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(Circulation. 2001;103:718.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Key Role of the P2Y₁ Receptor in Tissue Factor–Induced Thrombin-Dependent Acute Thromboembolism

Studies in P2Y₁-Knockout Mice and Mice Treated With a P2Y₁ Antagonist

Catherine Léon, PhD; Monique Freund, PhD; Catherine Ravanat, PhD; Anthony Baurand, MSc; Jean-Pierre Cazenave, MD, PhD; Christian Gachet, MD, PhD

From the Institut National de la Santé et de la Recherche Médicale U.311, Etablissement Français du Sang–Alsace, Strasbourg, France.

Correspondence to Dr C. Gachet, INSERM U.311, Etablissement Français du Sang–Alsace (EFS-Alsace), 10, rue Spielmann, BP N^o 36, 67065 Strasbourg Cédex, France. E-mail christian.gachet{at}etss.u-strasbg.fr

	Abstract

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Background—ADP plays a key role in hemostasis, acting through 2 platelet receptors: the P2Y₁ receptor and an unidentified P2 receptor, called P2cyc, coupled to adenylyl cyclase inhibition, which is the target of the antiplatelet drug clopidogrel. We showed that the P2Y₁ receptor is an essential cofactor in thrombotic states induced by intravenous infusion of collagen and epinephrine. The aim of the present study was to assess the role of this receptor in thrombin-dependent tissue factor–induced thromboembolism.

Methods and Results—Human thromboplastin was injected intravenously into wild-type or P2Y₁-deficient mice, and the effects on platelet count and mortality were determined and plasma thrombin–antithrombin III (TAT) complexes were quantified. P2Y₁-deficient mice were resistant to the thromboembolism induced by injection of thromboplastin. Whereas the platelet count decreased sharply in wild-type mice, there was no significant drop in platelets in P2Y₁-knockout mice. The platelet consumption in wild-type mice was probably due to thrombin generation, because it was abolished by hirudin. Thromboplastin also led to a rise in TAT complexes in plasma, again reflecting thrombin formation. This effect, however, was less important in P2Y₁-knockout mice than in wild-type mice, indicating that less thrombin was generated in the absence of P2Y₁. Similar results were obtained after intravenous administration of N⁶-methyl-2'-deoxyadenosine-3':5'-bisphosphate, a selective antagonist of the P2Y₁ receptor, to wild-type mice.

Conclusions—Our results demonstrate a role of the P2Y₁ receptor in thrombotic states involving thrombin generation and provide further evidence for the potential relevance of this receptor as a target for antithrombotic drugs.

Key Words: receptors • adenosine diphosphate • thrombosis • drugs • platelets

	Introduction

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Normal hemostasis and thrombotic cardiovascular disease result from complex interactions between the vascular wall and blood components, among which platelets and coagulation proteins are of major importance. Antiplatelet agents impairing platelet functions have proved effective in arterial thrombosis, in which high-flow conditions prevail, whereas anticoagulants act essentially by decreasing circulating thrombin concentrations, which are involved in many thrombotic events and particularly in venous thrombosis, in which stasis occurs. Activated platelets participate in thrombin generation through exposure of membrane receptors for blood coagulation proteins, through release of coagulation factors stored in their -granules,¹ and through formation of a procoagulant surface by rearrangement of their membrane phospholipids to expose negatively charged phosphatidylserine.^{1 2} Thus, antiplatelet agents may contribute in vivo to an inhibition of coagulation by decreasing thrombin generation at the surface of platelets. This has been demonstrated for anti–GP IIb/IIIa agents, which inhibit stasis-induced venous thrombosis in vivo³ and thrombin formation in vitro.⁴ Similarly, the thienopyridine compounds ticlopidine and clopidogrel, potent and selective inhibitors of ADP-induced platelet activation that display antiaggregatory and antithrombotic properties in both animals and humans,^{5 6 7} exhibit antithrombotic effects in experimental venous thrombosis.⁸ These compounds were recently shown to significantly inhibit the in vitro thrombin generation triggered by low concentrations of tissue factor in the presence of platelets by inhibiting platelet ADP receptors.⁹

