Dihydropyridine agonist Bay K 8644 inhibits platelet activation by competitive antagonism of thromboxane A2-prostaglandin H2 receptor

The dihydropyridine calcium channel antagonists, such as nifedipine, inhibit platelet aggregation in vitro and ex vivo, but the mechanism by which this occurs is uncertain. Bay K 8644 (BAY) is a substituted dihydropyridine that has effects on voltage-dependent calcium channels in cardiac and smooth muscle that are opposite the effects of nifedipine. To evaluate the mechanism responsible for dihydropyridine-induced inhibition of platelet function, we studied the in vitro effects of BAY on human platelet aggregation and secretion plus several related biochemical parameters, including cytoplasmic ionized calcium ([Ca2+]i). BAY exerted concentration-dependent effects on platelet aggregation and secretion of [14C]serotonin. BAY (1-10 microns) inhibited the second wave of platelet aggregation and secretion stimulated by adenosine diphosphate or epinephrine and blocked shape change, aggregation, and secretion induced by the thromboxane A2 (TXA2) mimic, U46619. BAY also inhibited U46619-induced phosphorylation of the approximately 40,000-dalton cytoplasmic protein substrate of protein kinase C (40K protein), formation of TXA2, and rise in [Ca2+]i, all biochemical consequences of platelet activation. The (+)-(R) enantiomer of BAY [BAY(+)] was predominantly responsible for the inhibitory effects of racemic BAY. Nifedipine had the same inhibitory effects on platelet function and biochemistry, except it was approximately 10 times less potent than BAY. Since these results suggested inhibition of the TXA2-prostaglandin H2 (PGH2) receptor, we measured binding of [3H]U46619 to intact platelets. BAY, BAY(+), and nifedipine all functioned as competitive antagonists of [3H]U46619 binding (BAY Ki = 1.47 microM). They did not inhibit binding of [3H]yohimbine to platelet alpha 2-adrenergic receptors. At 1-10 nM BAY, BAY(+) and the (-)-(S) enantiomer of BAY [BAY(-)] all resulted in slight stimulation of platelet function and biochemical events. No significant increase in [3H]U46619 binding was demonstrable, however. Therefore, dihydropyridines that function as either calcium channel agonists or antagonists in cardiac or smooth muscle exert concentration-dependent effects on platelet function. In nanomolar concentrations, they augment, and in micromolar concentrations, they inhibit platelet activation induced by TXA2 or U46619. These data indicate that dihydropyridines do not inhibit TXA2-induced platelet activation by an effect on voltage-dependent calcium channels; they define the mechanism of inhibition as competitive antagonism of the TXA2-PGH2 receptor. The mechanism responsible for augmentation of platelet activation is uncertain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thromboxane A2/prostaglandin endoperoxide (TXA2/PG-END) receptor binding properties in human platelets of ridogrel, a combined TXA2 synthase inhibitor--TXA2/PG-END receptor antagonist.

Ridogrel, a potent thromboxane A2 (TXA2) synthase inhibitor, also has thromboxane A2 prostaglandin endoperoxide (TXA2/PG-END) receptor antagonistic properties as documented in functional studies of human platelets. In the present study, the binding affinities of the TXA2 synthase inhibitors, ridogrel, dazoxiben, dazmegrel and pirmagrel, and the TXA2/PG-END receptor antagonists, GR32191, L670596, SQ29548, ICI159995, AH69212 and sulotroban, for the TXA2/PG-END receptor labelled with [3H]SQ29548 on intact human platelets were assessed. The potencies of the TXA2/PG-END receptor antagonists to inhibit specific [3H]SQ29548 binding to intact human platelets ranged between 1.2 nM and 6,200 nM and corresponded to the ability of the drugs to suppress human platelet aggregation induced by TXA2/PG-END receptor stimulation with U46619 and collagen. The TXA2 synthase inhibitors dazoxiben, dazmegrel and pirmagrel could not inhibit specific [3H]SQ29548 binding to intact human platelets, tested up to 10(-5) M, nor suppress human platelet aggregation, indicating lack of any receptor antagonistic properties. Ridogrel, however, directly bound to the TXA2/PG-END receptor with micromolar affinity (IC50 = 5.2 microM) and inhibited U46619-27, or collagen-induced platelet aggregation, with ED50-values of 27 microM and 4.7 microM respectively. The present study thus demonstrates that antagonism by ridogrel of TXA2/PG-END receptor activation on platelets as defined in functional tests, coincides with inhibition of specific ligand binding to the receptors.     

