title: Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets.
abstract: Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets-PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs.
authors: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors: Shauna L
authors: SL
authors: French
authors: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors: Antonia C
authors: AC
authors: Paramitha
authors: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors: Mitchell J
authors: MJ
authors: Moon
authors: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors: Ross A
authors: RA
authors: Dickins
authors: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors: Justin R
authors: JR
authors: Hamilton
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D018797:Cell Cycle Proteins; D020869:Gene Expression Profiling; D051379:Mice; D044462:Receptors, Proteinase-Activated / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; C115084:Pard3 protein, mouse; D044462:Receptors, Proteinase-Activated