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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1941.)
© 2003 American Heart Association, Inc.

Thrombosis

Differential Involvement of the P2Y₁ and P2Y₁₂ Receptors in Platelet Procoagulant Activity

Catherine Léon; Catherine Ravanat; Monique Freund; Jean-Pierre Cazenave; Christian Gachet

From the Institut National de la Santé et de la Recherche Médicale U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann, 67065 Strasbourg Cédex.

Correspondence to Dr. C. Gachet, INSERM U.311, Etablissement Français du Sang-Alsace (EFS-Alsace), 10, rue Spielmann, B.P. N°36, 67065 Strasbourg Cédex, France. E-mail christian.gachet{at}efs-alsace.fr

	Abstract

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Objective— In vivo, activated platelets contribute to the initiation of thrombin generation through the exposure of phosphatidylserine to form a procoagulant catalytic surface and through platelet-leukocyte interactions, which lead to the exposure of leukocyte tissue factor (TF). On the basis of observations that the platelet P2Y₁ and P2Y₁₂ receptors both contribute to thrombosis and thrombin formation in an in vivo model of TF-induced thromboembolism, we further characterized the role of these receptors in thrombin generation.

Methods and Results— By using the selective P2 antagonists MRS2179 and AR-C69931MX, the P2Y₁₂ receptor was found to be involved in thrombin-induced exposure of PS on isolated platelets and consequently in TF-induced thrombin formation in platelet-rich plasma. By contrast, the P2Y₁ receptor was not involved in phosphatidylserine exposure nor in thrombin generation in platelet-rich plasma. In addition, both receptors were found to contribute to the interactions between platelets and leukocytes mediated by platelet P-selectin exposure, which result in TF exposure at the surface of leukocytes.

Conclusions— Overall, these results point to a differential involvement of the 2 platelet ADP receptors in the generation of thrombin and provide further evidence for the relevance of molecules targeting these receptors as antithrombotic agents.

Key Words: P2 receptor • ADP • thrombin generation • thrombosis • platelets

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vivo, platelets contribute to the generation of thrombin by providing a procoagulant surface through the rearrangement of their membrane phospholipids to expose negatively charged phosphatidylserine (PS) and by releasing coagulation factors stored in their -granules.^1,2 Platelets also participate in thrombin generation by triggering exposure of tissue factor (TF) on the surface of leukocytes,^3,4 partly through platelet P-selectin exposure and the formation of platelet-leukocyte conjugates.^3,5 Thus, antiplatelet agents might be expected to decrease thrombin generation. Inhibitors of the platelet glycoprotein (GP) IIbIIIa have indeed been shown to decrease thrombin formation in vitro, both in a whole blood coagulation model⁶ and in platelet-rich plasma (PRP) as determined from the direct production of thrombin.^7,8 Moreover, in vivo administration of the anti-GPIIbIIIa antibody abciximab has been reported to decrease ex vivo platelet thrombus formation and fibrin deposition⁹ and levels of the thrombin generation marker F1.2 in patients undergoing coronary interventions.¹⁰

ADP plays a central role in platelet activation by acting as a cofactor in the platelet responses to all physiological agonists, including thromboxane A2, collagen, and thrombin. This crucial role of secreted ADP is demonstrated by the impaired aggregation responses of patients with storage pool deficiency. In addition, such patients display an impaired platelet prothrombinase activity, which can be corrected in vitro by addition of ADP, suggesting a role of ADP in thrombin generation.¹¹ ADP-induced platelet activation is initiated by the P2Y₁ receptor and amplified by the P2Y₁₂ receptor.¹² Hence, this latter receptor has become a major target for the management of thrombotic diseases. Ticlopidine and clopidogrel, which irreversibly inhibit the platelet P2Y₁₂ receptor, are currently used for the prevention of secondary events in acute cardiovascular disease^13,14 whereas AR-C69931MX, a potent and selective reversible antagonist of the receptor, has undergone phase II trials in patients with acute coronary syndromes.¹⁵

However, the role of the P2Y₁ receptor in thrombosis was more recently highlighted by studies of P2Y₁-deficient mice, which are resistant to the systemic intravascular thrombosis induced by intravenous injection of either ADP or collagen and adrenaline.^16,17 P2Y₁-deficient mice or wild-type mice treated with MRS2179, an antagonist of the P2Y₁ receptor, likewise display thromboresistance after intravenous injection of tissue factor (TF), a model that is highly dependent on thrombin generation.¹⁸ In this model, the absence or inhibition of the P2Y₁ receptor further results in reduced levels of thrombin-antithrombin complexes (TAT), suggesting that this receptor might also be involved in the generation of thrombin.

The aim of the present study was to characterize the respective roles of the P2Y₁ and P2Y₁₂ receptors in thrombin generation. In vivo studies were performed in P2Y₁-deficient mice treated or not treated with clopidogrel. The contributions of the P2Y₁ and P2Y₁₂ receptors to different aspects of thrombin formation were distinguished using their respective selective antagonists, MRS2179 and AR-C69931MX. The direct real-time in vitro thrombin generation in PRP was measured by the thrombogram method¹⁹ and the exposure of PS by annexin V binding.²⁰ Finally, the participation of the P2Y₁ and P2Y₁₂ receptors in (1) TF exposure at the surface of leukocytes, (2) the exposure of platelet P-selectin, and (3) interactions between platelets and leukocytes was determined in whole blood by flow cytometry. Overall, our data indicate that both ADP receptors are involved, although differentially, in the generation of thrombin in vivo and in vitro.

	Materials and Methods

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Materials
ADP, MRS2179, and fatty acid free human serum albumin were from Sigma-Aldrich (Saint Quentin-Fallavier, France). Purified human -thrombin was from EFS-Alsace (Strasbourg, France), thrombin receptor activating peptide (TRAP) from Neosystem (Strasbourg, France), clopidogrel (Plavix) from Sanofi-Synthélabo (Toulouse, France) and ^FITCAnnexin V from Roche (Meylan, France). Recombinant hirudin was kindly provided by Transgène (Strasbourg, France). Monoclonal antibodies against human antigens were ^FITCCD142 (TF, clone 4508) from American Diagnostica (Greenwitch, CT); ^PECD42b (GPIb, clone SZ2) and ^FITCCD62P (P-selectin, clone CLB-thromb/6) from Immunotech Coulter (Marseille, France); and ^PECD45 (clone 30-F11) and ^PECD42b (clone HIP1) from Pharmingen (France). Human thromboplastin (Thromborel S) and recombinant human TF (Innovin) were from Dade Behring (Mannheim, Germany). AR-C69931MX was from Astra Charnwood (Loughborough, UK) and the fluorogenic substrate Z-GGR-AMC from Bachem (Voisins-le-Bretonneux, France). Measurements of thrombin generation using the Thrombogram method were performed with a ThermoLabsystems Ascent reader (Helsinki, Finland) coupled to the Thrombinoscope software developed by Synapse (Utrecht, The Netherlands).

Methods
In Vivo Thrombosis Model
Wild-type and P2Y₁-deficient male mice were raised as described previously,¹⁷ had a C57BL/6 background, and were used at the F7 generation. Clopidogrel treatment was performed by oral administration of clopidogrel solubilized in arabic gum the day before and 2 hours before the experiment. Arabic gum alone was given orally to the control group. In vivo thrombosis was induced by injection of thromboplastin (200 µL/kg), and plasma levels of TAT were determined as previously described.¹⁸ Platelets were counted before and after thromboplastin injection to measure the percentage platelet consumption in each mouse.

Thrombin Generation in Human and Mouse PRP
Whole blood anticoagulated with citrate (3.8% for human and 3.15% for mouse blood) was centrifuged to prepare PRP (250g/16 minutes for 50 mL of human blood and 2300g/2 minutes for 10 mL of mouse blood). Human PRP (1x10⁸ platelets/mL) or mouse PRP (3x10⁸ platelets/mL) was incubated with MRS2179 (100 µmol/L), AR-C69931MX (10 µmol/L), or vehicle, and the fluorescent thrombin substrate Z-GGR-AMC (400 µmol/L) was added in Fluo buffer (20 mmol/L HEPES, 60 mg/mL bovine serum albumin, pH 7.35) containing CaCl₂ (17 mmol/L). AR-C69931MX, which is a potent P2Y₁₂ antagonist, was nevertheless used at a concentration of 10 µmol/L in all experiments so as to block all P2Y₁₂ receptors. At this concentration, AR-C69931MX is still selective with no effect on the P2Y₁ receptor. Thrombin generation was started by injection of recombinant human tissue factor (Innovin, 1/12, 000 final dilution) and recorded for 70 minutes as described.¹⁹ Concentrations of the thrombin generated were calculated from a calibration curve constructed with known amounts of calibrated thrombin.

