Intact Rabbit Platelets Research Articles

Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway

We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation. The present study was performed to investigate the effect of GTC on the arachidonic acid (AA) metabolism in order to elucidate a possible antiplatelet mechanism. GTC inhibited the collagen-, AA- and U46619-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC 50 values of 61.0±2.5, 105.0±4.9 and 67.0±3.2 μg/ml, respectively. Moreover, GTC administered orally into rats inhibited the AA-induced platelet aggregation ex vivo by 46.9±6.1% and 95.4±2.2% at the doses of 25 and 50 mg/kg, respectively. [ 3H]AA liberation induced by collagen in [ 3H]AA incorporated rabbit platelets was significantly suppressed by GTC compared to the control. GTC also significantly inhibited the thromboxane A 2 (TXA 2) and prostaglandin D 2 (PGD 2) generations induced by addition of AA in intact rabbit platelets. GTC significantly inhibited TXA 2 synthase activity in a concentration-dependent manner. Moreover, adenosine triphosphate (ATP) release from dense granule was inhibited by GTC in washed platelets. These results suggest that the antiplatelet activity of GTC may be due to the inhibition of TXA 2 formation through the inhibition of AA liberation and TXA 2 synthase.

Transcellular metabolism of leukotriene A4 by rabbit blood cells: lack of relevant LTC4-synthase activity in rabbit platelets

The objective of this study was to determine the transcellular metabolism of leukotriene A4 by rabbit blood cells. mixed peripheral blood leukocyte preparations with and without platelets in a ratio of 1:40 were challenged with the Ca(2+)-ionophore A23187. 5-Lipoxygenase metabolites production was assessed by RP-HPLC coupled with diode-array UV detection. In light of the observation that leukotriene C4 production in leukocyte-platelet coincubation was the same as with leukocytes alone, mixed coincubation of human and rabbit blood cells was tested. Rabbit leukocytes with human platelets resulted in a significant increase of leukotriene C4 production, while no changes were observed in human leukocytes with or without rabbit platelets. In agreement with these results, intact rabbit platelets or rabbit platelet lysates, unlike human platelets, were not able to convert synthetic leukotriene A4 free acid to leukotriene C4. These data provide evidence that rabbit leukocytes are able to make a significant amount of leukotriene A4 available for transcellular metabolism, while rabbit platelets, unlike human platelets, lack leukotriene C4-synthase activity.

Isolation of a Phospholipid Inhibitor of Platelet Activating Factor-Induced Activity from Perfused Rat Liver: Identification as Phosphatidylglycerol

An endogenous inhibitor of platelet activating factor action was isolated from perfused rat liver. It was purified by thin-layer chromatography and high-performance liquid chromatography and subjected to chemical modifications in order to identify its structure. On the basis of its fast atom bombardment-mass spectrum it was characterized as phosphatidylglycerol composed mainly of 16:0/18:1 and 16:0/20:2 fatty acyl chains ([M + H] + at m/ z 749 and 775, respectively) and very minor levels of 18:0/18:1 and 18:0/20:2. The purified compound exhibited inhibition on rabbit platelet aggregation induced by 5 × 10 −10 M platelet activating factor (PAF) at an EC 50 value near 2.5 × 10 −6 M and on the serotonin secretion at an EC 50 7 × 10 −6 M. Other phospholipids isolated from the liver preparations, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin (diphosphatidylglycerol), and phosphatidic acid, exhibited no inhibitory activity in the concentration range from 1 × 10 −4 to 1 × 10 −7 M nor did they induce any aggregation, or lysis, of the platelets. Of importance, phosphatidylglycerol could inhibit thrombin- and ADP-induced aggregation of rabbit platelets. These results suggested a possible site of inhibition common to the signal transduction pathway of these agonists. Preliminary binding experiments showed a noncompetitive type of inhibition on PAF binding to intact rabbit platelets.

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

The rabbit platelet metabolizes platelet-activating factor (PAF) intracellularly. PAF is deacetylated to produce lysoPAF which, in turn, can be acylated to produce 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl GPC). Some PAF receptor antagonists have been shown to inhibit this metabolic conversion. In the present study we examined whether the PAF receptor antagonists SRI 63-441 and WEB 2086 would inhibit the metabolism of PAF by intact rabbit platelets. In addition, we examined whether iloprost, a stable analogue of prostaglandin I2 (PGI2), and a potent inhibitor of platelet activation induced by a range of agonists, would also inhibit PAF metabolism. We found that SRI 63-441 and WEB 2086 caused an almost complete inhibition of the conversion of PAF to alkylacyl GPC. Iloprost caused up to a 50% inhibition of PAF metabolism compared to antagonist-free controls. Iloprost (and PGI2) is thought to inhibit platelet response by elevation of cAMP, while receptor antagonists act by blocking PAF binding to its receptor. Since iloprost caused partial inhibition of PAF metabolism, the results of this study suggest that inhibition of PAF metabolism does not occur solely due to competitive inhibition of PAF binding to its receptor.

