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• W1997659847 abstract "SummaryPrevious studies in experimental models revealed a role for the P2Y1 platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y1 receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl3, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y1-deficient mice and mice treated with the P2Y1 antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y1 deficiency with P2Y12 inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y1 receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs. Previous studies in experimental models revealed a role for the P2Y1 platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y1 receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl3, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y1-deficient mice and mice treated with the P2Y1 antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y1 deficiency with P2Y12 inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y1 receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs. In arteries, rupture of atheromatous plaques induces platelet activation and accumulation at the site of injury, which can lead to thrombus formation and vessel occlusion and hence to myocardial infarction or stroke among other outcomes. Venous thrombosis involves other mechanisms, being more dependent on activation of coagulation and thrombin generation than arterial thrombosis. However, platelets must also play a role in venous thrombosis since antiplatelet agents have an inhibitory effect, although moderate, in venous models of thrombosis [1Herbert J.M. Bernat A. Maffrand J.P. Importance of platelets in experimental venous thrombosis in the rat.Blood. 1992; 80: 2281-6Crossref PubMed Google Scholar]. ADP is one of the most important mediators of platelet activation. Two G protein-coupled receptors are involved in ADP-induced platelet activation and aggregation: the P2Y1 receptor is responsible for shape change and the initiation of aggregation, while the P2Y12 receptor is responsible for the amplification of platelet aggregation [2Gachet C. ADP receptors of platelets and their inhibition.Thromb Haemost. 2001; 86: 222-32Crossref PubMed Scopus (372) Google Scholar]. This latter receptor is the target of the antithrombotic drugs ticlopidine and clopidogrel, which irreversibly inhibit platelet activation and aggregation in response to ADP [3Savi P. Labouret C. Delesque N. Guette F. Lupker J. Herbert J.M. P2y(12), a new platelet ADP receptor, target of clopidogrel.Biochem Biophys Res Commun. 2001; 283: 379-83Crossref PubMed Scopus (211) Google Scholar]. Although these drugs are efficient in preventing arterial ischemic events, as has been demonstrated in several clinical trials [4CAPRIE Steering CommitteeA randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE)..Lancet. 1996; 348: 1329-39Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 5Solet D.J. Zacharski L.R. Plehn J.F. The role of adenosine 5′-diphosphate receptor blockade in patients with cardiovascular disease.Am J Med. 2001; 111: 45-53Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], this type of global antiplatelet therapy nevertheless prevents thrombotic recurrence in only 30% of cases [5Solet D.J. Zacharski L.R. Plehn J.F. The role of adenosine 5′-diphosphate receptor blockade in patients with cardiovascular disease.Am J Med. 2001; 111: 45-53Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 6Bennett J.S. Novel platelet inhibitors.Annu Rev Med. 2001; 52: 161-84Crossref PubMed Scopus (104) Google Scholar]. Hence research is being conducted to find new targets for alternative antithrombotic strategies. Previous work has shown that platelets from P2Y1-deficient mice display defective aggregation in response to ADP and low concentrations of stronger agonists such as collagen [7Léon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.J Clin Invest. 1999; 104: 1731-7Crossref PubMed Google Scholar, 8Fabre J.E. Nguyen M. Latour A. Keifer J.A. Audoly L.P. Coffman T.M. Koller B.H. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.Nat Med. 1999; 5: 1199-202Crossref PubMed Scopus (382) Google Scholar], suggesting an important role of this receptor in thrombosis. Recent studies have further demonstrated a role of the P2Y1 receptor in a systemic thrombosis model [7Léon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.J Clin Invest. 