The P2Y12 Antagonists, 2-Methylthioadenosine 5′-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors. ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors. Upon damage to the endothelial layer of the blood vessel wall, the underlying subendothelium is exposed to platelets in the blood, initiating a cascade of signaling events resulting in the transformation of “resting” platelets into “activated” platelets (1Coller B.S. N. Engl. J. Med. 1990; 322: 33-42Crossref PubMed Scopus (441) Google Scholar). One significant characteristic associated with these signaling events is the secretion of ADP from the platelet-dense granules (2Charo I.F. Feinman R.D. Detwiler T.C. J. Clin. Invest. 1977; 60: 866-873Crossref PubMed Scopus (238) Google Scholar). This released ADP acts to further amplify the platelet activation response by interacting with its G-protein-coupled receptors on the platelet surface, namely P2Y1 (coupled to Gq) and P2Y12 (coupled to Gi) (3Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 4Storey R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 5Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma Jr., F.J. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Crossref PubMed Scopus (378) Google Scholar). The consequence of platelet activation through ADP is a conformational change in the platelet membrane glycoprotein αIIbβ3 (6Nieswandt B. Schulte V. Zywietz A. Gratacap M.P. Offermanns S. J. Biol. Chem. 2002; 277: 39493-39498Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 7Jin J. Quinton T.M. Zhang J. Rittenhouse S.E. Kunapuli S.P. Blood. 2002; 99: 193-198Crossref PubMed Scopus (141) Google Scholar), which then binds to fibrinogen present in the plasma. The binding of fibrinogen with αIIbβ3 on the surface of adjacent platelets results in fibrinogen-platelet cross-linking and the formation of a hemostatic plug at the site of vascular injury (8Brass L.F. Manning D.R. Cichowski K. Abrams C.S. Thromb. Haemost. 1997; 78: 581-589Crossref PubMed Scopus (165) Google Scholar).Consequently, ADP is thought to play an integral role in the normal process of hemostasis. Of the two ADP-receptor signaling pathways in platelets, evidence has indicated that ADP-mediated P2Y12 signaling appears to play a more prominent role in platelet activation than ADP-mediated P2Y1 signaling (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar). For the most part, support for this notion derives from the use of the adenosine-based P2Y12 antagonists (i.e. 2MeSAMP 4The abbreviations used are: 2MeSAMP2-methylthioadenosine 5′-monophosphate triethylammonium saltPTXpertussis toxinPRPplatelet-rich abbreviations used are: 2MeSAMP2-methylthioadenosine 5′-monophosphate triethylammonium saltPTXpertussis toxinPRPplatelet-rich and ARC69931MX), which have a than P2Y1 antagonists or (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar). 2MeSAMP and ARC69931MX inhibit platelet in response to as R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar, R.T. Kunapuli S.P. J. Clin. Invest. PubMed Scopus Google Scholar, S. C. A. Quinton T.M. Prosser D.M. Jin J. Kunapuli S.P. Blood. 2002; 99: PubMed Scopus Google Scholar), P2Y1 antagonists the this for P2Y12 signaling to with that activation of platelet receptors through mechanisms PubMed Scopus Google Scholar, Google Scholar). Based on this in the of P2Y12 signaling to the platelet activation the present the that the of of this new of P2Y12 antagonists (i.e. and from an elevation in platelet cAMP data demonstrated that both 2MeSAMP and ARC69931MX in raise human platelet Furthermore, this is of signaling and appears to through activation of a separate platelet the results indicate that these adenosine-based P2Y12 antagonists inhibition of platelet through a cAMP-mediated is known to play an role in platelet activation by by platelet its secretion from the granules (3Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 4Storey R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 5Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma Jr., F.J. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Crossref PubMed Scopus (378) Google Scholar). It is that platelet in response to is on the secretion of ADP R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 7Jin J. Quinton T.M. Zhang J. Rittenhouse S.E. Kunapuli S.P. Blood. 