Activation of Phospholipase C as a Primary Target of the Thromboxane A(2)-mediated Amplification Mechanism in Thrombin-induced Rabbit Platelet Activation

The inhibitory effects of a cyclooxygenase inhibitor, indomethacin, and a thromboxane (TX) A(2) receptor antagonist, S-145, on thrombin-stimulated rabbit platelet responses were examined for their contribution to the TXA-mediated amplification mechanism. Although thrombin (0.01-0.3 U/ml) induced the dosedependent aggregation and release of TXB(2) from washed rabbit platelets, indomethacin (30 μM) inhibited only the aggregation induced by a threshold dose of thrombin, even though it completely inhibited the formation of TXB(2). Indomethacin inhibited both the secretion of ATP and the elevation of the intracellular concentration of Ca(2+) ([Ca(2+)](i)) induced by all doses of thrombin used and induced a considerable rightward shift of the dose-response curves. S-145 also significantly inhibited the elevation of [Ca(2+)](i). Thrombin caused rapid accumulation of [(3)H]-InsP(3) or of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. This accumulation was also inhibited by indomethacin to about 70% of the control level. STA(2), a stable analogue of TXA(2), and arachidonic acid caused accumulation of InsP, and that induced by the latter was completely inhibited by indomethacin (1 μM). Thrombin-induced aggregation peaked at a lower level of [Ca(2+)](i) than that required for the secretion of ATP. The apparent contribution of TXA(2) to aggregation therefore appears to be restricted to that induced by lower doses of thrombin. These results suggest that in thrombin-stimulated rabbit platelets, activation of phospholipase C, which is regulated by TXA(2) receptors, is a primary target of the TXA(2)-mediated amplification mechanism. Through its effect on the accumulation of Ins(1,4,5)P(3), this amplification mechanism may contribute to about 25-30% of the elevation of (Ca(2+)](i), in addition to the thrombin receptor-mediated mechanism.     

Pathogenesis of a bleeding disorder characterized by platelet unresponsiveness to thromboxane A2

A platelet disorder characterized by the absence of thromboxane A2 (TXA2)-induced platelet aggregation is a new clinical entity of platelet dysfunction. The platelets of three patients had the ability to bind exogenous TXA2, but synthetic TXA2 mimetic-induced postreceptor biochemical events, such as IP3 formation, Ca2+ mobilization, phosphatidic acid formation, and GTPase activities, were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. Gene analysis of the TXA2 receptor showed a substitution of Leu for Arg60 in the first cytoplasmic loop in all patients, and this mutation seemed to be responsible for this platelet disorder.     

Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non-aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.     

Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC- gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC- delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.     

Effect of losartan on human platelet activation by thromboxane A2

INTRODUCTION AND OBJECTIVES: Previous studies have demonstrated that losartan, an AT-1 receptor antagonist of angiotensin II (Ang II) could block the receptor of thromboxane A2 (TXA2) in the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. MATERIALS AND METHODS: Platelets were obtained from 15 healthy men between the age 26 and 40. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by a synthetic TXA2 analogue, U46619 (5 x 10(-6) mol/l). RESULTS: The U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-response manner. Only a high dose of EXP 3174 (5 10-5 mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I-converting inhibitor failed to modify U46619-induced platelet aggregation. Despite the platelets expressing AT-1 type receptors, of Ang II exogenous Ang II did not modify platelet aggregation induced by U46619. The binding of U46619 to platelets was competitively inhibited by losartan in dose-dependent manner. However, only a high dose of EXP 3174 reduced the binding of U46619. Captopril failed to modify the binding of U46619 to platelets. CONCLUSIONS: Losartan decreased platelet aggregation by a TXA2-dependent mechanism. EXP 3174 showed a lesser potency than losartan to reduce TXA2-platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced TXA2-dependent platelet activation independently of the blockade of AT-1 receptors.     

Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.     

Platelet receptors for adenine nucleotides and thromboxane A2

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.     

Beta-lactam antibiotic-induced platelet dysfunction: evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of [14C]-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with [3H]-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist [3H]-U46619 and antagonist [3H]-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ([Ca2+]i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in [Ca2+]i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.     

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.     

Disparate effects of the calcium-channel blockers, nifedipine and verapamil, on alpha 2-adrenergic receptors and thromboxane A2-induced aggregation of human platelets

Calcium-channel blockers inhibit human platelet aggregation in vitro and ex vivo. To further evaluate the mechanism(s) responsible for the inhibition induced by this structurally heterogeneous group of compounds, we studied the effect of nifedipine and verapamil on human platelet aggregation in vitro. Neither 10 microM nifedipine nor 10 microM verapamil consistently inhibited the aggregation response of platelet-rich plasma to threshold concentrations of ADP, sodium arachidonate, epinephrine, or collagen. However, both 10 microM nifedipine and 10 microM verapamil epinephrine-potentiated, thromboxane A2 (TXA2)-induced aggregation of aspirin-incubated, gel-filtered platelets. Aggregation of similarly prepared platelets induced by TXA2 alone was abolished by 10 microM nifedipine but not by 10 microM verapamil. Even 100 microM verapamil gave only partial and inconsistent inhibition of aggregation. Both drugs had essentially the same effects on platelet aggregation induced by the stable endoperoxide and TXA2 mimic, U46619, with or without epinephrine. Neither 10 microM nifedipine nor 10 microM verapamil elevated platelet cyclic AMP. Verapamil (10 microM) inhibited binding of [3H]-yohimbine (an alpha 2-adrenergic receptor antagonist) to intact human platelets (KD 10.5 nM vs 2.4 nM for control platelets) without altering the number of binding sites. In contrast, 10 microM nifedipine had no effect on KD or number of binding sites. These results indicate that nifedipine and verapamil inhibit epinephrine-potentiated, TXA2-induced human platelet aggregation by different mechanisms. Verapamil inhibits the epinephrine contribution to the aggregation response by blocking alpha 2-adrenergic receptor binding. Nifedipine blocks the platelet response to TXA2 without affecting alpha-adrenergic receptor binding. These observations have potential clinical implications with regard to the mechanisms by which calcium-channel blockers inhibit vascular spasm and myocardial ischemia.     

Difluorothromboxane A2 and stereoisomers: stable derivatives of thromboxane A2 with differential effects on platelets and blood vessels.

The present study reports on the selective effects on human platelets and canine saphenous veins of four stable difluorinated analogues and thromboxane A2 (TXA2), in which the characteristic 2,6-dioxa[3.1.1]bicycloheptane structure of TXA2 has been retained. The four compounds differ in their stereochemistry of the 5,6 double bond and/or the 15-hydroxyl group. Only 10,10-difluoro-TXA2 (compound I) with the natural stereochemistry of TXA2 was an agonist in both platelets and canine saphenous veins (EC50 = 36 +/- 3.6 nM and 3.7 +/- 0.8 nM, respectively). (15R)-10,10-Difluoro-TXA2 (compound II), (5E)-10,10-difluoro-TXA2 (compound III), and (5E,15R)-10,10-difluoro-TXA2 (compound IV) were antagonists of platelet aggregation stimulated by compound I (Kd = 98 +/- 46 nM, 140 +/- 42 nM, and 1450 +/- 350 nM, respectively). However, compounds II, III, and IV stimulated contraction of canine saphenous veins (EC50 = 36 +/- 4.4 nM, 31 +/- 6.8 nM, and 321 +/- 50 nM, respectively). All four compounds could displace the TXA2/prostaglandin H2 antagonist 9,11-dimethylmethano-11,12-methano-16-(3- 125I-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 from its platelet receptor (Kd values = 100 +/- 30 nM, compound I; 280 +/- 60 nM, compound II; 230 +/- 70 nM, compound III; and 1410 +/- 1020 nM, compound IV). These results support the existence of two subtypes of TXA2/prostaglandin H2 receptors and emphasize the importance of the stereochemical requirements of these TXA2 analogues for interaction with these receptors. These stable fluorinated TXA2 analogues should prove useful tools for the further characterization of these and other TXA2/prostaglandin H2 receptors.     

Modulation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/quisqualate receptors by phospholipase A2: a necessary step in long-term potentiation?

The effects of kainate (KA)-induced epileptic seizures on the binding properties of hippocampal glutamate receptors, on the modulation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/quisqualate receptor by phospholipase A2 (PLA2), and on the formation of long-term potentiation (LTP) were studied in hippocampal membranes and hippocampal slices. Systemic administration of KA (10 mg/kg; 15 hr survival) produced specific changes in the binding properties of the AMPA/quisqualate receptors and its regulation. Whereas the binding of various ligands to the N-methyl-D-aspartate receptors was not modified by KA treatment, there was a significant decrease in the maximal number of binding sites for [3H]AMPA. In addition, the increase in [3H]AMPA binding elicited by PLA2 treatment of hippocampal, but not cerebellar, membranes was markedly decreased after KA injection. LTP was also substantially reduced in area CA1 of hippocampal slices from KA-treated animals. The loss of LTP was not due to changes in postsynaptic responses elicited by the bursts that trigger the potentiation effect, thus suggesting that KA treatment disrupts processes that follow N-methyl-D-aspartate receptor activation. Systemic administration of KA was associated with calpain activation as the amount of spectrin breakdown products was increased severalfold in hippocampus but not in cerebellum. Pretreatment of telencephalic membranes with calpain greatly reduced the PLA2-induced increase in [3H]AMPA binding. The results provide evidence in favor of an essential role of PLA2 in the development of LTP and suggest that the order of activation of different calcium-dependent processes is critical for producing the final changes underlying LTP.     