1999; 104: 1731-7Crossref PubMed Google Scholar, 8Fabre J.E. Nguyen M. Latour A. Keifer J.A. Audoly L.P. Coffman T.M. Koller B.H. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.Nat Med. 1999; 5: 1199-202Crossref PubMed Scopus (382) Google Scholar, 9Léon C. Freund M. Ravanat C. Baurand A. Cazenave J.P. Gachet C. Key role of the P2Y(1) receptor in tissue factor-induced thrombin- dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.Circulation. 2001; 103: 718-23Crossref PubMed Google Scholar]. P2Y1 deficiency led to resistance to mortality in thromboembolism experiments, reflecting a decrease in pulmonary vessel occlusion, and to less platelet consumption, reflecting lower levels of platelet thrombi [7Léon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.J Clin Invest. 1999; 104: 1731-7Crossref PubMed Google Scholar, 8Fabre J.E. Nguyen M. Latour A. Keifer J.A. Audoly L.P. Coffman T.M. Koller B.H. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.Nat Med. 1999; 5: 1199-202Crossref PubMed Scopus (382) Google Scholar]. Intravenous administration of the P2Y1 antagonist MRS2179 [10Boyer J.L. Mohanram A. Camaioni E. Jacobson K.A. Harden T.K. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate.Br J Pharmacol. 1998; 124: 1-3Crossref PubMed Scopus (192) Google Scholar] also had inhibitory effects in thromboembolism experiments [9Léon C. Freund M. Ravanat C. Baurand A. Cazenave J.P. Gachet C. Key role of the P2Y(1) receptor in tissue factor-induced thrombin- dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.Circulation. 2001; 103: 718-23Crossref PubMed Google Scholar]. In this model, thrombosis is induced by injection of ADP or a mixture of collagen and epinephrine, agents that directly activate platelets, or by injection of tissue factor which stimulates coagulation. Thus the P2Y1 receptor might play a crucial role in thrombosis, since its absence or inhibition reduces thrombus formation in an acute systemic model. The aim of the present work was to assess the part played by the P2Y1 receptor in localized thrombosis. Models of localized thrombosis in fact better mimic physiopathological processes in humans and we performed experiments in both arterial and venous situations. In the arterial model [11Denis C. Methia N. Frenette P.S. Rayburn H. Ullman-Cullere M. Hynes R.O. Wagner D.D. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis.Proc Natl Acad Sci USA. 1998; 95: 9524-9Crossref PubMed Scopus (427) Google Scholar], thrombosis was induced locally in the mesenteric arteries of mice by injuring the wall of a selected arteriole with FeCl3, and its evolution was followed in real time by observing the deposition of fluorescent-labeled platelets on the injured vessel wall using intravital microscopy. Venous thrombosis was studied in a Wessler model [12Wessler S. Thrombosis in the presence of vascular stasis.Am J Med. 1962; 33: 648Abstract Full Text PDF PubMed Google Scholar] adapted to rats [13Freund M. Cazenave J.P. Courtney M. Degryse E. Roitsch C. Bernat A. Delebassee D. Defreyn G. Maffrand J.P. Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats.Thromb Haemost. 1990; 63: 187-92Crossref PubMed Scopus (0) Google Scholar]. Thrombosis was induced by a combination of stasis established between two ligatures placed on the inferior vena cava and activation of coagulation triggered by intravenous injection of tissue factor. The results presented demonstrate that the P2Y1 receptor is involved in both localized arterial and venous thrombosis. Anesthetic drugs ketamine (Imalgene 1000®) and xylazine were from Bayer (Puteaux, France) and Mérial (Lyon, France), respectively. Acid citrate dextrose (ACD) anticoagulant was from Braun SA (Boulogne, France) and CTAD (citrate/citric acid, theophylline, adenosine, dipyridamole) from Becton Dickinson Vacutainer Systems Europe. Triton 100X, NaCl, MgCl2 and CaCl2 were supplied by Merck (Darmstadt, Germany) and KCl, NaHCO3, NaH2PO4 and glucose by Prolabo (Paris, France). Calcein-AM was from Molecular Probes (Eugene, Oregon, USA), ADP from Sigma Chemicals (Saint Louis, MO, USA) and EDTA from Sigma-Aldrich (Saint-Quentin Fallavier, France). N6-Methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) was a generous gift from P. Raboisson and J.-J. Bourguignon (CNRS, Strasbourg, France) [14Baurand A. Raboisson P. Freund M. Léon C. Cazenave J.P. Bourguignon J.J. Gachet C. Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist.Eur J Pharmacol. 