2002; 99: 193-198Crossref PubMed Scopus (141) Google and the activation of receptors (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar, R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar). this are two of P2Y12 the and ticlopidine) and the adenosine-based (i.e. 2MeSAMP and the have been demonstrated to thromboembolic require in to and is a significant in of C. C. C. Thromb. Biol. 2003; PubMed Scopus Google Scholar). this the of P2Y12 namely the adenosine-based has been One of these is for its as an as as has been to and P2Y12 signaling pathways in platelets and the use of ARC69931MX and 2MeSAMP has to the that signaling is a activation and human platelet both in and in (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar, R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar, R.T. Kunapuli S.P. J. Clin. Invest. PubMed Scopus Google Scholar, C. C. C. Thromb. Biol. 2003; PubMed Scopus Google Scholar, C. B. B. C. C. Blood. PubMed Google Scholar, J. 2001; PubMed Scopus Google Scholar, K. Y. K. S. Br. J. PubMed Scopus Google Scholar). However, this notion of a on P2Y12 signaling to at with which demonstrated that activation of platelet receptors activation through pathways PubMed Scopus Google Scholar, Google Scholar). For was PubMed Scopus Google that platelets in ADP in response to in the of which on these the present experiments examined the underlying mechanisms by which 2MeSAMP and ARC69931MX inhibition of human platelet function. It was found that the inhibition of platelet by 2MeSAMP and ARC69931MX was to to platelet cAMP that cAMP play an role in the of these This notion was by the Specifically, was found that not the cAMP elevation by 2MeSAMP and ARC69931MX but to inhibit platelet by two separate (i.e. and the of 2MeSAMP and ARC69931MX to inhibit platelet in response to has as the for the notion that signaling is for platelet activation. The present results indicate that the of these compounds to inhibit platelet by than ADP derives from to platelet cAMP signaling by which 2MeSAMP and ARC69931MX platelet cAMP was The examined was inhibition of Gi this has been that the of ADP to platelet derives in from its to cAMP This is on the that ADP-mediated Gi signaling cAMP levels that have been by activation Y. Google Scholar). the has not been to that ADP is of cAMP levels in platelets, and the of Gi signaling to platelet cAMP levels and its in the initiation of platelet are in for 2MeSAMP and ARC69931MX to raise platelet cAMP levels through inhibition of the pathway, have to that cAMP levels are in the to to this inhibition of signaling with L.F. Rittenhouse S.E. J. Biol. Chem. Full Text PDF PubMed Google was It was found that although treatment of platelet with in of did not in in cAMP nor did or in cAMP that the of 2MeSAMP and ARC69931MX to cAMP levels to inhibition of the is that P2Y12 receptors couple to a separate signaling this experiments in human which P2Y12 receptors. both 2MeSAMP and ARC69931MX substantial in cAMP levels in this to further the notion that these compounds raise cAMP through a human platelets It was found that although these platelets functional P2Y12 receptor 2MeSAMP and ARC69931MX increased of these findings that 2MeSAMP and ARC69931MX a cAMP-mediated that is both and at the molecular by which a signaling activation of is known to of examined the cAMP elevation by 2MeSAMP and ARC69931MX is mediated through a G-protein-coupled It was found that a of that signaling did in the cAMP by 2MeSAMP and these results indicate that both 2MeSAMP and ARC69931MX platelet cAMP levels through with receptors that are presumably to this platelet receptors are known to through and inhibit platelet by cAMP K. S. E. 99: PubMed Scopus Google Scholar, S. S. C. C. E. J. Google Scholar). Furthermore, is known that platelets receptors, namely IP, DP, and to the of 2MeSAMP and ARC69931MX with these receptor It was that antagonists S. S. C. C. E. J. Google Scholar, N. A. A. 1997; 277: PubMed Scopus Google Scholar, M.L. Br. J. PubMed Scopus Google Scholar, A. A. A. PubMed Scopus Google Scholar, Br. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. C. PubMed Scopus Google against of these receptor on the of 2MeSAMP or that the unidentified receptor is not A2a, IP, DP, or the present results an by which and ARC69931MX inhibition of human platelet function. this derives from activation of a G protein-coupled signaling that not platelet P2Y12 receptors. appears that 2MeSAMP and ARC69931MX through two that although 2MeSAMP and ARC69931MX have the to this pathway, a of not to clopidogrel or a class of Upon damage to the endothelial layer of the blood vessel wall, the underlying subendothelium is exposed to platelets in the blood, initiating a cascade of signaling events resulting in the transformation of “resting” platelets into “activated” platelets (1Coller B.S. N. Engl. J. Med. 1990; 322: 33-42Crossref PubMed Scopus (441) Google Scholar). One significant characteristic associated with these signaling events is the secretion of ADP from the platelet-dense granules (2Charo I.F. Feinman R.D. Detwiler T.C. J. Clin. Invest. 