Calcium Mobilization in Human Platelets is Differentially Modulated by PAR‐1 and PAR‐4 through Gi/o and PI3K

Abstract

Protease activated receptors (PARs) on the platelet surface play an essential role in mediating hemostasis. Human platelets express PAR-1 and PAR-4, and these receptor subtypes are activated by thrombin. Knockout mice for the various G-alpha subunits of the heterotrimeric G-protein family have provided good tools for the study of PAR-4 signaling in platelets. However, differences in G-protein coupling between human and mouse PAR-4 could exist, and mouse platelets do not express PAR-1. Therefore, determining which G-alpha subunits PAR-1 and PAR-4 activate in human platelets is paramount in understanding the physiological effects of thrombin and its inhibitors. Both receptors have been shown to couple to Gq and G12/13 pathways. However, neither receptor has been shown to have a Gi/o signaling component. The main objective of our research was to determine the role of Gi/o in human platelet PAR-mediated calcium mobilization and integrin activation. We have found that PAR-1, but not PAR-4, directly couples to Gi/o family members to activate PI3K in human platelets using flow cytometry. We have also shown that this PI3K potentiates intraplatelet calcium mobilization in a conditional manner using Fura-2 loaded platelets, and also causes integrin glycoprotein IIbIIIa activation using flow cytometry. PAR-4 seems to lack this Gi/o pathway, and we are currently developing a cell permeable peptide based strategy to explore this possibility. Demonstration of a Gi/o signaling component downstream of PAR-1 in human platelets is a novel finding with potential therapeutic implications. This work was supported by the Training Grant in the Pharmacological Sciences (Vanderbilt-NIH).