(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

Nucleotide receptor signaling in platelets

Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet-dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein-coupled receptors, the Gq-coupled P2Y1 receptor, and the Gi-coupled P2Y12 receptor. ATP, on the other hand, acts through the ligand-gated channel P2X1. Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A2 generation. ADP-induced dense granule release depends on generated thromboxane A2. Furthermore, costimulation of both P2Y1 and P2Y12 receptors is required for ADP-induced platelet aggregation. ATP stimulation of P2X1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.     

Different G protein-coupled signaling pathways are involved in alpha granule release from human platelets.

Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause alpha granule release in platelets. Alpha granule release was measured by determining P-selectin surface expression in aspirin-treated washed platelets. ADP-induced P-selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR-C69931MX (a P2Y12 selective antagonist), suggesting a role for both Galpha(q) and Galpha(i) pathways in ADP-mediated alpha granule release. Consistent with these observations, the combination of serotonin (a Galpha(q) pathway stimulator) and epinephrine (a Galpha(z) pathway stimulator) also caused alpha granule release. Furthermore, U46619-induced P-selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR-C69931, indicating a dominant role for P2Y12 in U46619-mediated alpha granule release. Additionally, the Galpha(12/13)-stimulating peptide YFLLRNP potentiated alpha granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Galpha(i) and Galpha(12/13) pathways resulted in a significant dose-dependent increase in alpha granule release. We conclude that ADP-induced alpha granule release in aspirin-treated platelets occurs through costimulation of Galpha(q) and Galpha(i) signaling pathways. The P2Y12 receptor plays an important role in thromboxane A(2)-mediated alpha granule release, and furthermore activation of Galpha(12/13) and Galpha(q) signaling pathway can cause alpha granule release.     

αIIbβ3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause α-granule secretion by platelets

Summary.  The peptide LSARLAF (LSA) causes αIIbβ3-dependent platelet activation that results in α-granule secretion and aggregation. LSARLAF-induced, αIIbβ3-mediated outside-in signaling causing α-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Gαq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced αIIbβ3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause α-granule release by mediating their effects though Gαq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, αIIbβ3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Gαq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Gαq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced αIIbβ3-mediated signaling characterized in this study is α-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.     

Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon

In this study, we have identified a patient (OSP-1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP-1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets. Molecular genetic analysis revealed that OSP-1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild-type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP-1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real-time observations of thrombogenesis under a high shear rate (2000 s(-1)) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP-1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.     

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.     

Lipid rafts are required in Gαi signaling downstream of the P2Y12 receptor during ADP-mediated platelet activation

ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-beta-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Galpha(i) activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Galpha(z)-coupled agonist epinephrine. Finally, Galpha(i) preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Galpha(i) signaling downstream of P2Y12 activation, but not Galpha(q) or Galpha(z) signaling downstream of P2Y1 or alpha2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.     

Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic mice

Summary.  In order to investigate the role of the platelet P2Y₁ receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [³³P]2MeSADP in the presence or absence of the selective P2Y₁ antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y₁ receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y₁ receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y₁ receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y₁ expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y₁ receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y₁ receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.     

Effect of common agonists on cytoplasmic ionized calcium concentration in platelets. Measurement with 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin

Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.     

The platelet P2 receptors as molecular targets for old and new antiplatelet drugs

Platelet activation by ADP and ATP plays a crucial role in haemostasis and thrombosis, and their so-called P2 receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X1 and the 2 G protein-coupled P2Y1 and P2Y12 ADP receptors selectively contribute to platelet aggregation. The P2Y1 receptor is responsible for ADP-induced shape change and weak and transient aggregation, while the P2Y12 receptor is responsible for the completion and amplification of the response to ADP and to all platelet agonists, including thromboxane A2 (TXA2), thrombin, and collagen. The P2X1 receptor is involved in platelet shape change and in activation by collagen under shear conditions. Due to its central role in the formation and stabilization of a thrombus, the P2Y12 receptor is a well-established target of antithrombotic drugs like ticlopidine or clopidogrel, which have proved efficacy in many clinical trials and experimental models of thrombosis. Competitive P2Y12 antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y1 and P2X1 knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs.     

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.     

MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate], a potent, selective, and stable antagonist of the platelet P2Y1 receptor with strong antithrombotic activity in mice

The platelet P2Y(1) ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y(1) receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y(1) receptor antagonist MRS2500 [2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y(1) receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y(12) receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y(1) receptor in thrombosis and validate the concept that targeting the P2Y(1) receptor could be a relevant alternative or complement to current antiplatelet strategies.     

Physiological functions of platelets

Platelets are discoid, anucleate cells with a large number of secretory granules. Physiological agonists (thrombin, collagen, ADP, adrenaline, thromboxane A2, serotonin, vasopressin) interact with specific receptors on the platelet surface which causes the platelet responses shape change, aggregation, secretion of substances from three types of granules and liberation of arachidonate from membrane phospholipids. Some secreted substances and conversion products of arachidonate are platelet agonists and enhance platelet stimulation (positive feedback). The shape change and aggregation responses are of central importance for platelet adhesion to the subendothelium and formation of platelet thrombi. Dense granule secretion and the storage of ADP, ATP, Ca2+ and serotonin, a-granule secretion of platelet-specific, cationic, coagulation and carbohydrate-containing proteins as well as secretion of glycosidases are also shown to be important for platelet participation in haemostasis and thrombosis. Signal transduction mechanisms (phospholipase C activation, polyphosphoinositide metabolism, Ca2+ mobilization) and arachidonate oxygenation are central processes for the physiological functions of platelets.     