ADP plays a key role in hemostasis because it is itself an aggregating agent and because it is released from dense granules during platelet activation. Released ADP potentiates many in vitro platelet responses induced by other agents, including thrombin.¹⁰ ADP participates in the binding of fibrinogen to platelets stimulated by thrombin¹¹ and is involved in stabilizing the platelet aggregates induced by thrombin stimulation.¹² At the intracellular level, ADP has been shown to act synergistically with thrombin to activate PI 3-kinase¹³ and phospholipase D.¹⁴ Two ADP receptors involved in ADP-induced platelet responses have been described to date.¹⁵ The P2Y₁ receptor is responsible for platelet shape change, which occurs through an increase in intracellular calcium triggered by G_q/phospholipase C activation. The adenosine derivatives adenosine 2':5'-bisphosphate, adenosine 3':5'-bisphosphate,¹⁶ and N⁶-methyl-2'-deoxyadenosine-3':5'-bisphosphate (MRS2179)¹⁷ are selective antagonists of the P2Y₁ receptor. These compounds block shape change and aggregation in response to usual concentrations of ADP, demonstrating that the P2Y₁ receptor is necessary for platelet aggregation.^{18 19 20} The second, still unknown, platelet ADP receptor, called P2cyc, is coupled to adenylyl cyclase inhibition, probably through a G_i2 protein.²¹ It is the target of the thienopyridine drugs ticlopidine and clopidogrel and of ATP derivatives such as the AR-C compounds AR-C67085 and AR-C69931MX.^{22 23 24} The P2Y₁ and P2cyc receptors are both necessary to obtain full aggregation in response to ADP.^{18 25 26}

Recently, it was shown that P2Y₁-deficient mice are resistant to the acute thromboembolism induced by intravenous injection of either ADP or collagen and epinephrine.^{26 27} In vitro, aggregation in response to usual concentrations of ADP was totally abolished in platelets from P2Y₁-/- mice. Aggregation in response to collagen was also impaired, whereas thrombin-induced aggregation was affected only at low thrombin concentrations.²⁶ The aim of the present study was to assess the role of the P2Y₁ receptor in the thrombin-dependent acute thromboembolism induced by tissue factor administration. In this model, disruption of the P2Y₁ gene resulted in a marked reduction in mortality, in platelet consumption, and in levels of thrombin generation in the deficient mice. Moreover, the selective P2Y₁ antagonist MRS2179 effectively inhibited thrombin-dependent thromboembolism in wild-type mice.

	Methods

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Chemicals
The anesthetic drugs xylazine (Rompun) and ketamine (Imalgene 1000) were from Bayer and Mérial, respectively. Human thromboplastin (Thromborel) from Dade Behring was reconstituted according to the manufacturer’s instructions, and its activity in mouse plasma was checked by measuring the prothrombin time. According to the protocol described by the manufacturer, the prothrombin time was 8.72±0.037 seconds (mean±SEM) in mouse plasma and 11.92±0.22 seconds (mean±SEM) in human plasma. Hemogard C.T.A.D. (citrate/citric acid, theophylline, adenosine, dipyridamole) anticoagulant was from Becton Dickinson Vacutainer Systems Europe, and Leukoplate was from Sobodia. Recombinant hirudin rHV2-Lys47 (r-hirudin) was kindly provided by Transgène (Strasbourg, France). MRS2179¹⁷ was a generous gift from P. Raboisson and J-J. Bourguignon (CNRS, Strasbourg, France).

In Vivo Thrombosis Model
P2Y₁+/+ and P2Y₁-/- mice were produced as described previously,²⁶ and both wild-type (P2Y₁+/+) and mutant (P2Y₁-/-) mice were 50% 129/Sv–50% C57BL/6 at the F3 generation. C57BL/6 mice were bred at Iffa Credo (l’Arbresle, France). Male mice weighing 20 to 30 g were anesthetized by injection of 150 µL IP of a mixture of 0.2% xylazine base and 1% ketamine in physiological saline. The jugular vein was exposed surgically, and thromboplastin was injected at the indicated dose within an infusion time frame of 3 to 4 seconds. In some experiments, hirudin (50 mg/kg) or physiological saline (vehicle) was injected subcutaneously 1 hour before thromboplastin injection. Necropsies were performed on 5 mice that died, and the mice that recovered were killed 30 minutes after injection of thromboplastin, and the lungs were examined. In inhibition tests, the P2Y₁ antagonist MRS2179 (50 mg/kg) or physiological saline (100 µL/20 g) was injected into the jugular vein 30 seconds before injection of thromboplastin.