Platelet hypersensitivity in cholesterol-fed rabbits: enhancement of thromboxane A2-dependent and thrombin-induced, thromboxane A2-independent platelet responses

Hypercholesterolemia (mean plasma cholesterol: 15 mM) was induced in rabbits by the feeding of a chow diet enriched with a low amount (0.25%, w/w) of cholesterol only. Platelet size and protein content decreased significantly, but the whole blood platelet count did not change. The platelets became enriched in cholesterol, as indicated by a significant increase in the cholesterol:phospholipid molar (C/P) ratio. Specific responses of washed platelets stimulated with various agonists were studied to determine the effects of hypercholesterolemia on the various pathways of platelet aggregation in the absence of plasma components. In platelets from hypercholesterolemic rabbits compared with controls: aggregation induced by ADP was not altered; collagen-induced responses (aggregation, secretion of [14C]serotonin from prelabelled platelets, thromboxane A2 (TXA2) formation, mobilization of [3H]arachidonate from prelabelled platelets) were enhanced; with aspirin-treated platelets, aggregation induced by the TXA2 mimetic U46619 was enhanced: and thrombin-induced responses of both untreated platelets (aggregation, secretion of granule contents, TXA2 formation) and aspirin-treated platelets (aggregation) were enhanced. Thus, platelets from cholesterol-fed rabbits not only form more TXA2, but they aggregate more extensively when stimulated by its mimetic. In addition, it has not been previously recognized that these platelets are also hypersensitive to thrombin-induced aggregation that is independent of TXA2.     

Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets

Lysophosphatidic acid is a biologically active phospholipid able to induce cell proliferation and platelet aggregation. In this study we investigated the biochemical mechanisms of platelet activation by lysophosphatidic acid. We found that lysophosphatidic acid stimulated the binding of the photoreactive GTP-analog 4-azidoanilido-[α³²P]GTP to a 40-kDa protein on platelet membranes. Moreover, lysophosphatidic acid induced the rapid decrease of the intracellular concentration of cAMP in intact platelets, indicating that this lipid activates platelets by binding to a membrane receptor coupled to the inhibitory GTP-binding protein Gi. In agreement with a receptor-mediated action, we found that platelet activation by lysophosphatidic acid underwent homologous desensitization. In the absence of extracellular CaCl₂, lysophosphatidic acid did not induce platelet aggregation, and did not stimulate phospholipase C. However, under the same conditions, lysophosphatidic acid produced the rapid tyrosine phosphorylation of several platelet proteins. This effect was not mediated by the formation of thromboxane A₂. Our results demonstrate that, in lysophosphatidic acid-stimulated platelets, activation of protein-tyrosine kinases occurs in the absence of phospholipase C activation and platelet aggregation, and may be directly related to the activation of the G-protein-coupled lysophosphatidic acid-receptor.     

Identifying pathways involved in leptin-dependent aggregation of human platelets

OBJECTIVE: To investigate the role of phospholipase C (PLC), phospholipase A(2) (PLA(2)), calcium, and protein kinase C (PKC) in mediating leptin-enhanced aggregation of human platelets. DESIGN: In vitro, ex vivo study. SETTING: Outpatient's Service for Prevention and Treatment of Obesity at the University Hospital of Messina, Italy. SUBJECTS: In total, 14 healthy normal-weight male (age 31.4+/-1.9 y; body mass index 22.7+/-0.6 kg/m2) subjects. MEASUREMENTS: Adenosine diphosphate-(ADP-) induced platelet aggregation and platelet free calcium were measured after incubation of platelets with leptin alone (5-500 ng/ml), or leptin (50 and 100 ng/ml) in combination with anti-human leptin receptor long form antibody (anti-ObRb-Ab, 1:800-1:100 dilutions), PLC inhibitor U73122 (3.125-25 microM), PLA(2) inhibitor AACOCF3 (1.25-10 microM), or PKC inhibitor Ro31-8220 (1.25-10 microM). RESULTS: Platelet stimulation with leptin leads to a significant and dose-dependent increase in ADP-induced platelet aggregation and platelet free calcium concentrations. Leptin effects on both platelet aggregation and calcium mobilization were completely abated by the co-incubation with leptin and anti-ObRb-Ab. Leptin-induced platelet aggregation was dose-dependently inhibited by U73122, AACOCF3, or Ro31-8220. The effect of leptin on intracellular calcium was inhibited in a dose-dependent manner by incubation with U73122 and AACOCF3, but not with Ro31-8220. CONCLUSIONS: Our study confirms that leptin is able to enhance ADP-induced aggregation of human platelets, and raise the possibility that PLC, PKC, PLA(2), and calcium could play a relevant role in mediating the proaggregating action of leptin.     