Measurement of PI Exposure by ^FITCAnnexin V Binding
Washed platelet suspensions in Tyrode’s buffer were prepared from human and mouse blood as previously described.^17,21 Platelets (3x10⁸/mL) were incubated with MRS2179 (100 µmol/L), AR-C69931MX (10 µmol/L), or vehicle and activated with thrombin (1 U/mL) for 5 minutes at 37°C, without stirring. A 5-µL aliquot was then labeled with ^FITCAnnexin V in the presence of hirudin (10 U/mL) for 10 minutes at room temperature, diluted, and analyzed by flow cytometry.

Whole Blood Stimulation and Double-Labeling Flow Cytometry
Immediately after collection, a 70-µL sample of blood anticoagulated with hirudin (100 U/mL) was incubated with MRS2179 (100 µmol/L), AR-C69931MX (10 µmol/L), or vehicle at 37°C for 2 minutes without stirring. The sample was stimulated with ADP (100 µmol/L) or TRAP (10 µmol/L) at 37°C for 15 minutes. A 5-µL aliquot was then incubated with Tyrode’s buffer (45 µL) containing fluorescent antibodies (1/10 final dilution) for 20 minutes, diluted, and fixed in saline containing 0.5% (vol/vol) formaldehyde. All flow cytometric parameters were acquired on a logarithmic scale, and the forward and side scatter parameters were used to identify the leukocyte and isolated platelet populations. TF exposure on leukocytes was measured by recording a total of 1500 events in the leukocyte population gated as ^PECD45-positive and determining the percentage of ^FITCTF-positive cells in this population. Platelet-leukocyte aggregates were determined as the percentage of ^FITCCD42b-positive events in the leukocyte population. Platelet P-selectin expression was measured by recording 10, 000 events in the platelet population (^PECD42b-positive) and calculating the percentage of ^FITCCD62P-positive cells in this population. Fluorescein isothiocyanate- and PE-conjugated isotype controls were used to quantify the background labeling.

Statistical Analyses
Results were expressed as the mean (± SEM) and the data were compared by one-way analysis of variance or repeated measures one-way analysis of variance where appropriate, followed by a Dunnet post-test.

	Results

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Intravascular Thromboembolism
The role of the P2Y₁ and P2Y₁₂ receptors in systemic intravascular thrombosis was evaluated by measuring the platelet consumption caused by massive occlusion of the microcirculation of the lungs with platelet thromboemboli.²² Platelet consumption is a more sensitive marker of thrombosis than mortality and allows the simultaneous measurement of plasma TAT complexes.¹⁸ As previously reported,¹⁸ intravenous injection of TF induced less platelet consumption in P2Y₁-deficient mice (40.6±5.6%) than in wild-type mice (88.7±1.4%, P<0.01) (Figure 1A). Wild-type mice treated with clopidogrel displayed a significantly reduced platelet consumption similar to that of P2Y₁-deficient mice (38.6±7.6%, P<0.01 versus untreated wild-type mice). Finally, only 13.7±2.3% platelet consumption was observed in P2Y₁-deficient mice treated with clopidogrel, demonstrating an additive effect when the 2 ADP receptors were blocked (P<0.01 versus clopidogrel-treated wild-type mice or nontreated P2Y₁-deficient mice; Figure 1A).

View larger version (26K): [in this window] [in a new window]   Figure 1. TF-induced systemic intravascular thrombosis. Wild-type and P2Y1-deficient mice, treated or with clopidogrel (50 mg/kg/d) or untreated, were injected with thromboplastin (200 µL/kg). A, Platelet consumption expressed as the percentage (mean± SEM, n=9 to 10) of the initial platelet count. B, Plasma levels of TAT complexes (mean±SEM, n=8 to 10). *P<0.05, **P<0.01 versus control wild-type mice (open bar). P<0.01 versus clopidogrel-treated wild-type mice (gray bar) or nontreated P2Y1-deficient mice (hatched bar).

