title: Contributions of Protease-Activated Receptors PAR1 and PAR4 to Thrombin-Induced GPIIbIIIa Activation in Human Platelets.
abstract: Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.).
authors: Matthew T
authors: MT
authors: Duvernay
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.).
authors: Kayla J
authors: KJ
authors: Temple
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.).
authors: Jae G
authors: JG
authors: Maeng
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.).
authors: Anna L
authors: AL
authors: Blobaum
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.).
authors: Shaun R
authors: SR
authors: Stauffer
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.).
authors: Craig W
authors: CW
authors: Lindsley
authors: Department of Pharmacology (M.T.D., K.J.T., J.G.M., A.L.B., S.R.S., C.W.L., H.E.H.) and Vanderbilt Center for Neuroscience Drug Discovery (K.J.T., A.L.B., S.R.S., C.W.L.), Vanderbilt University Medical Center, Nashville, Tennessee; and Department of Chemistry, Vanderbilt University, Nashville, Tennessee (S.R.S., C.W.L.) heidi.hamm@vanderbilt.edu.
authors: Heidi E
authors: HE
authors: Hamm
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism*; D008024:Ligands; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors
chemical_list: D039661:Integrin beta3; D008024:Ligands; D019038:Platelet Glycoprotein GPIb-IX Complex; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C032193:glycoprotein receptor GPIb-IX; D013917:Thrombin; C112908:protease-activated receptor 4; D010975:Platelet Aggregation Inhibitors