Protein kinase C inhibits epidermal growth factor-dependent tyrosine phosphorylation of phospholipase C gamma and activation of phosphoinositide hydrolysis.

Recent studies have shown that the receptor for epidermal growth factor (EGF) can associate with and tyrosine-phosphorylate the gamma-isozyme of phosphoinositide (PtdIns)-specific phospholipase C (PLC gamma), suggesting a possible mechanism for activation of PtdIns hydrolysis by EGF. In the present study, the coupling between PtdIns hydrolysis and PLC gamma tyrosine phosphorylation in WB liver epithelial cells was examined. Peak levels of [P-Tyr]PLC gamma, measured by anti-P-Tyr immunoblotting, occurred at 0.5-2 min of EGF treatment and coincided with the onset of [3H]inositol phosphate production. The termination of PtdIns hydrolysis after EGF stimulation was accompanied by return of [P-Tyr]PLC gamma to near-basal levels. Activation of protein kinase C (PKC) with a phorbol ester inhibited (IC50 = 3-10 nM) both EGF-dependent PtdIns hydrolysis and PLC gamma phosphorylation by more than 90%. Both EGF-stimulated responses were potentiated in cells depleted of PKC by prolonged phorbol ester treatment. At physiological ionic strength, monoclonal antibodies to PLC gamma specifically precipitated (in addition to PLC gamma) the EGF receptor and at least six other [P-Tyr]proteins from extracts of EGF-treated cells. PKC activation had differential effects on the tyrosine phosphorylation of these coprecipitating proteins, i.e. the relative abundance of certain [P-Tyr] proteins decreased, whereas that of another protein increased. In conclusion, EGF-stimulated tyrosine phosphorylation of PLC gamma is broadly correlated with stimulation of PtdIns hydrolysis, consistent with a role for tyrosine phosphorylation in PLC activation. The attendant diacylglycerol release and activation of PKC may terminate PLC gamma activation, in part by inhibiting PLC gamma phosphorylation by the EGF receptor. Our results suggest further that PKC may exert regulatory effects by altering the relationship of PLC gamma to its associated [P-Tyr]proteins.     

Characterization of the vascular thromboxane A2/prostaglandin endoperoxide receptor in rabbit aorta. Regulation by dexamethasone

Recently, we have shown that dexamethasone treatment of rabbits specifically reduces vascular smooth muscle responsiveness to agonists that interact with the vascular thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. One potential site at which dexamethasone can influence prostanoid-mediated vasoconstriction may be at the level of the vascular TXA2/PGH2 receptor. Therefore, we characterized the vascular TXA2/PGH2 receptor in rabbit aortic membranes and examined the influence of dexamethasone treatment on vascular TXA2/PGH2 receptor affinity and number. The binding of [125I][1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R)4 alpha)]-7-[3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1] heptan-2-yl]-5-heptanoic acid ([125I]BOP), a potent TXA2/PGH2 receptor agonist, to rabbit aortic membranes was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding data disclosed one class of high affinity binding sites with a Kd of 0.44 +/- 0.13 nM and a Bmax of 114.4 +/- 5.2 fmol/mg protein (n = 7). Removal of the endothelium before membrane preparation did not significantly alter the affinity or number of binding sites for [125I]BOP. Kinetic analysis of the rates of [125I]BOP association/dissociation yielded a Kd of 0.62 nM. The ability of various agonists at the TXA2/PGH2 receptor to displace [125I]BOP from vascular membranes correlated well with their contractile potencies in rabbit aortic rings. Moreover, stereospecific displacement of [125I]BOP binding in aortic membranes and inhibition of U46619-mediated aortic contractions were obtained with the stereoisomers L657925(-) and L657926(+). Collectively, these data suggest that this binding site represents the functionally relevant vascular TXA2/PGH2 receptor. In functional experiments, [127I]BOP induced concentration-dependent contractions of the rabbit aorta, which were reduced by 52% in vessels from dexamethasone-treated rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s.

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.