The contribution of the receptors to thrombin generation was evaluated in this model by measuring levels of TAT complexes. After TF injection, levels of TAT were lower in P2Y₁-deficient mice than in wild-type mice (193.1±6.7 and 274.2±40.5 µg/L, respectively, P<0.01), confirming previous data (Figure 1B).¹⁸ Clopidogrel treatment of wild-type mice also reduced TAT formation (176.1±10.9 µg/L, P<0.01 versus untreated wild-type mice), but no additive effect on TAT complexes was observed in P2Y₁-deficient mice treated with clopidogrel (151.6±7.1 µg/L; Figure 1B). These results indicate that the P2Y₁ and P2Y₁₂ receptors both play a role in thrombin generation in vivo. To characterize the part played by each receptor, we next determined their involvement in the in vitro production of thrombin in PRP, the exposure of procoagulant phospholipids on platelets, and the exposure of TF on leukocytes.

In Vitro Thrombin Generation Induced by TF in Human and Mouse PRP
The respective roles of the P2Y₁ and P2Y₁₂ receptors in the thrombin production induced by TF in human and mouse PRP were investigated using the thrombogram method. The thrombin generation curve (thrombogram) is characterized by several parameters: (1) the lag phase reflecting the clotting time, (2) the peak thrombin height, which represents the maximum velocity of net thrombin production and reflects the maximum prothrombinase activity attained, (3) the time to this peak, and (4) the endogenous thrombin potential (ETP), which is the area under the curve representing the total amount of active thrombin generated, but is a less sensitive parameter than the peak thrombin production.¹⁹ The TF dilution (1/12, 000) was selected so that the formation of thrombin depended on the presence of platelets. Thus, the thrombin generated in platelet-poor plasma was undetectable under these conditions (data not shown). The effects on the thrombogram parameters of the P2Y₁₂ antagonist AR-C69931MX (10 µmol/L) and the P2Y₁ antagonist MRS2179 (100 µmol/L) in human PRP containing 1x10⁸ platelets/mL are shown in Figure 2A and Table 1. AR-C69931MX significantly delayed the thrombin burst because it increased the time to peak production (Table 1). AR-C69931MX also significantly reduced the peak height but did not significantly decrease the ETP. MRS2179 alone had no significant effect on the generation of thrombin, and no additive effect was observed in the presence of both antagonists. Similar results were obtained using PRP containing 2x10⁸ platelets/mL (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 2. Thrombin generation in human and mouse PRP. A, Effects of AR-C69931MX (10 µmol/L) and MRS2179 (100 µmol/L) on thrombin formation in human PRP (1x108 platelets/mL). B, Effects of AR-C69931MX on thrombin formation in PRP (3x108 platelets/mL) from wild-type and P2Y1-deficient mice. Results are from one experiment performed in quadruplicate, representative of at least 3 independent experiments.

View this table: [in this window] [in a new window]   Effects of MRS2179 and AR-C69931MX on Thrombin Generation in Human and Mouse PRP

Using mouse PRP, the lag time and time to peak thrombin production were shorter and the ETP and peak height lower in PRP from wild-type mice (3x10⁸ platelets/mL) as compared with human PRP (Table 1). No significant differences in the thrombogram parameters were observed between wild-type and P2Y₁-deficient PRP (Figure 2B and Table 1), confirming that the P2Y₁ receptor is not involved in the thrombin generation induced by TF in PRP. AR-C69931MX reduced the peak height in PRP from both wild-type and P2Y₁-deficient mice (Table 1), as well as the ETP, although not significantly (Figure 2B and Table 1). AR-C69931MX had no significant effect on the other parameters and no additive effect was observed when both receptors were blocked. Comparable results were obtained using mouse PRP containing 4.6 and 2x10⁸ platelets/mL (data not shown). Altogether, these findings point to the involvement of the P2Y₁₂ receptor, but not of the P2Y₁ receptor, in the thrombin generation induced by TF in PRP.

