Abstract
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 GPIbα promoter to drive expression of mouse PAR1 in platelets (GPIbα-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 (GPIbα-Tg-hPAR1). Once again we observed the expected frequency and distributing of the transgene. Human PAR1 expression was detected in platelets from the GPIbα-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 GPIbα-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 αIIb, to express human PAR1 (αIIb-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 αIIb-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.
Highlights
Protease activated receptors (PARs) are G-protein-coupled receptors (GPCR) that are activated by proteolytic cleavage of the N-terminus
Our experimental strategy was to generate the transgenic mice on the B6SJLF1/J background and backcross the mice to the C57BL6/J, which would allow us to breed the mice with the PAR3 knockout (PAR32/2) and PAR4 knockout (PAR42/2) mice
It has been well documented that human platelets express Protease Activated Receptor 1 (PAR1) and PAR4, whereas mouse platelets express PAR3 and PAR4
Summary
Protease activated receptors (PARs) are G-protein-coupled receptors (GPCR) that are activated by proteolytic cleavage of the N-terminus. Human platelets express PAR1 and PAR4 and both contribute to thrombin signaling [1]. Mouse platelets express PAR3 and PAR4, and thrombin signaling is mediated entirely by PAR4. PAR1 is expressed on human, monkey, and guinea pig platelets, but not on canine, rat, murine, and rabbit platelets; these species do express PAR1 in other tissues [5,6]. The expression of PAR3 complicates the translation to human disease since human platelets do not express PAR3 and, in mice, PAR3 influences PAR4 signaling [4]. There are well-described mouse models of thrombosis in which one could test the specific role of the interaction between PAR1 and PAR4 or PAR1 specific signaling. The selective expression of PAR1 has limited the opportunities for assessing the role of a PAR1 antagonist as antithrombotic agents with in vivo models
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