2001; 412: 213-21Crossref PubMed Scopus (140) Google Scholar]. Clopidogrel was a generous gift from Sanofi-Synthélabo (Toulouse, France). Apyrase (ATP diphosphohydrolase, EC3615) was purified from potatoes [15Cazenave J.P. Hemmendinger S. Beretz A. Sutter-Bay A. Launay J. Platelet aggregation: a tool for clinical investigation and pharmacological study. Methodology.Ann Biol Clin. 1983; 41: 167-79PubMed Google Scholar]. Human serum albumin was supplied by the Etablissement Français du Sang – Alsace (Strasbourg, France), FeCl3 by Prolabo (Fontenay Sous-Bois, France) and glutaraldehyde (25%) by Euromedex (Souffelweyersheim, France). Thromboplastin (Thromborel® S) was obtained from Dade Behring SA (Paris, France) and reconstituted according to the manufacturer's instructions. C57BL/6J@ICO mice and male Wistar Han rats were from Charles River (L'Abresles, France). P2Y1-deficient mice were raised as previously described [7Léon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.J Clin Invest. 1999; 104: 1731-7Crossref PubMed Google Scholar] and had a mixed genetic background (129/Sv 50% × C57BL/6 50%). All procedures for animal experiments were in accordance with the guide for the care and use of laboratory animals. Blood was drawn from the abdominal aorta of anesthetized mice into ACD anticoagulant (one volume for six blood volumes) and pooled and platelets were washed as described in [15Cazenave J.P. Hemmendinger S. Beretz A. Sutter-Bay A. Launay J. Platelet aggregation: a tool for clinical investigation and pharmacological study. Methodology.Ann Biol Clin. 1983; 41: 167-79PubMed Google Scholar]. Platelets were loaded with 5 µm calcein-AM (a fluorescein-derived marker) for 15 min at 37 °C during the first wash and the final suspension contained 0.02 U mL−1 of the ADP scavenger apyrase to prevent platelet desensitization [16Baurand A. Eckly A. Bari N. Léon C. Hechler B. Cazenave J.P. Gachet C. Desensitization of the platelet aggregation response to ADP. differential down-regulation of the P2Y1 and P2cyc receptors.Thromb Haemost. 2000; 84: 484-91Crossref PubMed Scopus (0) Google Scholar]. Aggregation was measured at 37 °C by a turbidimetric method to confirm that the labeled platelets were fully functional and not activated (data not shown). This suspension was then injected into the tail vein of 3–5-week-old mice weighing about 15 g (108 platelets/10 g of body weight) and the mice were allowed to recover for at least 12 h. The presence of fluorescent platelets in a sample of blood taken from the tail after the recovery period and analyzed by flow cytometry confirmed that the labeled platelets recirculated normally. Similar analysis of blood samples removed at different times showed that fluorescent platelets persisted for 3 days in the injected animals, which corresponds to the normal mean life span of mouse platelets (data not shown). The arterial model was an adaptation of the procedure described by Denis et al. [11Denis C. Methia N. Frenette P.S. Rayburn H. Ullman-Cullere M. Hynes R.O. Wagner D.D. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis.Proc Natl Acad Sci USA. 1998; 95: 9524-9Crossref PubMed Scopus (427) Google Scholar]. Mice 3–5 weeks old and weighing about 15 g were anesthetized by injection of a mixture of 0.2% xylazine base and 1% ketamine in physiological saline (50 µL/10 g of body weight). The mesentery was exposed by making a midline abdominal incision and the mouse was placed on a plate of Plexiglas. An arteriole (60–80 µm diameter) was selected and observed under an inverted fluorescence microscope (Leica DMIRB; Leica Microsystems SA, Westlar, Germany) coupled to a video camera (DAGE MTI, Michigan City, MI, USA) and images were recorded on DVD-Ram tapes (WDR 200; Matsushita Electric Industrial, Osaka, Japan). After application of 30 µL of a solution of FeCl3 (250 mmol L−1) to the upper side of the vessel, the evolution of thrombosis was followed for 10 min or until complete occlusion of the vessel. One arteriole was monitored in each mouse. At time points 0, 2, 5 and 10 min after vessel wall injury, the severity of thrombosis in the vessel was evaluated and the mouse ascribed one of the following scores: 0, normal platelet circulation; 1, < 50 single adherent platelets; 2, >50 single adherent platelets; 3, thrombus formed (diameter > 30 µm); 4, occlusive thrombus. The time to formation of the first thrombus (diameter >30 µm) was also recorded. Some mice were treated with an inhibitor of the P2Y1 or P2Y12 receptor as follows. The selective P2Y1 antagonist MRS2179 (50 mg kg−1) [10Boyer J.L. Mohanram A. Camaioni E. Jacobson K.A. Harden T.K. