title: Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.
abstract: OBJECTIVE
  Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use.
  
  
  RESULTS
  Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab.
  
  
  CONCLUSIONS
  Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Zubair A
authors.initials: ZA
authors.lastname: Karim
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Hari Priya
authors.initials: HP
authors.lastname: Vemana
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Fatima Z
authors.initials: FZ
authors.lastname: Alshbool
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Olivia A
authors.initials: OA
authors.lastname: Lin
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Abdullah M
authors.initials: AM
authors.lastname: Alshehri
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Payam
authors.initials: P
authors.lastname: Javaherizadeh
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA.
authors.forename: Enma V
authors.initials: EV
authors.lastname: Paez Espinosa
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA. fkhasawneh@westernu.edu.
authors.forename: Fadi T
authors.initials: FT
authors.lastname: Khasawneh
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D020212:Carotid Artery Injuries / Q000097:blood / Q000188:drug therapy; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D006470:Hemorrhage / Q000139:chemically induced; D006487:Hemostasis / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010718:Phosphatidylserines / Q000097:blood; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000378:metabolism; D058921:Purinergic P2Y Receptor Antagonists / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D058472:Receptors, Purinergic P2Y1 / Q000097:blood / Q000172:deficiency / Q000187:drug effects* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D013927:Thrombosis / Q000097:blood / Q000517:prevention & control; D013997:Time Factors
chemical_list: C495475:2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; D000906:Antibodies; D003838:Deoxyadenine Nucleotides; D000939:Epitopes; D005343:Fibrinolytic Agents; D008024:Ligands; C546382:P2RY1 protein, human; C546383:P2ry1 protein, mouse; D010718:Phosphatidylserines; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D058921:Purinergic P2Y Receptor Antagonists; D058472:Receptors, Purinergic P2Y1
keywords: P2Y1 receptor antagonists; antiplatelet agents; antithrombotic agents; blood platelets; platelet inhibitors