Serotonin organelles of rabbit platelets contain synaptophysin

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.

Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5′-[γ-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.

Modes of inhibitory action of 4β-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

The tumor-promoting phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC 50 vs thrombin = 0.5 n m, >90% inhibition at 10 n m. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 n m PMA. A non-tumor-promoting phorbol ester, 4- O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5′-(3- O-thio)triphosphate (GTPγS)-stimulated AA release, but not GTPγS- and AlF 4 −-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A 2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A 2 by PMA pretreatment is discussed.

K252a, a new inhibitor of protein kinase C, concomitantly inhibits 40K protein phosphorylation and seroton in secretion in a phorbol ester-stimulated platelets

K252a isolated from microbial origin was found to potently inhibit protein kinase C in vitro (1). This agent inhibits phosphorylation of 40,000 dalton protein (40K protein) induced by 12-0-tetradecanoylphorbol-13-acetate(TPA) in intact rabbit platelets. This indicates that K252a exhibits the inhibition of protein kinase C in intact cells. The serotonin secretion induced by TPA was inhibited by K252a at nearly equal concentrations required to inhibit the phosphorylation of 40K protein. This provides the evidence to support the cause-effect relationship between the protein phosphorylation and the secretion in TPA-stimulated platelets.

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of M r 80000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.

Tumor promoter chloroform is a potent protein kinase C activator

The major interaction site for tumor-promoting phorbol esters is the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), a key-element in signal transduction. Binding of phorbol esters results in enzyme activation which mediates, at least in part, the action of these agents. We have investigated the effects of tumor promoter chloroform on protein kinase C activity. Like thrombin and 12-0-tetradecanoylphorbol-13-acetate (TPA), chloroform was able to activate protein kinase C in intact rabbit platelets. In addition, chloroform stimulated enzyme activity as well as TPA binding capacity in cell-free system. Scatchard analysis of the data has shown that chloroform increased the number of phorbol ester binding sites. Structurally related compounds, carbon tetrachloride and methylene chloride, activated the enzyme similarly.

Mechanism of inhibitory effect of some amphiphilic drugs on platelet aggregation induced by collagen, thrombin or arachidonic acid

The effects of two representative groups of amphiphilic drugs, lysophosphatidylcholine species (myristoyl, palmitoyl and stearoyl) and phenothiazine neuroleptics (promethazine, chlorpromazine and trifluoperazine), on the morphology of intact rabbit platelets, arachidonate release from membrane phospholipids, pseudopod formation and the resulting aggregation of stimulated platelets were examined and compared with those of two known cycloxygenase inhibitors, aspirin and indomethacin. Collagen-induced aggregation was inhibited by relatively low concentrations of amphiphilic drugs in parallel with the prevention of arachidonate release from the membrane phospholipids. Thrombin- and arachidonate-induced aggregations were inhibited by these drugs at higher concentrations, at which they transform intact platelets from discoid to spiny and spherical shape, respectively, and consequently suppress formation of native pseudopods induced by these stimuli. Washing the drug-treated platelets with BSA-Tyrode solution abolished all the effects of drugs. In contrast, the cycloxygenase inhibitors blocked both collagen- and arachidonate-induced aggregations without causing membrane shape change of the intact platelets, although they inhibited thrombin-induced aggregation at extremely high concentrations, at which they did elicit membrane shape change. These observations suggest that these amphiphilic drugs act on the plasma membrane of platelets and impair membrane-linked functions. At lower concentrations they specifically inhibit the arachidonate

On the role of the major platelet membrane glycoproteins in platelet adhesion to collagen

The effect of modification of intact rabbit platelets with chymotrypsin, trypsin or thrombin on platelet adhesion to collagen fibers was determined. Experiments were performed in vitro with washed platelets suspended in a medium containing EGTA to prevent platelet aggregation. Platelet adhesion was unaffected by chymotrypsin or thrombin treatment but was reduced upon trypsin treatment. The latter effect could be attributed to the ADP released upon trypsin treatment. In the presence of high concentrations of AMP, adhesion was restored to control values. Release also occurred on thrombin treatment but this did not influence adhesion. The effect of the enzymes on platelet membrane glycoproteins was monitored by gel electrophoresis. Chymotrypsin caused the loss, at least, of the regions susceptible to lactoperoxidase-catalyzed iodination and to periodic acid-Schiff (PAS) staining from the major membrane glycoproteins I, II and III of rabbit platelets. Such degradation also occurred to glycoproteins I and II with trypsin, but thrombin was without detectable effect on the major membrane glycoproteins. The results indicate that degradation of the major membrane glycoproteins does not affect adhesion to collagen. Adhesion appears to be mediated either by protease-resistant parts of these molecules or by other membrane entities not readily attacked by proteases.

Adjuvant arthritic rat serum enhances HETE1 biosynthesis.

