Platelet Specific Promoters Are Insufficient to Express Protease Activated Receptor 1 (PAR1) Transgene in Mouse Platelets

The in vivo study of protease activated receptors (PARs) in platelets is complicated due to species specific expression profiles. Human platelets express PAR1 and PAR4 whereas mouse platelets express PAR3 and PAR4. Further, PAR subtypes interact with one another to influence activation and signaling. The goal of the current study was to generate mice expressing PAR1 on their platelets using transgenic approaches to mimic PAR expression found in human platelets. This system would allow us to examine specific signaling from PAR1 and the PAR1-PAR4 heterodimer in vivo. Our first approach used the mouse GPIb alpha promoter to drive expression of mouse PAR1 in platelets (GPIb alpha-Tg-mPAR1). We obtained the expected frequency of founders carrying the transgene and had the expected Mendelian distribution of the transgene in multiple founders. However, we did not observe expression or a functional response of PAR1. As a second approach, we targeted human PAR1 with the same promoter (GPIb alpha-Tg-hPAR1). Once again we observed the expected frequency and distributing of the transgene. Human PAR1 expression was detected in platelets from the GPIb alpha-Tg-hPAR1 mice by flow cytometry, however, at a lower level than for human platelets. Despite a low level of PAR1 expression, platelets from the GPIb alpha-Tg-hPAR1 mice did not respond to the PAR1 agonist peptide (SFLLRN). In addition, they did not respond to thrombin when crossed to the PAR4(-/-) mice. Finally, we used an alternative platelet specific promoter, human a(IIb), to express human PAR1 (a(IIb)-Tg-hPAR1). Similar to our previous attempts, we obtained the expected number of founders but did not detect PAR1 expression or response in platelets from alpha(IIb)-Tg-hPAR1 mice. Although unsuccessful, the experiments described in this report provide a resource for future efforts in generating mice expressing PAR1 on their platelets. We provide an experimental framework and offer considerations that will save time and research funds.