The Association of PAR4 Polymorphism with Human Platelet Reactivity in Japanese
Authors: Morikawa Yoichiro , Kato Hisashi , Akuta Keigo , Kashiwagi Hirokazu , Honda Shigenori , Kanakura Yuzuru , Tomiyama Yoshiaki 
Published: October 2019

Abstract
Background & objectives:
Thrombin is the most important physiological agonist for platelet thrombus formation. In human platelets, two thrombin receptors, protease activated receptor 1 (PAR1) and PAR4, elicit intraplatelet signals through G-protein for platelet activation. Although PAR1 has been established as a major thrombin receptor, the role of PAR4 in human platelet activation is so far not clear. Genetically modified mice are critical tool for human platelet research. However, the different thrombin receptor system in murine platelets which have PAR3 and PAR4 prevents applying the information of murine platelets for human platelets. To explore the role of PAR4, we investigated inter-individual variation of platelet activation induced by PAR4 and PAR4 dependent signaling in Japanese subjects.
Methods:
Human platelets obtained from healthy volunteers were stimulated by PAR1-activating peptide (PAR1-AP) and PAR4-AP, and detailed aggregation responses were analyzed by aggregometer. Platelet integrin αIIbβ3 activation, granule release and calcium mobilization were measured by flow cytometry. Platelet signaling induced by PAR4-AP was analyzed by Western blotting. Sequencing analysis of PAR4 was performed using RNA and genomic DNA isolated from peripheral mononuclear cells. PAR4 expression vector was transfected to 293T cells, and calcium mobilization was analyzed by flow cytometry.
Results:
Sequencing analysis between control subjects revealed heterogeneity of PAR4 polymorphism c.533G>A (p.Ala120Thr). The further sequencing analysis of PAR4 in 143 Japanese revealed that the frequencies of 120A/A, A/T, and T/T were 53.8, 40.6, and 5.6% respectively. Subjects who have PAR4-120T required significantly lower concentrations of PAR4-AP to induce irreversible aggregation compared to subjects with PAR4-120A (n=24, p<0.01). However, the concentration of PAR1-AP for induction of irreversible aggregation was not different between PAR4-120A and -120T. In addition, when platelets were stimulated with low concentrations of PAR4-AP, platelets with PAR4-120T showed enhanced αIIbβ3 activation, granule release, and calcium mobilization compared to those with PAR4-120A. Western blot analysis also showed stronger phosphorylation of extracellular signal-regulated kinase (ERK) in PAR4-120T platelets stimulated with low concentration of PAR4-AP than in -120A platelets. To confirm that these results observed in human platelets are associated with the PAR4 polymorphism, expression vector of PAR4-120A or -120T was transfected to 293T cells and determined PAR4-induced calcium mobilization. As observed in human platelets, 293T cells transfected with PAR4-120T showed more ehnanced calcium mobilization than those with -120A by PAR4-AP stimulations.
Conclusions:
We demonstrate the significant difference in PAR4-AP induced platelet aggregation, αIIbβ3 activation, granule release, calcium mobilization, and ERK phosphorylation between platelets with PAR4-120A and -120T. Our results suggest the association of PAR4 polymorphism with human platelet reactivity in response to PAR4-AP in Japanese.

Disclosures
Kanakura: Eisai: Research Funding; Alexionpharma: Research Funding; Chugai Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Shionogi: Research Funding; Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding.