indicating that the material was ADP. Thus, the mechanism by which thrombin induces aggregation and release with human platelets in the presence of agents which destroy ADP and block the formation of thromboxane A2 is a direct effect of thrombin on platelets and does not involve a substance freed from platelets. In contrast, when washed rabbit platelets were treated with thrombin in the presence of indomethacin and the released ADP was removed, material remained in the supernate which caused aggregation and release from washed rabbit platelets but was without effect on washed human platelets. The activity of this material (MW > 10,000) was not abolished by dialysis or boiling. Therefore rabbit platelets differ from human platelets because they have a mechanism in addition to released ADP, thromboxane A2 and the direct effect of thrombin on platelets that can cause aggregation and release.

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A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom

Biochemical Journal ◽

10.1042/bj2770351 ◽

1991 ◽

Vol 277 (2) ◽

pp. 351-357 ◽

Cited By ~ 53

Author(s):

T F Huang ◽

J R Sheu ◽

C M Teng

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Ionic Exchange ◽

Dependent Manner ◽

Single Chain ◽

Platelet Suspension ◽

Trimeresurus Flavoviridis ◽

Platelet Membranes ◽

Fibrinogen Binding ◽

Induced Aggregation

The interaction of fibrinogen with its receptors on platelet surfaces leads to platelet aggregation. A snake-venom peptide, trigramin, has previously been demonstrated to inhibit platelet aggregation by acting as a fibrinogen-receptor antagonist. By means of gel filtration, ionic-exchange chromatography and reverse-phase h.p.l.c., a potent platelet-aggregation inhibitor, triflavin, has now been purified from the venom of Trimeresurus flavoviridis. The purified triflavin is a single-chain polypeptide, consisting of about 71 amino acid residues with a molecular mass of 7600 Da, and its N-terminal sequence is Gly-Glu-Glu-Cys-Asp. Triflavin dose-dependently inhibited human platelet aggregation stimulated by ADP, adrenaline, collagen, thrombin or prostaglandin endoperoxide analogue U46619 in preparations of platelet-rich plasma, platelet suspension and whole blood. Its IC50 ranged from 38 to 84 nM, depending on the aggregation inducer used and the platelet preparation. However, triflavin apparently did not affect the platelet shape change and ATP-release reactions caused by these agonists. Triflavin inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner, indicating that it directly interferes with the binding of fibrinogen to its receptors on platelet membranes exposed by elastase treatment. Additionally, triflavin dose-dependently blocked 125I-labelled fibrinogen binding to ADP-activated platelets. In conclusion, triflavin inhibits platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors on platelet membranes.

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The effect of inhibitors of platelet aggregation on the metabolism of platelet-activating factor (PAF) in washed rabbit platelets

Lipids ◽

10.1007/bf02536493 ◽

1991 ◽

Vol 26 (12) ◽

pp. 1011-1014 ◽

Cited By ~ 2

Author(s):

C. O'Neill ◽

A. J. Ammit ◽

R. Korth ◽

S. Fleming ◽

X. Wells

Keyword(s):

Platelet Aggregation ◽

Platelet Activating Factor ◽

Rabbit Platelets ◽

Effect Of Inhibitors

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Degranulation of Rabbit Platelets with PAF-Acether: A New Procedure for Unravelling the Mode of Action of Platelet-Activating Substances

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657218 ◽

1982 ◽

Vol 48 (01) ◽

pp. 067-071 ◽

Cited By ~ 5

Author(s):

B Boris Vargaftig ◽

Danielel Joseph ◽

Guy Marlas ◽

L-G Chevance

Keyword(s):

Snake Venom ◽

Mode Of Action ◽

Dense Body ◽

Platelet Activating Factor ◽

Thromboxane A2 ◽

Venom Component ◽

Rabbit Platelets ◽

Body Components ◽

Membrane Changes ◽

Membrane Components

SummaryAggregation and secretion of ATP induced by thrombin, collagen, the snake venom component convulxin and platelet-activating factor (PAF-acether) were studied after the exposure of rabbit platelets to 1 μM of PAF-acether. This concentration, which is around 6 orders of magnitude above the concentration needed to induce full aggregation, was required to remove most of the releasable ATP from the platelets. The depleted platelets aggregated to PAF-acether, to thrombin and to convulxin under conditions where only very low amounts of ATP were secreted, confirming that these agents do not require the release of dense body components to trigger aggregation. Furthermore, when exposure to PAF-acether was associated to inactivation of platelet cyclooxygenase with aspirin, aggregation to thrombin persisted, validating the claim that thrombin induces aggregation by a third pathway unrelated to ADP and to thromboxane A2. Aggregation by collagen was markedly reduced by exposure of the platelets to PAF-acether or to aspirin; when both procedures were associated, aggregation was suppressed. Failure to desensitize the rabbit platelets to PAF-acether upon exposure to high amounts of it indicates the absence of irreversible membrane changes due to PAF-acether, and allows its use as a depleting procedure for the dense body materials, which does not affect platelet membrane components as is the case for thrombin.

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Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.1.c47 ◽

1998 ◽

Vol 274 (1) ◽

pp. C47-C57 ◽

Cited By ~ 14

Author(s):

Masahiro Suzuki ◽

Junko Sugatani ◽

Mitsuhiro Ino ◽

Masahiko Shimura ◽

Masaki Akiyama ◽

...

Keyword(s):

Platelet Aggregation ◽

Cell Surface ◽

Platelet Activating Factor ◽

Signaling System ◽

Platelet Disaggregation ◽

Rabbit Platelets ◽

Paf Receptor ◽

Aggregation Of Platelets ◽

Web 2086

Human and rabbit platelets fully aggregated by platelet-activating factor (PAF) underwent slow disaggregation but were rapidly disaggregated by the PAF receptor antagonists WEB-2086, Y-24180, SM-12502, and CV-3988. Whereas the 1-alkyl-2-[3H]acetyl- sn-glycero-3-phosphocholine ([3H]acetyl-PAF) specifically bound to platelet receptors underwent slow and spontaneous dissociation, it dissociated promptly from its receptor when WEB-2086 was added, in parallel with platelet disaggregation and disappearance of P-selectin on the cell surface. Extracellular [3H]acetyl-PAF was rapidly deacetylated by normal rabbit platelets; some of the [3H]acetyl-PAF was bound to the cells and a very small amount of [3H]acetate was detected in the cells. In contrast, when 1-[3H]alkyl-2-acetyl- sn-glycero-3-phosphocholine was added to the platelets, the radioactivity was rapidly incorporated into the 1-alkyl-2-acyl- sn-glycero-3-phosphocholine fraction. These results indicate that 1) continuous binding of PAF to its receptor is necessary for prolonged platelet aggregation, which may be mediated through an unknown signaling system for a long-term cell response rather than a transient signaling system, and 2) most of the [3H]acetyl-PAF bound to platelets is metabolized extracellularly by ecto-type PAF acetylhydrolase, with the lyso-PAF generated being incorporated rapidly into the cells and converted to 1-alkyl-2-acyl- sn-glycero-3-phosphocholine.

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Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets

Biochemical Journal ◽

10.1042/bj2670689 ◽

1990 ◽

Vol 267 (3) ◽

pp. 689-696 ◽

Cited By ~ 20

Author(s):

H Salari ◽

V Duronio ◽

S Howard ◽

M Demos ◽

S L Pelech

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Platelet Activating Factor ◽

Kinase C ◽

Camp Analogue ◽

Rabbit Platelets ◽

Rapid Hydrolysis ◽

Hydrolysis Of ◽

Pkc Activation

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.

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