Platelet-specific P2Y1 receptor deficient mice have suppressed leukocyte recruitment in response to lipopolysaccharide.

Pan, D.; Oralman, T.; Evans, R.; Baker, O.; Cocks, G.; Page, C. P.; Pitchford, S. C.

doi:10.1101/2024.11.04.621858

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bioRxiv · 10.1101/2024.11.04.621858

Platelet-specific P2Y1 receptor deficient mice have suppressed leukocyte recruitment in response to lipopolysaccharide.

Pan, D.·Oralman, T.·Evans, R.·Baker, O.·Cocks, G.·Page, C. P.·Pitchford, S. C.

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Abstract

A role for the P2Y1 receptor in inflammation has been established using a pharmacological approach over an acute 4 hour time span. However, nucleotide-structure P2Y1 receptor antagonists have limited experimental use due to inadequate pharmacokinetics, and inability to decipher global versus cell specific effects in vivo. The creation of a conditional knock out (platelet) P2Y1 transgenic mouse model was designed to overcome these restrictions. A homozygous P2Y1 LoxP mouse colony was created using CRISPR/Cas9 technology, and crossed with a hemizygous P2Y1 LoxP with PF4-cre to provide offspring that are homozygous for P2Y1 LoxP flanked allele, and hemizygous for the PF4cre (platelet P2Y1-/-) and offspring homozygous for P2Y1 LoxP flanked allele, but non-carriers for PF4cre (control mice). Animals were intranasally administered LPS to induce pulmonary inflammation to assess the influence of phenotype on leukocyte recruitment. 24 hours post intranasal LPS administration; pulmonary neutrophil and platelet recruitment were significantly suppressed, despite the fact that neutrophils retained the ability to migrate to fMLP ex vivo. Circulating platelet and leukocyte numbers were not different between control and platelet P2Y1-/- animals. Tail bleeding times revealed the platelet P2Y1-/- mice had a severe bleeding phenotype. This is the first demonstration of a platelet specific P2Y1-/- mouse model to confirm the importance of platelet P2Y1 receptors in the regulation of inflammatory responses with a 60-70% inhibition of leukocyte recruitment over an extended time period compared to previous pharmacological studies. platelet P2Y1-/- mice will help further elucidate the mechanisms by which P2Y1 receptors regulate platelet activation during inflammation. Key PointsO_LIMice selectively deficient in the platelet P2Y1 have suppressed leukocyte and platelet recruitment during inflammation. C_LIO_LIWe provide a methodology to determine mechanistic relevance that is otherwise limited by pharmacological approaches. C_LI

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Pan, D., Oralman, T., Evans, R., Baker, O., Cocks, G., Page, C. P., Pitchford, S. C.. 2024-11-06. Platelet-specific P2Y1 receptor deficient mice have suppressed leukocyte recruitment in response to lipopolysaccharide.. https://doi.org/10.1101/2024.11.04.621858

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