Effects of P2 Years₁ and P2 Tears₁₂ Antagonists on PS Exposure on Human and Mouse Platelets
The part played by the P2Y₁ and P2Y₁₂ receptors in the exposure of PS at the surface of platelets was investigated by measuring the binding of ^FITCAnnexin V by flow cytometry. Human platelets were stimulated with thrombin (1 U/mL) in the presence or absence of the P2Y₁ antagonist MRS2179 or the P2Y₁₂ antagonist AR-C69931MX. Thrombin treatment increased the percentage of ^FITCAnnexin V-labeled platelets from 1.9±0.8% in the resting state to 31.4±4.8% after stimulation, reflecting the exposure of negative membrane phospholipids. Conversely, no significant increase was observed when platelets were stimulated with ADP (100 µmol/L) or collagen (10 µg/mL; data not shown). AR-C69931MX (10 µmol/L) significantly decreased thrombin-induced PS exposure (12.6±2.8% ^FITCAnnexin V-labeled platelets, P<0.01; Figure 3A), whereas MRS2179 (100 µmol/L) had no significant effect (25.9±3.5% labeled platelets).

View larger version (34K): [in this window] [in a new window]   Figure 3. Exposure of PS as detected by FITCAnnexin V binding at the surface of platelets. A, Human platelets stimulated with thrombin (1 U/mL), effects of AR-C69931MX (10 µmol/L, ARC) and MRS2179 (100 µmol/L, MRC), n=4. B, P2Y1+/+ and P2Y1-/- mouse platelets stimulated with thrombin (1 U/mL), effects of AR-C69931MX (n=3). Values represent the percentage of annexin V-labeled isolated platelets or CD42b-positive cells (mean±SEM). *P<0.05, **P<0.01 versus vehicle (human) or vehicle/wild-type (mouse).

Similarly to human platelets, wild-type mouse platelets stimulated with thrombin (1 U/mL) displayed a significant increase in ^FITCAnnexin V binding (42.3±11.7% labeled platelets versus 0.7±0.1% in the resting state). P2Y₁-deficient mouse platelets had a PS exposure comparable to that of wild-type platelets (37.1±11.0% versus 1.0±0.1% in the resting state; Figure 3B). AR-C69931MX decreased the percentage of labeled wild-type or P2Y₁-deficient mouse platelets to 16.5±3.0% (P<0.05) and 19.3±4.2% (P<0.05), respectively. These results suggest that in isolated human or mouse platelets, the P2Y₁₂ receptor is involved in PS exposure after thrombin stimulation whereas the P2Y₁ receptor is not, which probably explains the thrombogram observations.

Platelet P-Selectin Exposure in Whole Blood
The involvement of the P2Y₁ and P2Y₁₂ receptors in platelet P-selectin exposure was evaluated in whole blood stimulated with either ADP (100 µmol/L) or TRAP (10 µmol/L). Double-labeling flow cytometry was used to determine the percentage of P-selectin-positive events in the isolated platelet population (CD42b positive). In the resting state, less than 0.05% of platelets expressed P-selectin, whereas ADP weakly increased this expression to up to 6.6%. The effect of ADP was blocked by either MRS2179 (0.8±0.4% P-selectin-positive platelets, P<0.01) or ARC69931MX (0.6±0.2% P-selectin-positive platelets, P<0.01; Figure 4A). TRAP markedly increased P-selectin exposure to 41.0±5.5% of platelets. Blockade of the P2Y₁ receptor decreased TRAP-induced P-selectin expression by 50% (19.3±4.5% P-selectin-positive platelets, P<0.01), whereas blockade of the P2Y₁₂ receptor reduced this expression by more than 90% (2.9±0.5% P-selectin-positive platelets, P<0.01; Figure 4B). Thus, the 2 ADP receptors would both appear to be involved in platelet P-selectin exposure.

View larger version (27K): [in this window] [in a new window]   Figure 4. Platelet P-selectin exposure in whole blood stimulated with ADP (100 µmol/L; A) or TRAP (10 µmol/L; B). Effects of MRS2179 (100 µmol/L, ARC), AR-C69931MX (10 µmol/L, MRS), or both antagonists (MRS/ARC). Values represent the percentage of P-selectin-positive platelets (mean±SEM, n=4 to 6). **P<0.01 versus vehicle.

