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.
Highlights
Anti-platelet drugs are the primary therapy for heart attack and stroke prevention, yet improvements are continually sought
When taken together with earlier studies using distinct approaches, this study indicates that forced expression of PAR1 in mouse platelets will be difficult to achieve and suggests that other options will be required for reliable preclinical screening of any future protease-activated receptors (PARs) antagonists
The stability and surface expression of the predicted fusion protein was confirmed in HEK293T cells transiently transfected with vectors expressing the same sequence as that remaining after Cre-mediated excision of the targeted allele, with the predicted Par3/PAR1 fusion protein expressed at similar levels to native PAR1 (S1 Fig)
Summary
Anti-platelet drugs are the primary therapy for heart attack and stroke prevention, yet improvements are continually sought. Due to the importance of thrombin-induced platelet activation during thrombosis, targeting platelet thrombin receptors has received significant clinical attention, and PAR antagonists are one of the most promising of the emerging anti-thrombotic approaches [1,2,3]. Vorapaxar has limited clinical utility due to an increase in major bleeding events associated with its use [6, 7]. These limitations on the first PAR1 inhibitor have ignited interest in evaluating the clinical potential of PAR4 antagonists, and these agents are in early clinical development (NCT02208882) [8]
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