Lung Histology
The lungs were removed 2 minutes after injection of thromboplastin (100 µL/kg) and fixed in 4% formaldehyde. Paraffin sections 5 µm thick were stained with hematoxylin/eosin, and the occluded vessels were quantified under a phase-contrast microscope. At least 5 fields were examined at a total magnification of x200, and the identifiable occluded vessels (diameter >50 µm) were counted in each field. Lung histology was performed in 5 wild-type and 6 P2Y₁-knockout mice and in mortality experiments in 5 mice that died and 5 that recovered.

Platelet Counts
Whole blood was diluted 80-fold with Leukoplate, and platelets (300 to 400 platelets for control mice) were counted with a hemocytometer under phase-contrast microscopy.

Quantification of Thrombin–Antithrombin III Complexes
Blood was drawn from the abdominal aorta into Hemogard C.T.A.D. 2 minutes after thromboplastin injection and centrifuged (13 000g, 10 minutes, 4°C) to obtain platelet-poor plasma. Mouse thrombin–antithrombin III complexes (TAT) in plasma were determined with a commercial ELISA kit (Enzygnost TAT, Behringwerke AG) and calibration standards of human origin (2 to 60 ng/mL TAT).²⁸

Statistical Analyses
The ² test was used to determine the significance of the difference in mortality in the sudden death assay. Student’s 2-tailed unpaired t test was used to evaluate the significance of the differences in platelet count, TAT levels, and number of occluded vessels.

	Results

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P2Y₁-Deficient Mice Are Resistant to Thromboplastin-Induced Thromboembolism
Human thromboplastin 200 µL/kg was injected intravenously, and the mice were observed for 30 minutes. In most cases, thromboplastin injection led to transient respiratory arrest and a decreased heart rate, followed by either definitive respiratory arrest within 3 to 5 minutes or recovery. Among wild-type mice, 72% died of widespread pulmonary vascular thrombosis and cardiac arrest. In contrast, only 36% of P2Y₁-/- mice died under the same conditions (Figure 1). In histological investigations, the mice were injected with thromboplastin 100 µL/kg, and their lungs were removed 2 minutes later. Large and small vessels, including arteries, in the lungs of wild-type mice were obstructed by platelet-fibrin thrombi, and large zones of atelectasis were observed (Figure 2c). Conversely, mainly small vessels were obstructed in the lungs of P2Y₁-/- mice (Figure 2d). Quantification revealed that the number of occluded vessels was 3 to 4 times higher in wild-type mice than in P2Y₁-deficient mice (mean±SEM, 20.12±1.34 and 5.73±0.56 occluded vessels per microscopic field, respectively, P<0.0001). No obstructed vessels were detected in the lungs of either genotype injected with physiological saline (Figure 2a and 2b). Necropsy of the lungs of mice that had died in mortality experiments showed no significant difference between P2Y₁+/+and P2Y₁-/- mice. In all cases, the vessels were obstructed by occlusive thrombi, and there was atelectasis and large infarct zones. Conversely, only a few small vessels were obstructed in the lungs of mice that had recovered, whatever the genotype, with very few or no atelectasis or infarct zones.

View larger version (14K): [in this window] [in a new window]   Figure 1. Mortality resulting from thromboplastin injection in P2Y1+/+ and P2Y1-/- mice. Thromboplastin (200 µL/kg IV) was injected, and mice were observed for 30 minutes. Death occurred within 3 to 5 minutes. Results are expressed as percentage mortality. n=22 mice for both genotypes. **P=0.0077 by 2 test.

