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.
Approach and 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.
Keywords:
P2Y1 receptor antagonists; antiplatelet agents; antithrombotic agents; blood platelets; platelet inhibitors.
© 2015 American Heart Association, Inc.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Antibodies / metabolism
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Antibodies / pharmacology*
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Antibodies / toxicity
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Binding Sites
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Binding, Competitive
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Blood Platelets / drug effects*
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Blood Platelets / metabolism
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Carotid Artery Injuries / blood
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Carotid Artery Injuries / drug therapy
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Deoxyadenine Nucleotides / metabolism
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Disease Models, Animal
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Dose-Response Relationship, Drug
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Epitopes
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Fibrinolytic Agents / metabolism
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Fibrinolytic Agents / pharmacology*
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Fibrinolytic Agents / toxicity
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Hemorrhage / chemically induced
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Hemostasis / drug effects
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Humans
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Ligands
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Mice, Inbred C57BL
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Mice, Knockout
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Phosphatidylserines / blood
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Platelet Activation / drug effects*
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Platelet Aggregation / drug effects
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Platelet Aggregation Inhibitors / metabolism
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Platelet Aggregation Inhibitors / pharmacology*
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Platelet Aggregation Inhibitors / toxicity
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Platelet Glycoprotein GPIIb-IIIa Complex / antagonists & inhibitors
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Platelet Glycoprotein GPIIb-IIIa Complex / metabolism
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Purinergic P2Y Receptor Antagonists / metabolism
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Purinergic P2Y Receptor Antagonists / pharmacology*
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Purinergic P2Y Receptor Antagonists / toxicity
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Receptors, Purinergic P2Y1 / blood
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Receptors, Purinergic P2Y1 / deficiency
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Receptors, Purinergic P2Y1 / drug effects*
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Receptors, Purinergic P2Y1 / genetics
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Receptors, Purinergic P2Y1 / immunology
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Receptors, Purinergic P2Y1 / metabolism
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Secretory Vesicles / drug effects
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Secretory Vesicles / metabolism
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Thrombosis / blood
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Thrombosis / prevention & control
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Time Factors
Substances
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2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate
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Antibodies
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Deoxyadenine Nucleotides
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Epitopes
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Fibrinolytic Agents
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Ligands
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P2RY1 protein, human
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P2ry1 protein, mouse
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Phosphatidylserines
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Platelet Aggregation Inhibitors
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Platelet Glycoprotein GPIIb-IIIa Complex
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Purinergic P2Y Receptor Antagonists
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Receptors, Purinergic P2Y1