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate.Br J Pharmacol. 1998; 124: 1-3Crossref PubMed Scopus (192) Google Scholar, 14Baurand A. Raboisson P. Freund M. Léon C. Cazenave J.P. Bourguignon J.J. Gachet C. Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist.Eur J Pharmacol. 2001; 412: 213-21Crossref PubMed Scopus (140) Google Scholar] or vehicle (physiological saline) was injected into the jugular vein just before induction of thrombosis with FeCl3. The P2Y12 inhibitor clopidogrel (50 mg kg−1) or vehicle (5% arabic gum) was administered orally the preceding day and at least 2 h before the experiment, a protocol ensuring maximum inhibition of platelet aggregation in response to ADP [17Savi P. Herbert J.M. ADP receptors on platelets and ADP-selective antiaggregating agents.Med Res Rev. 1996; 16: 159-79Crossref PubMed Scopus (0) Google Scholar]. Operators were unaware of the mouse genotype or treatment when analyzing the results of these experiments. The venous model was a Wessler thrombosis model [12Wessler S. Thrombosis in the presence of vascular stasis.Am J Med. 1962; 33: 648Abstract Full Text PDF PubMed Google Scholar] adapted to rats [13Freund M. Cazenave J.P. Courtney M. Degryse E. Roitsch C. Bernat A. Delebassee D. Defreyn G. Maffrand J.P. Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats.Thromb Haemost. 1990; 63: 187-92Crossref PubMed Scopus (0) Google Scholar]. Male Wistar rats weighing 250–300 g were anesthetized by injection of a mixture (1 : 4 v/v) of 2% xylazine and 10% ketamine (1 mL kg−1 of body weight). A midline abdominal incision was made and the inferior vena cava exposed, two loose ligatures 10-mm apart were placed on it and all collateral veins were ligated. MRS2179 (50 mg kg−1) or vehicle (physiological saline) was then injected into the jugular vein. Tissue thromboplastin (Thromborel® S) was reconstituted according to the manufacturer's instructions and diluted in saline solution and all experiments were carried out using the same batch of thromboplastin to eliminate product variations. Dilute thromboplastin solution (2 mL kg−1 of body weight) was injected for 30 s into the jugular vein. Two doses of thromboplastin were used, corresponding to administration of 40 or 80 µL of pure thromboplastin per kilogram of rat body weight. Ten seconds after completion of the injection, stasis was established by tightening first the proximal and then the distal ligature on the inferior vena cava and 10 min later, the resultant thrombus was removed by excision of the ligated segment. The thrombus was rinsed in physiological saline, fixed overnight in 2.5% glutaraldehyde and weighed. A Student's two-tailed unpaired t-test or anova was used to compare the means of the thrombosis scores at each time point [18Bolton S. Pharmaceutical Statistics-Practical and Clinical Applications. Drugs and the Pharmaceutical Science. Marcel Dekker, 1984Google Scholar], results being calculated as the mean ± SEM for each group of animals. The times to formation of the first thrombus were compared by means of a Student's one-tailed unpaired t-test. In the case of animals presenting no thrombus formation during the 10 min of observation, the time to formation of the first thrombus was considered to be 601 s (10 min and 1 s). Thrombi weights were also analyzed with a Student's one-tailed unpaired t-test and all tests were performed using Prism™ software. P2Y1-deficient mice and control mice shared a mixed 129/Sv X C57BL/6 genetic background. The thrombosis score in control mice was 3 ± 0.13 (mean ± SEM) at time point 2 min and 3.1 ± 0.16 at time points 5 and 10 min (n = 11). The evolution of thrombosis was therefore very rapid, as the situation did not deteriorate between the two latter time points where the mean scores were identical. Complete occlusion of the vessel did not occur in all cases. Deficiency of the P2Y1 receptor resulted in a significant decrease in thrombosis at all time points (Fig. 1a). Thus, P2Y1-deficient mice displayed thrombosis scores of 2.3 ± 0.22 at 2 min (P = 0.02) and of 2.5 ± 0.19 at 5 and 10 min (P = 0.03) after vessel wall injury (n = 12). The time to formation of the first thrombus of diameter >30 µm (Fig. 1b) was also significantly increased in P2Y1 knockout mice (291.7 ± 