title: Targeting the GPIbα binding site of thrombin to simultaneously induce dual anticoagulant and antiplatelet effects.
abstract: Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis-Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin.
authors.affiliation: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , Richmond, Virginia 23219, United States.
authors.forename: Akul Y
authors.initials: AY
authors.lastname: Mehta
authors.forename: Jay N
authors.initials: JN
authors.lastname: Thakkar
authors.forename: Bassem M
authors.initials: BM
authors.lastname: Mohammed
authors.forename: Erika J
authors.initials: EJ
authors.lastname: Martin
authors.forename: Donald F
authors.initials: DF
authors.lastname: Brophy
authors.forename: Takao
authors.initials: T
authors.lastname: Kishimoto
authors.forename: Umesh R
authors.initials: UR
authors.lastname: Desai
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000925:Anticoagulants / Q000494:pharmacology*; D000991:Antithrombins / Q000494:pharmacology; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008031:Lignin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010974:Platelet Aggregation / Q000187:drug effects*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008031:Lignin; D011493:Protein Kinase C; D013917:Thrombin