Persistent production of platelet thromboxane A2 in patients chronically treated with aspirin

BACKGROUND: Patients treated with aspirin may have a reduced sensitivity to its antiplatelet effect. The mechanism accounting for such a reduced sensitivity might involve an impaired interaction of aspirin with cyclooxygenase-1 (COX)-1. OBJECTIVE: We sought to investigate whether platelets from patients under chronic treatment with aspirin still produce TxA2 and whether there is any relationship between the eventual persistent TxA2 formation and platelet aggregation. Finally, whether platelet-derived TxA2 can be inhibited by in vitro addition of aspirin. METHODS: Collagen-induced platelet aggregation and thromboxane-A2 (TxA2) were measured in 196 patients treated with aspirin (100-330 mg day(-1)) because of previous vascular events or presence of risk factors of atherosclerosis. RESULTS: Collagen-induced TxA2 production of the entire cohort was 128.7 +/- 21.6 pg 10(-8) cells, and was significantly correlated with platelet aggregation (Spearman's correlation coefficient = 0.44; P < 0.0001). Patients in the highest quartile of TxA2 showed higher platelet response to collagen (P < 0.0001) when compared with those in the lowest quartile. In a subgroup of 96 patients, platelets were treated in vitro with a TxA2 receptor antagonist (13-azaprostanoic acid) or aspirin before stimulation with collagen. 13-APA acid significantly inhibited platelet aggregation. Aspirin reduced (-72.9%) TxA2 production in patients with TxA2 values above the median but it was ineffective in those with TxA2 values below the median. CONCLUSION: In some patients chronically treated with aspirin platelet production of TxA2 may persist and account for enhanced platelet aggregation. Incomplete inhibition of COX-1 seems to be implicated in persistent TxA2 production.     

P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.     

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor.

BACKGROUND: Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. OBJECTIVE: The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A(2) (TxA(2)) generation and on receptor antagonism, and to analyze the structural requirements for such effects. METHODS: The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA(2) generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA(2) receptors. RESULTS: Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA(2) synthesis. These compounds were identified as specific ligands of the TxA(2) receptor in the micromol L(-1) range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA(2) receptor relies on structural features such as the presence of the double bond in C2-C3, and a keto group in C4. CONCLUSIONS: The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA(2) receptor. Therefore, antagonism of this TxA(2) receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.     

P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis.

BACKGROUND: The platelet adenosine 5'-diphosphate (ADP) receptor P2Y(12) plays a crucial role in haemostasis. Only a few patients with haemorrhagic diathesis due to molecular defects in the P2Y(12) receptor have been described so far. We report a novel molecular defect in the gene coding for P2Y(12) in a patient with a history of epistaxis, easy bruising and excessive posttraumatic blood loss. METHODS: Platelet aggregation studies, perfusion studies, in which patient blood was perfused over collagen surfaces at arterial shear rates, and PCR and sequencing were used. RESULTS: Platelet aggregation studies showed impaired ADP and collagen-induced aggregation for patient G.S. Perfusion of patient blood over collagen surfaces showed small thrombi consisting of spread platelets overlayered with non-spread platelets. These thrombi were identical to control thrombi formed in the presence of a P2Y(12) antagonist. DNA analysis of the P2Y(12) gene revealed a novel heterozygous base pair C-->A substitution in exon 3, changing codon 258 from proline to threonine in the third extracellular loop of the P2Y(12) receptor. CONCLUSIONS: We conclude that perfusion studies with patient blood are of added value in the diagnostic process, which resulted in identification of a novel molecular defect in the P2Y(12) gene of a patient with haemorrhagic diathesis.     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

Significance of testing platelet functions in vitro

Abstract. Platelets respond through discrete receptors to a number of physiological agonists and foreign surfaces with a sequence of measurable responses: shape change, aggregation, secretion and arachidonate liberation. Three secretory responses are distinguished: exocytosis of substances from (1) dense granules, (2) α-granules and (3) lysosomes. Free arachidonate, liberated from phospholipids by phospholi-pase A₂, is rapidly converted (by oxygenation) to prostaglandins and thromboxanes which, together with secreted ADP and close cell contact, will cause further platelet activation through ‘positive feedback’ (autocrine stimulation). Some agonists are classified as ‘weak’ (ADP, vasopressin, platelet-activating factor [PAF], serotonin) because they depend on autocrine stimulation to promote the full sequence of responses, while others are ‘strong’ agonists (thrombin, collagen) and activate all responses directly without autocrine stimulation. Adrenaline, long thought to be a platelet agonist per se, most probably acts by amplifying the activation brought about by other, proper, agonists. Such synergistic interaction among agonists is very typical for platelet activation and most likely takes place in vivo. Shape change, aggregation and secretion(s) may be tested by flow cytometry or electron microscopy in vitro under conditions that probably reflect the in vivo situation. However, the aggregation response to weak agonists in vitro is dependent on the extracellular [Ca²⁺], with biphasic aggregation at the low [Ca²⁺] present when citrate is used as anticoagulant (or in suspension of washed platelets) but not at the physiological [Ca²⁺] present in platelet-rich plasma from heparinized blood.