Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.     

Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C

Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induce activation of phospholipase C as reflected by formation of inositol phosphates and phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed.     

A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.     

Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization

The effect of ethanol on signal generation in collagen-stimulated human platelets was evaluated. Incubation of washed human platelets with physiologically relevant concentrations of ethanol (25-150 mM) resulted in a dose-dependent inhibition of aggregation and secretion in response to collagen (0.5-10 micrograms/ml), but did not inhibit shape change. In platelets labeled with [3H]arachidonic acid, ethanol significantly inhibited the release of arachidonic acid from phospholipids, in both the presence and the absence of indomethacin. Thromboxane B2 formation was also inhibited in proportion to the reduction in free arachidonic acid. There was a close correlation between the extent of inhibition of arachidonic acid release and secretion. The inhibition of platelet aggregation and secretion by ethanol was partially overcome by the addition of exogenous arachidonic acid. In the presence of indomethacin, ethanol had no effect on the activation of phospholipase C by collagen as determined by the formation of inositol phosphates and phosphatidic acid. Moreover, ethanol had no effect on the mobilization of intracellular calcium by collagen and only minimally inhibited the early phases of the phosphorylation of myosin light chain (20 kDa) and a 47-kDa protein, a known substrate for protein kinase C. Arachidonic acid formation was also inhibited by ethanol in response to ionomycin under conditions where phospholipase C activation was prevented. The results suggest that the functional effects of ethanol on collagen-stimulated platelets are due, at least in part, to an inhibition of phospholipase A2.     

Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase.

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.     

The inhibitory effects of okadaic acid on platelet function.

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation.     

Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases.

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.     

Lysophosphatidic acid can activate platelets without increasing 32P-labelling of phosphatidic acid

Stimulation of platelets by thrombin produced a rise in [32P]phosphatidic acid labelling of platelets which was greater in medium without added calcium than in medium with 2.5 mM calcium. A rise in [32P]lysophosphatidic acid was also seen in platelets stimulated by thrombin in the presence of 2.5 mM extracellular calcium, though it was of lesser magnitude (average 35%) than the rise in phosphatidic acid. In platelets resuspended without added calcium no change in [32P]lysophosphatidic acid was seen in response to thrombin. Lysophosphatidic acid can itself induce platelet aggregation. Similarly to the calcium ionophore A23187, lysophosphatidic acid produced minimal change (in medium with no added calcium) to no change (in medium with 2.5 mM external calcium) in [32P]lysophosphatidic acid. The endoperoxide analog U46619 produced changes in 32P-labelling of platelet phosphatidic and lysophosphatidic acid similar to those produced by thrombin but of lesser magnitude. The results of these studies show that the action of lysophosphatidic acid on platelets differs from the action of thrombin, U46619 and platelet-activating factor, which produce a rapid rise in [32P]phosphatidic acid, and suggests that lysophosphatidic acid, like A23187, largely bypasses the initial receptor-coupled breakdown of phosphoinositides leading to formation of diacylglycerols and phosphatidic acid.     

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

The activation of protein kinase C in human platelets by phorbol-12, 13- dibutyrate (PDBu) results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.     

Neomycin inhibits platelet functions and inositol phospholipid metabolism upon stimulation with thrombin, but not with ionomycin or 12-O-tetradecanoyl-phorbol 13-acetate

Gel-filtered human platelets that had been pre-labelled with [32P]Pi were stimulated with thrombin, ionomycin or the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The effect of the hexacationic aminoglycoside antibiotic, neomycin, on platelet physiological responses, such as aggregation and secretion, as well as changes in phosphoinositide metabolism was studied. Neomycin strongly inhibited thrombin-induced aggregation and secretion whereas the antibiotic had no effect on ionomycin- or TPA-induced platelet functions. The thrombin-induced enhancement of inositol phospholipid metabolism was strongly inhibited by the presence of neomycin whereas the TPA- or ionomycin-induced increase in inositol [32P]polyphospholipids remained unaffected. The inhibitory effect of some other aminoglycoside antibiotics was compared to that of neomycin and the data demonstrate that the inhibition of platelet secretion and phosphatidic acid production was dependent on the cationic charge of the antibiotic. It is suggested that neomycin inhibits signal transduction in platelets at a level prior to the inositol-phospholipid-specific phosphodiesterase.     

Decanoyl lysophosphatidic acid induces platelet aggregation through an extracellular action. Evidence against a second messenger role for lysophosphatidic acid.

Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into its corresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. 1-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3 microM upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 microM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 microM-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

The role of phospholipase C in platelet responses

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.     

Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion.

It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.     

A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets

Stimulation of washed human platelets with alpha-thrombin was accompanied by aggregation, formation of inositol phosphates and phosphatidic acid, liberation of arachidonic acid, mobilization of intracellular Ca2+ stores, and influx of Ca2+ from the extracellular medium. Each of these responses was potentiated by a short pretreatment with epinephrine, although alone this agent was ineffective. A prolonged (5 min) stimulation with alpha-thrombin desensitized both phospholipase C and Ca2+ mobilization to a further thrombin challenge. Epinephrine added following thrombin desensitization restored both the ability of thrombin to release Ca2+ stores and stimulate inositol phospholipid hydrolysis. Resensitization was mediated by alpha 2-adrenergic receptors and lasted about 3 min, after which the Ca2+ levels returned again to basal levels. Pretreatment of platelets with phorbol dibutyrate at concentrations which specifically activate protein kinase C increased the rate of desensitization of the thrombin-induced release of Ca2+ stores and abolished the ability of epinephrine to restore the thrombin response. The protein kinase C inhibitor, staurosporine, blocked the inhibitory effect of phorbol ester and also reduced the rate of desensitization of thrombin and subsequent epinephrine action. These results suggest that thrombin activation of protein kinase C phosphorylates and inactivates a signaling protein which is common to both thrombin and alpha 2-adrenergic receptors. This protein is involved in thrombin stimulation of phospholipase C but is not directly stimulatory since epinephrine alone does not activate this enzyme. We searched for a known second messenger protein common to both thrombin and alpha 2-adrenergic receptors which was phosphorylated in intact platelets by protein kinase C in parallel with thrombin-induced desensitization. The alpha subunit of the inhibitory GTP-binding protein, Gi, was the only candidate which fulfilled all of these criteria as shown by immunoprecipitation. Therefore, we suggest that alpha i maintains the thrombin receptor in a state which can couple to phospholipase C when activated with thrombin. This permissive state of alpha i is blocked by phosphorylation by thrombin-activated protein kinase C.     

Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Inhibition of platelet aggregation by S-(1,2-dicarboxyethyl)glutathione, intrinsic tripeptide in liver, heart, and lens

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or baker's yeast showed strong inhibitory effects on blood coagulation and platelet aggregation. The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate. DCE-GS did not show chelating activity. As for ADP- or thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation. DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen. The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol-13-acetate. Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different.     

Shape change induced in human platelets by platelet-activating factor. Correlation with the formation of phosphatidic acid and phosphorylation of a 40,000-dalton protein

Washed human platelets that have been separated from plasma in the presence of prostacyclin are activated by the addition of platelet activating factor (PAF). Activation (shape change, serotonin release, and aggregation) correlates closely with the formation of phosphatidic acid and the phosphorylation of a 40,000-dalton protein. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation precede aggregation and are induced at lower concentrations of PAF than those required to induce release of serotonin and platelet aggregation. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation induced by PAF are not affected by trifluoperazine or indomethacin. This indicates that these responses are independent of the liberation of arachidonic acid from platelet phospholipids and the metabolism of arachidonic acid via cyclooxygenase and lipoxygenase. These responses are, however, inhibited by prostacyclin. Platelet shape change is the first measurable physiologic response to platelet agonists and may be associated with the stimulation of phospholipase C, inducing formation of 1,2-diacylglycerol and its phosphorylated product, phosphatidic acid. Transient formation of 1,2-diacylglycerol may also induce the specific activation of the protein kinase C that phosphorylates a 40,000-dalton protein.     

Differential effect of retinoic acid on ADP and collagen induced platelet aggregation

Retinoic acid (RA) was found to inhibit ADP induced but not collagen induced aggregation of human platelets and the differential action is related to intraplatelet Ca2+ reflux. RA was active at concentrations as low as 10(-7) M and required 20 min prior incubation with platelet suspension in order to inhibit aggregation by ADP. All the steps in ADP induced but not collagen induced platelet activation, viz. hydrolysis of phosphatidyl inositol, phosphorylation of 20, 47 and 250 kDa proteins as well as increased association of actin with Triton X-100 insoluble cytoskeletal matrix were inhibited by RA. RA when used as an agent for differentiation induction of cell progenitor is likely to affect the platelet aggregation and thereby the haemostatic process.