Platelet-Leukocyte Aggregates (PLAs)
PLAs were determined as the percentage of CD42b-positive events in the leukocyte population (CD45 positive). Whereas very few aggregates were observed in the resting state (less than 4%), stimulation with ADP increased the percentage of PLA to 26.4±3.2%. ADP-induced PLA formation was not significantly decreased by either MRS2179 or AR-C69931MX alone, but a combination of the 2 inhibitors reduced the percentage of aggregates to near basal levels (6.3±1.4% PLA, P<0.01; Figure 5A). TRAP increased PLA formation to about 30% of leukocytes. MRS2179 had no significant effect on TRAP-induced PLA formation, whereas AR-C69931MX decreased it by 50% (17.9±3.5% PLA, P<0.05). In this case, no additive effect was observed when combining the 2 antagonists (Figure 5B).

View larger version (36K): [in this window] [in a new window]   Figure 5. Platelet-leukocyte conjugate formation in whole blood stimulated with ADP (100 µmol/L; A) or TRAP (10 µmol/L; B). Effects of MRS2179 (100 µmol/L, MRS), AR-C69931MX (10 µmol/L, ARC), or both antagonists (MRS/ARC). Values represent the percentage of CD42b-positive leukocytes (mean±SEM, n=5 to 8). *P<0.05, **P<0.01 versus vehicle.

Leukocyte TF Expression
Leukocyte TF expression was determined as the percentage of TF-positive events in the leukocyte population (CD45 positive). In the absence of stimulation, less than 5% of leukocytes were TF positive, whereas ADP (100 µmol/L) increased TF exposure to 21.0±2.9% TF-positive leukocytes. MRS2179 and ARC-69931MX both inhibited ADP-induced TF expression (6.5±1.4%, P<0.01, and 5.1±0.8%, P<0.01, respectively; Figure 6A). TRAP stimulation led to 23.2±2.9% TF-positive leukocytes, and this percentage decreased to 9.9±0.7% (P<0.01) or 3.4±0.2% (P<0.01) in the presence of MRS2179 or AR-C69931MX, respectively. When both ADP receptors were blocked, no further decrease was observed using either agonist (Figure 6B).

View larger version (35K): [in this window] [in a new window]   Figure 6. Leukocyte TF exposure in whole blood stimulated with ADP (100 µmol/L; A) or TRAP (10 µmol/L; B). Effects of MRS2179 (100 µmol/L, MRS), AR-C69931MX (10 µmol/L, ARC), or both antagonists (MRS/ARC). Values represent the percentage of TF-positive leukocytes (mean±SEM, n=5 to 8). **P<0.01 versus vehicle.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of the present study was to characterize the involvement of the 2 platelet ADP receptors in thrombin generation. In vivo, the blocking of either the P2Y₁ or the P2Y₁₂ receptor was equally effective with regard to both platelet consumption and thrombin generation as measured by TAT complex formation. However, the blocking of both receptors had an additive effect on platelet consumption but not on thrombin generation, suggesting that separate pathways are responsible for these 2 phenomena.

Activated platelets contribute to fibrin formation by providing a catalytic surface for the assembly of enzyme complexes involved in the sequential reactions leading to thrombin formation.² The P2Y₁₂ receptor appeared to play a significant part in the exposure of a procoagulant surface on platelets because the P2Y₁₂ antagonist AR-C69931MX decreased the exposure of PS and delayed the course of thrombin generation in PRP. The total amount of thrombin generated (ETP), although delayed, was nevertheless not significantly affected by AR-C69931MX in either human or mouse PRP. Similarly, when platelet GPIIbIIIa is absent as in Glanzmann’s thrombasthenia or inhibited with abciximab at a therapeutic dose, the kinetics of thrombin formation are strongly retarded, the lag time, peak time, and peak height being affected, whereas the endogenous thrombin potential is not or only slightly decreased (C. Ravanat, unpublished data, 2002).^8,23 This is consistent with previous observations showing that blocking platelet activation affects primarily the kinetics of the reaction rather than the total amount of thrombin produced, unlike direct anticoagulants, which considerably decrease the total thrombin generation.²⁴ These results also confirm earlier studies demonstrating a role of the P2Y₁₂ receptor in the exposure of negative phospholipids and the formation of thrombin in PRP.^7,25,26 In contrast, the P2Y₁ receptor did not appear to be significantly involved in either PS exposure on isolated platelets or global thrombin generation in PRP. Such results highlight the differential roles of the 2 platelet ADP receptors. Substantial PS exposure is obtained with high concentrations of strong activating agents,¹ and this could explain why the P2Y₁ receptor, through its weak coupling to Gq, played no significant part in isolated platelets or PRP. Conversely, the P2Y₁₂ receptor, which is coupled to Gi and the PI3kinase pathway¹² and contributes to the amplification of most platelet responses, made an important contribution to the exposure of a procoagulant surface.

