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.

Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury

“…Furthermore, an evaluation of the origin of circulating MP based on the presence of unique markers derived from parental cells revealed that microglial MP displayed the greatest increase (approximately twofold) following TBI, accounting for nearly 15% of total circulating MP. Although P2Y12 is also expressed on platelets [ 40 ], the detected P2Y12-positive MP were co-stained with CD45, a unique myeloid cell marker that is not expressed on platelets [ 41 , 42 ], indicating that the P2Y12-positive MP enriched in the circulation following TBI were not derived from platelets. These data indicate that microglial-derived MP are released by the TBI brain and reach the systemic circulation.…”

Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia

“…33,34 Our findings are in contrast to endotoxemia models in PAR-1deficient mice, in which no effects on the LPS-induced inflammatory response or mortality were observed. 9 However, the profile of PAR expression in animals differs from humans, which may explain the differences in results, 10 but also the dose of endotoxin was several million-fold higher in the lethal mouse model (10 mg/kg vs. 2 ng/kg). The disagreeing results emphasize the importance of disease models performed in humans.…”

“…9 However, the expression of PAR sub-types on cells differs relevantly between species. 10 Vorapaxar, a reversible, orally active, low-molecular weight, competitive antagonist of PAR-1 binds to the receptor with high affinity, inhibits thrombin-induced PAR-1 activation without affecting other platelet receptors and stops further intracellular signalling events. 11 Vorapaxar reduced the risk of experiencing major adverse cardiovascular events, while it also increased the bleeding rate in two phase III trials that included patients with non-ST-elevation myocardial infarction (MI) or patients with a history of MI, stroke or peripheral arterial disease.…”

Platelets in Sepsis: An Update on Experimental Models and Clinical Data

“…To overcome this problem, mice expressing human receptors have been generated. While this was successful in some cases (FcγRIIA) (194), other attempts failed so far to lead to functional receptor expression (PAR-1) (195, 196). However, the role of FcγRIIA was never addressed in a murine sepsis model.…”