View larger version (146K): [in this window] [in a new window]   Figure 2. Microscopic examination of lungs of mice challenged with thromboplastin. Sections were stained with hematoxylin-eosin. a, P2Y1+/+ mice injected with saline; b, P2Y1-/- mice injected with saline; c, P2Y1+/+ mice injected with thromboplastin (100 µL/kg); d, P2Y1-/- mice injected with thromboplastin (100 µL/kg). Bar=0.1 mm.

Effect of Thromboplastin Injection on Platelet Count
A lower dose of thromboplastin (100 µL/kg) was used to study the effects on platelet count in blood drawn 2 minutes after injection. As shown in Figure 3, although the platelet count remained normal in control wild-type mice injected with physiological saline, it decreased sharply in wild-type mice receiving thromboplastin (mean±SEM, 1 168 514±570 03 and 451 280±119 217 platelets/µL, respectively, P<0.0001). Conversely, no significant decrease in platelet count was observed in P2Y₁-knockout mice injected with thromboplastin compared with control P2Y₁-/-mice (1 027 086±676 57 and 1 176 000±813 02 platelets/µL, respectively). Subcutaneous injection of hirudin 50 mg/kg 1 hour before intravenous injection of thromboplastin 100 µL/kg abolished its effect on platelet count (Figure 3), indicating that platelet consumption was due to thrombin generation.

View larger version (18K): [in this window] [in a new window]   Figure 3. Platelet counts after thromboplastin injection in P2Y1+/+ and P2Y1-/- mice. Thromboplastin (100 µL/kg IV) was injected, and blood was drawn 2 minutes later. Results are expressed as mean platelet count±SEM. Open bar, control mice receiving physiological saline; solid bar, mice receiving thromboplastin; hatched bar, mice treated with hirudin (50 mg/kg) before thromboplastin injection. n=number of mice in each group.*P<0.05; ***P<0.0001.

Effect of Thromboplastin Injection on TAT Formation In Vivo
To further assess thromboplastin-induced thrombin generation, we measured levels of TAT in plasma after injection of thromboplastin or saline. Injection of thromboplastin 100 µL/kg led to an increase in TAT in the plasma of both P2Y₁-/- and P2Y₁+/+ mice, reflecting in vivo thrombin generation. Plasma TAT concentrations were high in either case but nevertheless significantly lower in P2Y₁-deficient mice than in wild-type mice (mean±SEM, 136.7±15.8 and 191.8±16.2 ng TAT/mL, respectively, P=0.027) (Figure 4). Under the same conditions, basal levels after injection of physiological saline were 9.11±1.37 and 10.78±1.50 ng TAT/mL for P2Y₁-/- and P2Y₁+/+ mice, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effect of thromboplastin injection on plasma thrombin–antithrombin III complexes. Thromboplastin (100 µL/kg) was injected intravenously into P2Y1+/+ and P2Y1-/- mice, blood was drawn 2 minutes later, and TAT was determined in platelet-poor plasma. Results are expressed as mean±SEM. *P=0.027. n=number of mice in each group.

Effect of MRS2179 on Acute Tissue Factor–Induced Thromboembolism
C57BL/6 mice were injected intravenously with physiological saline or the P2Y₁ antagonist MRS2179 (50 mg/kg) 30 seconds before administration of thromboplastin 100 µL/kg. MRS2179 totally prevented death, whereas 90% of the control mice receiving saline died within 3 to 5 minutes (P=0.0008) (Figure 5). In another control group, the platelet count had decreased by 80% 2 minutes after thromboplastin injection (mean±SEM, 214.06±17.94 platelets/µL). No such decrease in platelets was observed in mice receiving MRS2179 (mean±SEM, 1155.60±52.84 platelets/µL for mice pretreated with MRS2179 followed by thromboplastin injection, whereas control mice injected with saline alone displayed 1140.29±45.50 platelets/µL) (Figure 6a). Similarly, as in P2Y₁-deficient mice, TAT levels were 35% lower in mice treated with MRS2179 than in the control group (mean±SEM, 247.90±13.70 and 378.40±34.16 ng/mL, respectively, P=0.0032) (Figure 6b). It is noteworthy that C57BL/6 mice were more sensitive to thromboplastin administration than mice having a mixed genetic background (C57BL/6-129/Sv), because a dose of 100 µL/kg thromboplastin leads to 90% mortality for C57BL/6 mice. For comparison, 72% mortality for the mixed genetic background (C57BL/6-129/Sv) is achieved with a dose of 200 µL/kg thromboplastin.

