Characterization and Comparison of Two Known Mutations in Protease-Activated Receptors (PAR4 Ala120Thr and PAR2 Ala118Thr)

Abstract

Introduction: Protease-activated receptors (PARs) play an essential role in the human bloodstream. They are involved in coagulation and barrier function as well as in upregulation of inflammation. Four PARs are known in humans, namely PAR1-4. A wellknown mutation located in PAR4, Ala120Thr, seems to be important in coagulation as it was shown that people with this single nucleotide polymorphism have an increased activation of blood platelets by the protease thrombin and leading to more cardiovascular events. Interestingly, a similar mutant is known in PAR2 Ala118Thr that is in contrast to PAR4 not expressed on platelets. Both receptors appear to be involved in vascular inflammation and thus increased or decreased vascular inflammation is suspected in carriers of the mutations. The PAR4 mutation has already been studied thoroughly regarding platelet activation, while its effect on vascular inflammation is not fully understood. On the contrary, the PAR2 mutant has not been studied at all. Accordingly, the main focus in this paper lies on PAR2 and PAR4. This thesis examined whether the mutations PAR4 Ala120Thr and PAR2 Ala118Thr lead to an increased or decreased proteolytic cleavage efficiency by thrombin of the receptors compared to the wild-type (PAR4 Ala120, PAR2 Ala118). Additionally, the aim of the study was to find out what effect the variation of thrombin concentrations and the addition of the cofactor thrombomodulin (TM) have on the cleavage efficiency by thrombin.
Methods: For this comparison of the cleavage efficiency by thrombin an experimental study design was chosen. In order to construct the mutations Ala120Thr in PAR4 and Ala118Thr in PAR2, the corresponding primers were prepared first. Cloning took place via site-specific mutagenesis followed by transformation in E. coli (Escherichia coli). In a further step, the cleavage assay was carried out. Human 293T cells (human embryonic kidney cells) were transfected with the wild-type and mutated plasmids via lipiddroplets. The cleavage efficiency of the receptors by thrombin was made visible using a secreted alkaline phosphatase (SEAP) assay. Receptor expression was quantified by counting the cells and measuring the cell activity by a cell viability assay. With the help of various antibodies, the expression of the receptors into the cell membrane was made visible by using a cell surface enzyme-linked immunosorbent assay (ELISA). Results: The PAR4 mutation did not reveal any different cleavage efficiency by thrombin compared to the wild-type receptor. Quite unexpectedly, there was no direct cleavage by thrombin in the mutated PAR2, although the receptors were expressed and carried to the cell membrane, which was confirmed by the cell surface ELISA. Hence, the PAR2 mutation showed a lower cleavage efficiency by thrombin than the wild-type PAR2. A higher cleavage efficiency by thrombin was measured in both receptors, PAR4 and PAR2, with an increased concentration of the protease thrombin as well as when adding the cofactor TM.
Conclusion: The results of the PAR4 examinations lead to the assumption that the observed clinical effects in carriers of the PAR4 mutation may be caused by different expression or activation on platelets rather than by the cleavage efficiency by thrombin. Furthermore, the present study on PAR2 revealed quite unexpectedly no direct cleavage by thrombin of the PAR2 mutation compared to the wild-type and thereby provides a new basis for approaching further research. An even better understanding of the cleavage and activation of the PARs would be essential to develop, for example, new treatment methods of PAR-related coagulation and inflammatory diseases.