The effect of serum from adjuvant arthritic rats in the chronic inflammatory stage, on lipoxygenase activity in either rat peritoneal macrophages, intact rabbit platelets or the cytoplasmic fraction of horse platelet homogenates was studied. All preparations produced significantly more HETE from exogenous arachidonic acid in the presence of arthritic serum than normal serum. This effect was known not to be associated with the lower albumin level in arthritic serum, and it was specific for the lipoxygenase pathway of arachidonic acid metabolism as there was no effect on cyclo-oxygenase activity. The presence of an arthritic serum factor(s), with a molecular weight in the range of 100,000 as indicated by gel-filtration experiments, which specifically enhances the synthesis of HETE, is proposed.

Investigation of possible mechanisms of pyridoxal 5'-phosphate inhibition of platelet reactions.

<h2>Abstract</h2> Pyridoxal 5′-phosphate (PLP) inhibits ADP-induced platelet shape change and aggregation. PLP also causes rapid deaggregation of platelets aggregated by ADP. Effects on platelet aggregation and coagulation are demonstrable following administration to man. Several suggestions concerning the possible mechanism of inhibition have been investigated. PLP did not inhibit the nucleoside diphosphokinase activity of intact rabbit platelets nor of isolated platelet membranes. PLP had no measurable effect on rabbit platelet cyclic AMP either alone or in the presence of prostaglandin E₁ and caffeine. ADP-induced aggregation and ¹²⁵I-fibrinogen binding of rabbit platelets were inhibited concomitantly; however, fibrinogen-induced aggregation of chymotrypsin-treated platelets was only slightly inhibited by PLP. ¹²⁵I-fibrinogen binding to chymotrypsintreated platelets was partially inhibited by PLP; when PLP was added to these platelets, bound fibrinogen was displaced. With chymotrypsin-treated platelets, PLP inhibited the ADP component of the synergistic aggregating effect of the combination of fibrinogen and ADP. It seems unlikely that the inhibitory effects of PLP are due to interference with nucleoside diphosphokinase, increasing platelet cyclic AMP or inhibition of fibrinogen binding. The mechanism of inhibition remains to be established.

Oestrogen and progesterone change the binding characteristics of α-adrenergic and serotonin receptors on rabbit platelets

The binding characteristics of the α-adrenergic and serotonin receptors on intact rabbit platelets were identified using [ 3H]-dihydroegocryptine and [ 3H]-5-hydroxytryptamine respectively. In untreated female rabbits a single α-adrenoreceptor and two serotonin receptors were identified with ligand affinities similar to these in human platelets. The effects of oestrogen and/or progesterone on the α-adrenergic and serotonin receptors of rabbit platelets were studied. Oestrogen alone increased the capacity of serotonin receptors but reduced that of the α-adrenoceptor. Oestrogen plus progesterone reduced the capacity of both receptors and reduced the affinity of the α-adrenoceptor. The possible mechanism of the steriod-induced changes in the implications of these with regard to the effects of the oral contraceptive pill are discussed.

Effects of rabbit platelets on vascular smooth muscle cell DNA synthesis

During platelet aggregation a factor is released which stimulates DNA synthesis in vascular smooth muscle cells. We have examined the capacity of plasma free suspensions of intact rabbit platelets to stimulate DNA synthesis in vascular smooth muscle cells in tissue culture. Platelet-rich plasma was obtained by low-speed centrifugation of citrated blood and passed through sterile columns of sepharose 2B beads. The resultant plasma-free platelet suspension was adjusted to 400,000 platelets/mm 3. An aliquot was completely aggregated with thrombin, centrifuged and the supernatant examined for ability to stimulate incorporation of [ 3H]thymidine into DNA by cultured vascular smooth muscle cells. Sonication of an aliquot of platelet suspension released an equivalent amount of this stimulating factor. Another aliquot of platelet suspension of identical volume was added directly to the test smooth muscle cells. Stimulation by intact platelets co-cultured with smooth muscle cells was more than twice as great as that of the thrombin-aggregated supernatant. The latter was not increased by increasing the thrombin concentration nor by increasing the time of exposure. The potency of intact platelets was decreased only slightly by segregating them in Millipore chambers indicating that direct contact was not responsible for its enhancement.

Dependence of Platelet Adenyl Cyclase System on Oxidative Phosphorylation

The radioactive adenosine 3',5'-monophosphate (cyclic AMP) level derived from 8-14C adenine in intact rabbit platelets decreased in the presence of mitochondrial inhibitor (potassium cyanide) or uncoupler (sodium azide), and markedly increased by the addition of NaF, monoiodoacetic acid (MIA), or 2-deoxy-D-glucose. The stimulative effect of the glycolytic inhibitors was distinctly enhanced by the simultaneous addition of sodium succinate. MIA did neither directly stimulate the adenyl cyclase activity nor inhibit the phosphodiesterase activity. These results suggest that cyclic AMP synthesis in platelets is closely linked to mitochondrial oxidative phosphorylation.