Role of ADP Receptor P2Y12 in Platelet Adhesion and Thrombus Formation in Flowing Blood

ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y1 and P2Y12. We have investigated the role of P2Y12 in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y12. Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient’s thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y12 antagonist, N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-&bgr;,&ggr;-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y1 antagonist (adenosine-3′,5′-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y12 interaction is essential for normal thrombus buildup on collagen. The patient’s blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3′,5′-diphospate. This suggested that P2Y12 and P2Y1 were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.

[1]  J. Sixma,et al.  Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment γ316–322, part of the fibrin‐specific region * , 2002, British journal of haematology.

[2]  J. Cazenave,et al.  Platelets in Thrombotic and Non-thrombotic Disorders: Pharmacology of ticlopidine and clopidogrel , 2002 .

[3]  B. Coller.  Anti-GPIIb/IIIa Drugs: Current Strategies and Future Directions , 2001, Thrombosis and Haemostasis.

[4]  C. Gachet.  ADP Receptors of Platelets and their Inhibition , 2001, Thrombosis and Haemostasis.

[5]  M. Moroi,et al.  Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change. , 2001, European journal of biochemistry.

[6]  C. Legrand,et al.  Platelets in Suspension Require Preactivation to Adhere to Immobilized Fibrinogen , 2001, Annals of the New York Academy of Sciences.

[7]  J. Sixma,et al.  Absence of Fibrinogen in Afibrinogenemia Results in Large but Loosely Packed Thrombi under Flow Conditions , 2001, Thrombosis and Haemostasis.

[8]  David Julius,et al.  Identification of the platelet ADP receptor targeted by antithrombotic drugs , 2001, Nature.

[9]  J. Herbert,et al.  Identification and Biological Activity of the Active Metabolite of Clopidogrel , 2000, Thrombosis and Haemostasis.

[10]  M. Faraci,et al.  High dose therapy and autologous hematopoietic stem cell transplantation in poor risk solid tumors of childhood. , 2000, Haematologica.

[11]  Michael L. Gonzales,et al.  Prevention of arterial thrombosis by intravenously administered platelet P2T receptor antagonist AR-C69931MX in a canine model. , 2000, The Journal of pharmacology and experimental therapeutics.

[12]  G. Mariani,et al.  Immune tolerance by intermittent factor VIII boluses in two high responder hemophilia A patients. , 2000, Haematologica.

[13]  J. Sixma,et al.  Platelet thrombus formation on collagen at high shear rates is mediated by von Willebrand factor-glycoprotein Ib interaction and inhibited by von Willebrand factor-glycoprotein IIb/IIIa interaction. , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[14]  J P Cazenave,et al.  Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice. , 1999, The Journal of clinical investigation.

[15]  Beverly H. Koller,et al.  Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice , 1999, Nature Medicine.

[16]  A. Bailey,et al.  Antagonists of the platelet P2T receptor: a novel approach to antithrombotic therapy. , 1999, Journal of medicinal chemistry.

[17]  J. Sixma,et al.  A new perfusion chamber to detect platelet adhesion using a small volume of blood. , 1998, Thrombosis research.

[18]  A. Kimura,et al.  Cloning and characterization of the gene encoding the murine glycoprotein V: the conserved thrombin-cleavable protein on platelet surface. , 1998, Thrombosis research.

[19]  G. Dubyak,et al.  The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes. , 1998, Blood.

[20]  J. Sixma,et al.  Role of glycoprotein IIb:IIIa in the adhesion of platelets to collagen under flow conditions. , 1997, Blood.

[21]  M. Humbert,et al.  Ultrastructural studies of platelet aggregates from human subjects receiving clopidogrel and from a patient with an inherited defect of an ADP-dependent pathway of platelet activation. , 1996, Arteriosclerosis, thrombosis, and vascular biology.

[22]  Brian Savage,et al.  Initiation of Platelet Adhesion by Arrest onto Fibrinogen or Translocation on von Willebrand Factor , 1996, Cell.

[23]  L. McIntire,et al.  Adhesion of platelets to surface-bound fibrinogen under flow. , 1996, Blood.

[24]  P. Nurden,et al.  An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function. , 1995, The Journal of clinical investigation.

[25]  K. Sakariassen,et al.  Axial Dependence of Collagen–Induced Thrombus Formation in Flowing Non-Anticoagulated Human Blood , 1995, Thrombosis and Haemostasis.

[26]  G. Kieffer,et al.  Clopidogrel – a Platelet Inhibitor which Inhibits Thrombogenesis in Non-Anticoagulated Human Blood Independently of the Blood Flow Conditions , 1994, Thrombosis and Haemostasis.

[27]  N. Kirschbaum,et al.  Characterization of adhesion of resting and stimulated platelets to fibrinogen and its fragments. , 1993, Annals of the New York Academy of Sciences.

[28]  Anika Niambi Al-Shura.  Leukocytes. , 1994, Current opinion in hematology.

[29]  P. D. de Groot,et al.  Platelet adhesion to fibrin(ogen). , 1993, Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.

[30]  P. Mannucci,et al.  Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. , 1992, Blood.

[31]  J. Hubbell,et al.  ADP Receptor Antagonists and Converting Enzyme Systems Reduce Platelet Deposition onto Collagen , 1992, Thrombosis and Haemostasis.

[32]  J. Moake,et al.  Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation. , 1992, Blood.

[33]  H. Weiss,et al.  Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules. , 1986, Blood.

[34]  M. Petitou,et al.  Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. , 1983, Biochemical and biophysical research communications.

[35]  B. Koch,et al.  Characterization of specific receptors for vasopressin in the pituitary gland. , 1983, Biochemical and biophysical research communications.

[36]  G. Born,et al.  Effect of ADP‐utilizing enzymes on the arterial bleeding time in rats and rabbits , 1982, British journal of haematology.

[37]  H. Baumgartner,et al.  Enzymatic Removal of ADP from Plasma: Unaltered Platelet Adhesion but Reduced Aggregation on Subendothelium and Collagen Fibrils , 1976, Thrombosis and Haemostasis.

[38]  H. Weiss,et al.  Hereditary Defect in the Platelet Release Reaction Caused by a Deficiency in the Storage Pool of Platelet Adenine Nucleotides , 1970, British journal of haematology.