Protease-activated Receptor-4 (PAR4) Variant Influences on Platelet Reactivity Induced by PAR4-activating Peptide Through Altered Ca2+ Mobilization and ERK Phosphorylation in Healthy Japanese Subjects

Background: Thrombin belongs to the most potent platelet agonists and activates human platelets through GPIb alpha and two protease activated receptors (PARs), PAR1 and PAR4. However, the details of thrombin receptor system, especially the role of PAR4 on human platelet activation is still not clear. Objectives: We found a significant difference in PAR4-activating peptide (PAR4-AP)-induced, but not PAR1-AP, platelet aggregation between healthy Japanese subjects. Sequencing analysis revealed a single nucleotide change in PAR4 gene F2RL3 (SNP rs773902) leading to Ala120Thr variant. To elucidate the role of PAR4 in human platelet activation, we examined if platelet activation induced by PAR4-AP may be associated with PAR4 genotype. Methods: Platelets from 202 healthy Japanese volunteers were genetically analyzed and determined the genotype frequency of rs773902. Agonist induced platelet aggregation, integrin alpha IIb beta 3 activation, granule release, Ca2+ mobilization, and activation of ERK and MLC were evaluated. The specificity of effects observed in platelets was confirmed in 293T cells transfected PAR4-Thr120 or Ala120. Results: The frequencies of PAR4 variant Thr/Thr120, Ala/Thr120, and Ala/Ala 120 were 5.9, 37.1, and 57.0%, respectively. Platelets with Thr/Thr120 showed significantly higher reactivity in PAR4-AP-induced platelet aggregation, alpha IIb beta 3 activation and granule release compared to platelets with Ala/Ala120. PAR4-AP induced higher Ca2+ mobilization and ERK activation in platelets with Thr/Thr120 than Ala/Ala120. Ca2+ mobilization and ERK activation were also increased in 293T cells transfected with PAR4-Thr120 compared to Ala120. Conclusion: Our data suggested that PAR4-AP-induced platelet reactivity between PAR4 rs773902 was associated with altered intensity of Ca2+ mobilization and ERK activation.