1977; 60: 866-873Crossref PubMed Scopus (238) Google Scholar). This released ADP acts to further amplify the platelet activation response by interacting with its G-protein-coupled receptors on the platelet surface, namely P2Y1 (coupled to Gq) and P2Y12 (coupled to Gi) (3Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 4Storey R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 5Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma Jr., F.J. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Crossref PubMed Scopus (378) Google Scholar). The consequence of platelet activation through ADP is a conformational change in the platelet membrane glycoprotein αIIbβ3 (6Nieswandt B. Schulte V. Zywietz A. Gratacap M.P. Offermanns S. J. Biol. Chem. 2002; 277: 39493-39498Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 7Jin J. Quinton T.M. Zhang J. Rittenhouse S.E. Kunapuli S.P. Blood. 2002; 99: 193-198Crossref PubMed Scopus (141) Google Scholar), which then binds to fibrinogen present in the plasma. The binding of fibrinogen with αIIbβ3 on the surface of adjacent platelets results in fibrinogen-platelet cross-linking and the formation of a hemostatic plug at the site of vascular injury (8Brass L.F. Manning D.R. Cichowski K. Abrams C.S. Thromb. Haemost. 1997; 78: 581-589Crossref PubMed Scopus (165) Google Scholar). ADP is thought to play an integral role in the normal process of hemostasis. Of the two ADP-receptor signaling pathways in platelets, evidence has indicated that ADP-mediated P2Y12 signaling appears to play a more prominent role in platelet activation than ADP-mediated P2Y1 signaling (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar). For the most part, support for this notion derives from the use of the adenosine-based P2Y12 antagonists (i.e. 2MeSAMP 4The abbreviations used are: 2MeSAMP2-methylthioadenosine 5′-monophosphate triethylammonium saltPTXpertussis toxinPRPplatelet-rich abbreviations used are: 2MeSAMP2-methylthioadenosine 5′-monophosphate triethylammonium saltPTXpertussis toxinPRPplatelet-rich and ARC69931MX), which have a than P2Y1 antagonists or (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar). 2MeSAMP and ARC69931MX inhibit platelet in response to as R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar, R.T. Kunapuli S.P. J. Clin. Invest. PubMed Scopus Google Scholar, S. C. A. Quinton T.M. Prosser D.M. Jin J. Kunapuli S.P. Blood. 2002; 99: PubMed Scopus Google Scholar), P2Y1 antagonists the this for P2Y12 signaling to with that activation of platelet receptors through mechanisms PubMed Scopus Google Scholar, Google Scholar). Based on this in the of P2Y12 signaling to the platelet activation the present the that the of of this new of P2Y12 antagonists (i.e. and from an elevation in platelet cAMP 5′-monophosphate triethylammonium salt 5′-monophosphate triethylammonium salt data demonstrated that both 2MeSAMP and ARC69931MX in raise human platelet Furthermore, this is of signaling and appears to through activation of a separate platelet the results indicate that these adenosine-based P2Y12 antagonists inhibition of platelet through a cAMP-mediated is known to play an role in platelet activation by by platelet its secretion from the granules (3Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 4Storey R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 5Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma Jr., F.J. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Crossref PubMed Scopus (378) Google Scholar). It is that platelet in response to is on the secretion of ADP R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 7Jin J. Quinton T.M. Zhang J. Rittenhouse S.E. Kunapuli S.P. Blood. 2002; 99: 193-198Crossref PubMed Scopus (141) Google and the activation of receptors (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar, R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar). this are two of P2Y12 the and ticlopidine) and the adenosine-based (i.e. 2MeSAMP and the have been demonstrated to thromboembolic require in to and is a significant in of C. C. C. Thromb. Biol. 2003; PubMed Scopus Google Scholar). this the of P2Y12 namely the adenosine-based has been One of these is for its as an as as has been to and P2Y12 signaling pathways in platelets and the use of ARC69931MX and 2MeSAMP has to the that signaling is a activation and human platelet both in and in (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar, R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar, R.T. Kunapuli S.P. J. Clin. Invest. PubMed Scopus Google Scholar, C. C. C. Thromb. Biol. 2003; PubMed Scopus Google