79.32 s) as compared to control mice (100.1 ± 50.45 s; P = 0.03). Hence the P2Y1 receptor seemed to play a role in localized arterial thrombosis. C57BL/6 mice were injected intravenously with MRS2179 (50 mg kg−1), a selective P2Y1 antagonist, or with physiological saline immediately before superfusion of FeCl3. Administration of this P2Y1 antagonist resulted in a significant decrease in thrombus formation as compared to control mice. Thrombosis scores were 2.7 ± 0.14 at 2 min, 3 ± 0.23 at 5 min and 3.27 ± 0.24 at 10 min in controls (n = 12), but decreased to 2 ± 0.21 at 2 min (P = 0.01), 2.33 ± 0.19 at 5 min (P = 0.03) and 2.42 ± 0.19 at 10 min (P = 0.01) in animals treated with MRS2179 (n = 11) (Fig. 2a). The time to formation of the first thrombus was significantly increased in animals receiving MRS2179 (446.9 ± 69.52 s) as compared to controls (194.7 ± 75.12 s, P= 0.01) (Fig. 2b). It has to be noticed that the thrombus formation time was also greater in control C57BL/6 mice (194.7 ± 75.12 s) than in control 129/Sv X C57BL/6 mice (100.1 ± 50.45 s). This was due to their different genetic background. The genetic background is known to play a role in platelet functions and thrombin generation [19Zumbach A. Marbet G.A. Tsakiris D.A. Influence of the genetic background on platelet function, microparticle and thrombin generation in the common laboratory mouse.Platelets. 2001; 12: 496-502Crossref PubMed Scopus (13) Google Scholar], and indeed we measured significant differences between the genotypes (data not shown). These data confirm the involvement of the P2Y1 receptor in localized arterial thrombosis and suggest that P2Y1 antagonists might be effective as antiplatelet drugs. Clopidogrel is to date the antiplatelet drug of reference among inhibitors of ADP-induced platelet activation. It was therefore of interest to determine whether our model was sensitive to clopidogrel and if so to compare the effect of this drug to that of the P2Y1 antagonist MRS2179. C57BL/6 mice were treated orally with 50 mg kg−1 clopidogrel, a dose producing maximal inhibition of ADP-induced platelet aggregation [17Savi P. Herbert J.M. ADP receptors on platelets and ADP-selective antiaggregating agents.Med Res Rev. 1996; 16: 159-79Crossref PubMed Scopus (0) Google Scholar], or with vehicle (5% gum arabic). Thrombosis scores were significantly reduced in mice treated with clopidogrel as compared to those receiving vehicle at all time points (Fig. 3a). Thus, control mice (n = 11) displayed thrombosis scores of 2.5 ± 0.34 at 2 min and 2.8 ± 0.26 at 5 min, whereas treated mice (n = 17) had scores of only 1.6 ± 0.23 at 2 min (P = 0.02) and 1.7 ± 0.22 at 5 min (P < 0.01) and thrombosis was still significantly less in these animals (P < 0.01) 10 min after vessel wall injury. The time to formation of the first thrombus of diameter > 30 µm was also significantly increased in animals treated with clopidogrel (437 ± 61.17 s) as compared to controls (222.5 ± 74.3 s, P= 0.02) (Fig. 3b). Hence this model was responsive to the P2Y12 inhibitor clopidogrel and the inhibition of arterial thrombosis was of the same order as that observed with the P2Y1 antagonist MRS2179. Since P2Y1 deficiency as well as P2Y12 inhibition by clopidogrel led to a decreased localized arterial thrombosis, we wondered if P2Y1 receptor deficiency associated with P2Y12 receptor inhibition would evidence potential additive effects. Thus clopidogrel (50 mg kg−1) was administered to P2Y1-deficient mice. A combination of both P2Y1 deficiency and P2Y12 inhibition led to an additive decrease in thrombosis (Fig. 4). Indeed, for each time point, the thrombosis score was significantly reduced in P2Y1-deficient mice treated with clopidogrel (n = 10) as compared with P2Y1 knockout mice (n = 12) or 129 X C57BL/6 mice treated with clopidogrel only (n = 9) (P < 0.05 at 2 min, P < 0.01 at 5 and 10 min). Again, it could be noted that thrombosis scores of clopidogrel treated mice are greater in the mixed genetic background mice (Fig. 3) than in C57BL/6 mice (Fig. 4). Mice deficient in the P2Y1 receptor or treated with the P2Y1 antagonist MRS2179 are resistant to the systemic thromboembolism induced by an injection of tissue factor [9Léon C. Freund M. Ravanat C. Baurand A. Cazenave J.P. Gachet C. Key role of the P2Y(1) receptor in tissue factor-induced thrombin- dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.Circulation. 