View larger version (9K): [in this window] [in a new window]   Figure 5. Effect of MRS2179 on mortality (%) resulting from thromboplastin injection in C57BL/6 mice. n=number of mice in each group. Left bar, mice injected with saline only; solid (middle) bar, mice injected with saline followed by thromboplastin (100 µL/kg); right bar, mice injected with MRS2179 (50 mg/kg) followed by thromboplastin (100 µL/kg). ***P<0.0001.

View larger version (22K): [in this window] [in a new window]   Figure 6. Effect of MRS2179 on thromboplastin-induced thromboembolism in C57BL/6 mice. n=number of mice in each group. a, platelet count (mean±SEM); b, plasma TAT (mean±SEM). Open bars, mice injected with saline only; solid bars, mice injected with saline followed by thromboplastin (100 µL/kg); hatched bars, mice injected with MRS2179 (50 mg/kg) followed by thromboplastin (100 µL/kg). **P<0.01; ***P<0.0001.

	Discussion

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Recently, we and others demonstrated that the P2Y₁ receptor, necessary for ADP-induced platelet shape change and activation, plays a role in the thromboembolism resulting from intravenous injection of collagen and epinephrine or collagen and ADP.^{26 27} These in vivo effects could be correlated with the in vitro properties of P2Y₁-deficient platelets, which exhibit strongly reduced aggregation in response to ADP or collagen.

In the present study, we investigated the role of the P2Y₁ receptor in the acute thrombin-dependent thromboembolism induced by intravenous injection in the jugular vein of thromboplastin. P2Y₁-deficient mice were more resistant to thromboplastin-induced thromboembolism than wild-type mice. Mortality, which probably resulted from lung occlusion and cardiac arrest, reflected at least in part the pulmonary vessel occlusion seen on all necropsy specimens. No thrombi were found in the kidneys after thromboplastin injection, although we cannot rule out the possibility that thrombi may have been generated in other vascular systems. A sharp decrease in platelet count was observed in the wild-type mice treated with thromboplastin, consistent with platelet thromboembolism of the lung microcirculation. Conversely, no decrease in platelet count was observed in the thrombosis-resistant P2Y₁-deficient mice. When hirudin was injected before thromboplastin challenge, wild-type mice displayed no drop in platelets, suggesting that mortality was a consequence of thrombin generation. In addition, plasma TAT concentrations were strongly increased in mice receiving thromboplastin, directly reflecting thrombin formation. TAT levels were less enhanced in P2Y₁-knockout mice than in wild-type mice, indicating involvement of the P2Y₁ receptor in the in vivo generation of thrombin. Plasma TAT nevertheless remained high whatever the genotype of the mice, in contrast to the situation in thromboembolism induced by injection of collagen and epinephrine into the jugular vein, in which no significant increase in TAT was observed (mean±SEM, 11.51±2.42 and 12.86±3.45 ng TAT/mL for P2Y₁+/+ and P2Y₁-/-, respectively, after injection of collagen 0.25 mg/kg and epinephrine 60 µg/kg, whereas basal levels were 9.51±1.41 and 7.53±1.08 ng TAT/mL, respectively, after physiological saline administration) (C.L., unpublished data, 1999). Stimulation with collagen and epinephrine resembles arterial thrombosis, however, in which platelets are activated mainly by the subendothelium under conditions of high shear stress,^{29 30} whereas in the present model, thrombin is the main inducer. Plasma TAT levels were strongly increased in our model of thromboembolism, being close to 400 ng TAT/mL, which is comparable to the levels observed in humans after trauma or sepsis. In these patients, the increase in tissue factor expression leads to a dramatic rise in plasma TAT, to up to 665 ng/mL after trauma or close to 80 ng/mL in sepsis.³¹