Scholar, C. B. B. C. C. Blood. PubMed Google Scholar, J. 2001; PubMed Scopus Google Scholar, K. Y. K. S. Br. J. PubMed Scopus Google Scholar). However, this notion of a on P2Y12 signaling to at with which demonstrated that activation of platelet receptors activation through pathways PubMed Scopus Google Scholar, Google Scholar). For was PubMed Scopus Google that platelets in ADP in response to in the of which on these the present experiments examined the underlying mechanisms by which 2MeSAMP and ARC69931MX inhibition of human platelet function. It was found that the inhibition of platelet by 2MeSAMP and ARC69931MX was to to platelet cAMP that cAMP play an role in the of these This notion was by the Specifically, was found that not the cAMP elevation by 2MeSAMP and ARC69931MX but to inhibit platelet by two separate (i.e. and the of 2MeSAMP and ARC69931MX to inhibit platelet in response to has as the for the notion that signaling is for platelet activation. The present results indicate that the of these compounds to inhibit platelet by than ADP derives from to platelet cAMP signaling by which 2MeSAMP and ARC69931MX platelet cAMP was The examined was inhibition of Gi this has been that the of ADP to platelet derives in from its to cAMP This is on the that ADP-mediated Gi signaling cAMP levels that have been by activation Y. Google Scholar). the has not been to that ADP is of cAMP levels in platelets, and the of Gi signaling to platelet cAMP levels and its in the initiation of platelet are in for 2MeSAMP and ARC69931MX to raise platelet cAMP levels through inhibition of the pathway, have to that cAMP levels are in the to to this inhibition of signaling with L.F. Rittenhouse S.E. J. Biol. Chem. Full Text PDF PubMed Google was It was found that although treatment of platelet with in of did not in in cAMP nor did or in cAMP that the of 2MeSAMP and ARC69931MX to cAMP levels to inhibition of the is that P2Y12 receptors couple to a separate signaling this experiments in human which P2Y12 receptors. both 2MeSAMP and ARC69931MX substantial in cAMP levels in this to further the notion that these compounds raise cAMP through a human platelets It was found that although these platelets functional P2Y12 receptor 2MeSAMP and ARC69931MX increased of these findings that 2MeSAMP and ARC69931MX a cAMP-mediated that is both and at the molecular by which a signaling activation of is known to of examined the cAMP elevation by 2MeSAMP and ARC69931MX is mediated through a G-protein-coupled It was found that a of that signaling did in the cAMP by 2MeSAMP and these results indicate that both 2MeSAMP and ARC69931MX platelet cAMP levels through with receptors that are presumably to this platelet receptors are known to through and inhibit platelet by cAMP K. S. E. 99: PubMed Scopus Google Scholar, S. S. C. C. E. J. Google Scholar). Furthermore, is known that platelets receptors, namely IP, DP, and to the of 2MeSAMP and ARC69931MX with these receptor It was that antagonists S. S. C. C. E. J. Google Scholar, N. A. A. 1997; 277: PubMed Scopus Google Scholar, M.L. Br. J. PubMed Scopus Google Scholar, A. A. A. PubMed Scopus Google Scholar, Br. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. C. PubMed Scopus Google against of these receptor on the of 2MeSAMP or that the unidentified receptor is not A2a, IP, DP, or the present results an by which and ARC69931MX inhibition of human platelet function. this derives from activation of a G protein-coupled signaling that not platelet P2Y12 receptors. appears that 2MeSAMP and ARC69931MX through two that although 2MeSAMP and ARC69931MX have the to this pathway, a of not to clopidogrel or a class of ADP is known to play an role in platelet activation by by platelet its secretion from the granules (3Daniel J.L. Dangelmaier C. Jin J. Ashby B. Smith J.B. Kunapuli S.P. J. Biol. Chem. 1998; 273: 2024-2029Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 4Storey R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 5Foster C.J. Prosser D.M. Agans J.M. Zhai Y. Smith M.D. Lachowicz J.E. Zhang F.L. Gustafson E. Monsma Jr., F.J. Wiekowski M.T. Abbondanzo S.J. Cook D.N. Bayne M.L. Lira S.A. Chintala M.S. J. Clin. Invest. 2001; 107: 1591-1598Crossref PubMed Scopus (378) Google Scholar). It is that platelet in response to is on the secretion of ADP R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: 197-209Crossref PubMed Scopus (114) Google Scholar, 7Jin J. Quinton T.M. Zhang J. Rittenhouse S.E. Kunapuli S.P. Blood. 