2001; 103: 718-23Crossref PubMed Google Scholar], which triggers thrombin formation. Thus it was of interest to test P2Y1 receptor inhibition in a model of localized venous thrombosis, where thrombus formation is more dependent than in arteries on thrombin generation and plasma coagulation. In rats injected with thromboplastin (40 µL kg−1 of body weight), administration of MRS2179 (50 mg kg−1) resulted in a significant decrease in thrombus weight (9.24 mg ± 1.1, n= 15) as compared to control rats receiving physiological saline (14.85 mg ± 1.4, n= 13, P < 0.01) (Fig. 5). When the dose of thromboplastin was doubled (80 µL kg−1 of body weight), the thrombus weights did not differ, significantly (15.18 mg ± 2.4, n= 6 vs. 14.4 mg ± 2.40, n= 6, P > 0.05). These results provide further evidence that the P2Y1 receptor could play a role in venous thrombosis. Since recent studies have demonstrated a role of the P2Y1 receptor in the systemic thrombosis induced in mice by intravenous injection of tissue factor or a mixture of collagen and epinephrine [7Léon C. Hechler B. Freund M. Eckly A. Vial C. Ohlmann P. Dierich A. LeMeur M. Cazenave J.P. Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.J Clin Invest. 1999; 104: 1731-7Crossref PubMed Google Scholar, 8Fabre J.E. Nguyen M. Latour A. Keifer J.A. Audoly L.P. Coffman T.M. Koller B.H. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice.Nat Med. 1999; 5: 1199-202Crossref PubMed Scopus (382) Google Scholar, 9Léon C. Freund M. Ravanat C. Baurand A. Cazenave J.P. Gachet C. Key role of the P2Y(1) receptor in tissue factor-induced thrombin- dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.Circulation. 2001; 103: 718-23Crossref PubMed Google Scholar], we decided to investigate whether this receptor was involved in localized arterial and venous thrombosis. Mice deficient in the P2Y1 receptor presented significantly less arterial thrombosis, supporting the concept that this receptor plays a major part in the development of arterial thrombosis. The effects observed in C57BL/6 mice after administration of MRS2179, the selective antagonist of the P2Y1 receptor, were of similar order, the thrombosis scores being significantly reduced at all time points in both cases. On the other hand, clopidogrel, so far the ‘standard’ antiplatelet drug which inhibits ADP-induced platelet aggregation through inhibition of the P2Y12 receptor, also induced inhibition of thrombosis in this localized arterial model. Interestingly, clopidogrel-induced inhibition of thrombosis was of similar order of magnitude as compared to MRS2179-induced inhibition. Thus, inhibition of the P2Y1 receptor might be as effective as inhibition of the P2Y12 receptor. These results thus confirm the P2Y1 receptor as a potential target for new antithrombotic compounds. Furthermore, combination of P2Y1 deficiency with P2Y12 inhibition lead to an additive effect since no thrombus formation was evidenced under these conditions for all the duration of the experiment. Arterial thrombosis is particularly dependent on platelet activation, whereas venous thrombosis depends mainly on thrombin generation. However, platelet activation has recently been shown to play a crucial role in thrombin generation during blood coagulation induced by tissue factor in humans [20Butenas S. Cawthern K.M. Van't Veer C. DiLorenzo M.E. Lock J.B. Mann K.G. Antiplatelet agents in tissue factor-induced blood coagulation.Blood. 2001; 97: 2314-22Crossref PubMed Scopus (87) Google Scholar]. Our previous studies have demonstrated that mice with P2Y1 deficiency or treated with the selective P2Y1 antagonist MRS2179 are resistant to the vascular thromboembolism triggered by injection of tissue factor, which results from intravascular thrombin formation [9Léon C. Freund M. Ravanat C. Baurand A. Cazenave J.P. Gachet C. Key role of the P2Y(1) receptor in tissue factor-induced thrombin- dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.Circulation. 2001; 103: 718-23Crossref PubMed Google Scholar]. The effects of P2Y1 inhibition were further evaluated here in a model of localized venous thrombosis, using a Wessler procedure [12Wessler S. Thrombosis in the presence of vascular stasis.Am J Med. 1962; 33: 648Abstract Full Text PDF PubMed Google Scholar] adapted to rats [13Freund M. Cazenave J.P. Courtney M. Degryse E. Roitsch C. Bernat A. Delebassee D. Defreyn G. Maffrand J.P. Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats.Thromb Haemost. 