Thromboplastin injection, which mimics in vivo tissue factor exposed or released from damaged vessel wall or circulating cells, activates factor VII and triggers the coagulation cascade, leading to thrombin formation.³² Thrombin activates platelets and endothelial cells, resulting in the development of procoagulant activities and explosive thrombin generation. In this process, platelets have been estimated to accelerate thrombin formation by 5 to 6 orders of magnitude.¹ Platelets from P2Y₁-deficient mice are less responsive in vitro to threshold doses of thrombin than normal platelets,²⁶ suggesting that circulating platelets in P2Y₁-knockout mice may be less strongly activated than those of wild-type mice after thromboplastin injection. This phenomenon could at least partly account for the thromboresistance of P2Y₁-/- mice. This, however, might not be the only reason for the enhanced resistance of P2Y₁-knockout mice to thrombosis. In thrombin-dependent thromboembolism models, circulating microthrombi result in pulmonary vascular thrombosis.^{33 34} Such models are very sensitive to vasoactive agents, because vasodilatory compounds have been shown to reduce thrombin-induced mortality.³⁴ In this context, thrombin not only activates coagulation and platelets but also causes pulmonary vasoconstriction,³⁵ which may contribute to organ failure and death in these models. Moreover, because TAT levels remained very high even in P2Y₁-deficient mice, it may be possible that their thromboresistance is not entirely due to the difference in thrombin generation resulting from the absence of P2Y₁ receptors on platelets. P2Y₁ receptors are expressed in a wide range of tissues, and in the cardiovascular system, purinergic nucleotides have been found to exert pronounced although relatively complex effects on coronary tone and mechanical activity of the heart.^{36 37} Hence, the prevention of mortality may also be related to blockade of these functions either in the cardiovascular system or indirectly in the bronchopulmonary tree.

MRS2179 has been reported to be a selective P2Y₁ antagonist with no effect on P2Y₂, P2Y₄, or P2Y₆ receptors.¹⁷ In vitro, MRS2179 inhibited ADP-induced platelet shape change and aggregation (pA₂=6.55±0.05) but did not affect the ADP-induced adenylyl cyclase pathway, whereas K_d for the binding of [³³P]MRS2179 to human platelets was 109±18 nmol/L.³⁸ MRS2179 also inhibited aggregation induced by threshold concentrations of thrombin (0.01 U/mL) in a manner resembling the behavior of platelets from P2Y₁-deficient mice.²⁶ In this work, a dose of 50 mg/kg MRS2179 was injected 30 seconds before the thromboplastin challenge. Assuming 2 mL of blood per 20 g of mouse, 50 mg/kg MRS2179 should correspond to a final blood concentration of 1 mmol/L, although it is probably difficult to estimate the true concentration of circulating MRS2179, which will also depend on its clearance. According to the K_d value of 109 nmol/L, this concentration should nevertheless be sufficient to inhibit all P2Y₁ receptors. At this dose, MRS2179 prevented thrombin-dependent thromboembolism in C57BL/6 mice. Platelet consumption was also reduced, and less TAT was produced than in control mice receiving no MRS2179. These results confirm our findings in P2Y₁-knockout mice as to the role played by the P2Y₁ receptor in the contribution of platelet activation to the generation of thrombin.

The thromboplastin injection model can be related to a model of disseminated intravascular coagulation. One pathogenesis of disseminated intravascular coagulation is endotoxin shock, which is triggered in particular by tissue factor expression.^{39 40} Drugs acting on the platelet P2cyc receptor have already proved effective in models of endotoxin-induced disseminated intravascular coagulation,⁴¹ and our results indicate that P2Y₁ receptors may also play a role in this pathological condition. The fact that the P2Y₁ receptor is involved in thrombin-dependent thromboembolism, together with previous data demonstrating its importance in thrombosis induced by collagen, strongly suggests that this receptor will prove to be a useful target in a wide range of thrombotic diseases.

	Acknowledgments

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

Received April 20, 2000; revision received August 7, 2000; accepted August 14, 2000.

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