2002; 99: 193-198Crossref PubMed Scopus (141) Google and the activation of receptors (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar, R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar). this are two of P2Y12 the and ticlopidine) and the adenosine-based (i.e. 2MeSAMP and the have been demonstrated to thromboembolic require in to and is a significant in of C. C. C. Thromb. Biol. 2003; PubMed Scopus Google Scholar). this the of P2Y12 namely the adenosine-based has been One of these is for its as an as as has been to and P2Y12 signaling pathways in platelets and the use of ARC69931MX and 2MeSAMP has to the that signaling is a activation and human platelet both in and in (9Kunapuli S.P. Ding Z. Dorsam R.T. Kim S. Murugappan S. Quinton T.M. Curr. Pharm. Des. 2003; 9: 2303-2316Crossref PubMed Scopus (88) Google Scholar, 10Storey R.F. Sanderson H.M. White A.E. May J.A. Cameron K.E. Heptinstall S. Br. J. Haematol. 2000; 110: 925-934Crossref PubMed Scopus (262) Google Scholar, R.F. Newby L.J. Heptinstall S. Platelets. 2001; 12: PubMed Scopus Google Scholar, R.T. Kunapuli S.P. J. Clin. Invest. PubMed Scopus Google Scholar, C. C. C. Thromb. Biol. 2003; PubMed Scopus Google Scholar, C. B. B. C. C. Blood. PubMed Google Scholar, J. 2001; PubMed Scopus Google Scholar, K. Y. K. S. Br. J. PubMed Scopus Google Scholar). However, this notion of a on P2Y12 signaling to at with which demonstrated that activation of platelet receptors activation through pathways PubMed Scopus Google Scholar, Google Scholar). For was PubMed Scopus Google that platelets in ADP in response to in the of which Based on these the present experiments examined the underlying mechanisms by which 2MeSAMP and ARC69931MX inhibition of human platelet function. It was found that the inhibition of platelet by 2MeSAMP and ARC69931MX was to to platelet cAMP that cAMP play an role in the of these This notion was by the Specifically, was found that not the cAMP elevation by 2MeSAMP and ARC69931MX but to inhibit platelet by two separate (i.e. and the of 2MeSAMP and ARC69931MX to inhibit platelet in response to has as the for the notion that signaling is for platelet activation. The present results indicate that the of these compounds to inhibit platelet by than ADP derives from to platelet cAMP The signaling by which 2MeSAMP and ARC69931MX platelet cAMP was The examined was inhibition of Gi this has been that the of ADP to platelet derives in from its to cAMP This is on the that ADP-mediated Gi signaling cAMP levels that have been by activation Y. Google Scholar). the has not been to that ADP is of cAMP levels in platelets, and the of Gi signaling to platelet cAMP levels and its in the initiation of platelet are in for 2MeSAMP and ARC69931MX to raise platelet cAMP levels through inhibition of the pathway, have to that cAMP levels are in the to to this inhibition of signaling with L.F. Rittenhouse S.E. J. Biol. Chem. Full Text PDF PubMed Google was It was found that although treatment of platelet with in of did not in in cAMP nor did or in cAMP that the of 2MeSAMP and ARC69931MX to cAMP levels to inhibition of the is that P2Y12 receptors couple to a separate signaling this experiments in human which P2Y12 receptors. both 2MeSAMP and ARC69931MX substantial in cAMP levels in this to further the notion that these compounds raise cAMP through a human platelets It was found that although these platelets functional P2Y12 receptor 2MeSAMP and ARC69931MX increased of these findings that 2MeSAMP and ARC69931MX a cAMP-mediated that is both and at the molecular by which a signaling activation of is known to of examined the cAMP elevation by 2MeSAMP and ARC69931MX is mediated through a G-protein-coupled It was found that a of that signaling did in the cAMP by 2MeSAMP and these results indicate that both 2MeSAMP and ARC69931MX platelet cAMP levels through with receptors that are presumably to Gs. this platelet receptors are known to through and inhibit platelet by cAMP K. S. E. 99: PubMed Scopus Google Scholar, S. S. C. C. E. J. Google Scholar). Furthermore, is known that platelets receptors, namely IP, DP, and to the of 2MeSAMP and ARC69931MX with these receptor It was that antagonists S. S. C. C. E. J. Google Scholar, N. A. A. 1997; 277: PubMed Scopus Google Scholar, M.L. Br. J. PubMed Scopus Google Scholar, A. A. A. PubMed Scopus Google Scholar, Br. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. C. PubMed Scopus Google against of these receptor on the of 2MeSAMP or that the unidentified receptor is not A2a, IP, DP, or the present results an by which and ARC69931MX inhibition of human platelet function. this derives from activation of a G protein-coupled signaling that not platelet P2Y12 receptors. appears that 2MeSAMP and ARC69931MX through two that although 2MeSAMP and ARC69931MX have the to this pathway, a of not to clopidogrel or a class of are to for ARC69931MX and for