1990; 63: 187-92Crossref PubMed Scopus (0) Google Scholar] and animals treated with MRS2179. Inhibition of the P2Y1 receptor led to a significant decrease in the weight of thrombi formed in response to the lower dose of thromboplastin injected and these effects of MRS2179 were comparable to those reported by Herbert et al. for clopidogrel in the same model [1Herbert J.M. Bernat A. Maffrand J.P. Importance of platelets in experimental venous thrombosis in the rat.Blood. 1992; 80: 2281-6Crossref PubMed Google Scholar]. MRS2179 itself had no anticoagulant activity as could be verified by measuring the prothrombin time in rat plasma in vitro (data not shown). Its impact on venous thrombosis might therefore be due to inhibition of P2Y1-mediated platelet activation. One cannot however, rule out a contribution from inhibition by MRS2179 of the P2Y1 receptor on other cells, since this receptor is expressed on endothelial cells and leukocytes, which are known to be involved in tissue factor expression [21Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Nucleotide receptors: an emerging family of regulatory molecules in blood cells.Blood. 2001; 97: 587-600Crossref PubMed Scopus (620) Google Scholar]. Previous work in mice had also demonstrated that blockade of the P2Y1 receptor with MRS2179 lead to a significant decrease in thrombin–antithrombin (TAT) complex formation when thromboembolism was induced by high doses of thromboplastin [9Léon C. Freund M. Ravanat C. Baurand A. Cazenave J.P. Gachet C. Key role of the P2Y(1) receptor in tissue factor-induced thrombin- dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.Circulation. 2001; 103: 718-23Crossref PubMed Google Scholar]. Further studies will be required to assess the respective roles of the various cell types in thrombin generation in vivo. ADP-induced platelet aggregation has been found to contribute to thrombin generation in plasma and Hérault et al. recently reported decreased thrombin formation in the platelet rich plasma of rats treated with clopidogrel [22Herault J.P. Dol F. Gaich C. Bernat A. Herbert J.M. Effect of clopidogrel on thrombin generation in platelet-rich plasma in the rat.Thromb Haemost. 1999; 81: 957-60Crossref PubMed Scopus (62) Google Scholar]. The involvement of the P2Y12 receptor in the procoagulant role of platelets has already been reported [23Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.Br J Haematol. 2000; 110: 925-34Crossref PubMed Scopus (265) Google Scholar, 24Léon C, Ravanat C, Cazenave JP, Gachet C. Additive effect of clopidogrel in P2Y1-knockout mice in collagen- and thromboplastin-induced models of thromboembolism. CD Rom Abstracts XVIII Congress of the ISTH. Paris, France, 612 July 2001.Google Scholar]. Recent studies showed that P2Y1 invalidation and selective P2Y12 inhibition by clopidogrel were equally effective in thromboplastin-induced thromboembolism experiments ([24Léon C, Ravanat C, Cazenave JP, Gachet C. Additive effect of clopidogrel in P2Y1-knockout mice in collagen- and thromboplastin-induced models of thromboembolism. CD Rom Abstracts XVIII Congress of the ISTH. Paris, France, 612 July 2001.Google Scholar], unpublished data). Furthermore, protection against thromboembolism was potentiated by combination of P2Y1 invalidation and P2Y12 inhibition, thus providing further evidences of additive effects. It remains, however, to be assessed whether the P2Y1 and P2Y12 receptors play equivalent roles in thrombin generation. In conclusion, this study provides further convincing evidence in support of the P2Y1 receptor as a potential target for new antithrombotic drugs. P2Y1 inhibition results in a resistance to localized arterial and venous thrombosis which in the models employed is comparable to that conferred by P2Y12 inhibition, and combination of both P2Y1 deficiency and P2Y12 inhibition lead to additive effects. Whether such association would be beneficial remains to be assessed and the bleeding risk should be evaluated. This work was supported by ARMESA (Association de Recherche et Développement en Médecine et Santé Publique), INSERM (Institut National de la Santè et de la Recherche Médicale) and EFS-Alsace (Etablissement Français du Sang – Alsace). The authors would like to thank C. Schwartz, D. Cassel and J. Dambach for expert technical assistance and J.N. Mulvihill for reviewing the English of the manuscript." @default.
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