However, the in vivo thromboembolism studies supporting a role of the P2Y₁ receptor in thrombin formation suggest that either the in vivo context is important in highlighting the involvement of this receptor or that other mechanisms could account for its participation in thrombin generation. We therefore investigated in whole blood the role of the P2Y₁ and P2Y₁₂ receptors in interactions between platelets and leukocytes. Activation of platelets leads to the expression of adhesion molecules, among which P-selectin has been shown to be involved in the formation of conjugates with leukocytes resulting in TF exposure, the physiological initiator of thrombin generation, at the surface of these conjugates.^3,4 Whole blood was stimulated with TRAP or ADP. ADP was used at 100 µmol/L to compensate for nucleotide degradation resulting from the presence of ectonucleotidase activities in blood. The exposure of P-selectin at the surface of platelets increased within minutes, and P2Y₁ and P2Y₁₂ antagonists inhibited not only ADP-induced but also TRAP-induced P-selectin exposure. Similar effects were observed when examining platelet TF exposure and aggregate formation, the P2Y₁₂ antagonist having a more pronounced effect than the P2Y₁ antagonist. These results are consistent with previous studies showing that the blocking of the P2Y₁₂ receptor with clopidogrel or AR-C69931MX decreases both P-selectin expression and PLA formation in response to TRAP or ADP.^27–30 Our findings confirm the potentiating role of the P2Y₁₂ receptor in such responses and suggest that this receptor is further involved in the generation of thrombin through platelet-leukocyte interactions. In addition, our results also demonstrate that the P2Y₁ receptor contributes to these interactions and to the exposure of TF at the surface of the platelet-leukocyte conjugates, in accordance with our in vivo observations. However, the P2Y₁ antagonist failed to decrease the formation of PLA while it inhibited platelet P-selectin exposure, suggesting alternative mechanisms for the role of the P2Y₁ receptor in TF exposure. P-selectin exposure is a measure of -granule secretion and therefore platelet-released products, such as chemokines, known to attract leukocytes and to potentiate platelet activation, may contribute to TF exposure.^31,32 Moreover, although it is admitted that platelets are involved in the rapid TF exposure on leukocytes without de novo transcription, it is not yet clear whether TF originates from platelets or from leukocytes or both and by what mechanisms it may be transferred from one cell to another. In our whole blood experiments, we could not come to a conclusion regarding the origin of the TF exposed at the surface of the platelet-leukocytes aggregates. However, recent evidences have shown that TF may also be of intraplatelet origin and may transfer this TF to leukocytes.^33–35 Both P2Y₁ and P2Y₁₂ receptors participate in the exposure of TF at the platelet surface on activation (C. Léon, unpublished data, 2003) probably contributing also to the in vivo generation of thrombin. However, because the P2Y₁ receptor is also expressed by endothelial cells and other blood cells,³⁶ we nevertheless cannot exclude a role of endothelial and leukocyte P2Y₁ receptors, which might participate to and reinforce these interactions, leading to platelet activation and TF exposure.

In conclusion, we show here that the P2Y₁ and P2Y₁₂ receptors are differentially involved in the generation of thrombin. Both receptors participate in leukocyte TF exposure in whole blood, whereas mostly the P2Y₁₂ receptor plays a role in the TF-induced formation of thrombin in PRP, probably by mediating negative PS exposure on platelets. These results, together with the increased thromboresistance observed in mice when both ADP receptors are blocked, provide further evidence for the relevance of molecules targeting these receptors as antithrombotic agents.

	Acknowledgments

 
This work was supported by INSERM and ARMESA. The authors would like to thank M. Ehret, C. Schwartz, and J. Weber for expert technical assistance and J.N. Mulvihill for reviewing the English of the manuscript.

	Footnotes

 
Presented in part at the ISTH Meeting, Paris, France, July 6–12, 2001, and at the ADP 2002 Congress, Cagliari, Italy, October 3–5, 2002.

Received July 23, 2003; accepted August 8, 2003.

	References

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