Protease-Activated Receptor 4 and Its Contribution to Immune Thrombotic Thrombocytopenia Disorders
Abstract
Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor (GPCR) and one of two thrombin receptors expressed on human platelets. It is responsible for the latter portion of the biphasic cytosolic calcium flux observed after platelets are activated by thrombin, which has been suggested to be a key factor in thrombosis. It is not known, however, whether PAR4 plays a role in immune thrombotic thrombocytopenia (ITT) disorders. In ITT, circulating IgG immune complexes activate platelets and certain leukocytes via the Fc receptor for IgG IIA (FcγRIIA) which initiates platelet activation and a small amount of thrombin generation. Here we investigate the biology of human PAR4 and its role in ITT pathogenesis. In vivo studies of PAR4 are currently precluded by the lack of an appropriate mouse model. While human and mouse platelets both express PAR4, sequence alignment reveals differences in critical functional domains that may impact receptor activation and negative regulation. Here we show that our novel transgenic mice, which express human but not mouse PAR4 (PAR4 tg/KO), exhibited shortened tail bleeding time and their platelets were more responsive to PAR4 activating peptide (PAR4-AP) as assessed by α-granule release and integrin activation, compared with wild-type (WT). Additionally, PAR4 tg/KO platelets exhibited greater calcium area under the curve and more robust extracellular vesicle release than WT platelets stimulated with PAR4-AP. Genetic deletion of the negative regulator GPCR kinase (GRK) 6 had no effect on PAR4 tg/KO platelets, unlike WT platelets. We also show that WT platelets are more responsive to thrombin than PAR4 tg/KO platelets, but that human PAR4 is more sensitive to thrombin cleavage than mouse Par4 in a heterologous cell system. To study PAR4 in the context of ITT, we first performed ex vivo studies using human platelets and specific PAR inhibitors. Interestingly, we found that upon dual stimulation with an FcγRIIA agonist (αCD9) and thrombin, both PAR1 and PAR4 contributed to phosphatidylserine (PS) exposure and microvesicle release, two markers of the procoagulant phenotype; the reliance appeared to shift toward PAR4 at the higher concentration of thrombin that was used. We also observed that the two PARs combined with FcγRIIA to result in considerable increases in cytosolic calcium. We further demonstrated that humanized FcγRIIA transgenic mice lacking endogenous mouse Par4 (IIA-Par4–/–), therefore having platelets insensitive to thrombin, were protected from severe disease in an in vivo model of ITT where mice were injected with αCD9. Compared with IIA-Par4+/+ mice, IIA-Par4–/– mice experienced significantly less severe thrombocytopenia and were fully protected from hypothermia and fibrin-rich clot formation in the lungs. Together our results suggest that 1) human PAR4 is distinct from mouse Par4, 2) the combination of mouse Par3 and Par4 behaves in a manner like that of human PAR1, 3) PAR4 plays an important role in the procoagulant phenotype upon dual stimulation of human platelets through FcγRIIA and the PARs, and 4) PAR signaling is necessary for ITT pathogenesis in vivo.
Subject Area
Biology|Cellular biology
Recommended Citation
Renna, Stephanie A, "Protease-Activated Receptor 4 and Its Contribution to Immune Thrombotic Thrombocytopenia Disorders" (2022). ProQuest ETD Collection - Thomas Jefferson University. AAI29399905.
https://jdc.jefferson.edu/dissertations/AAI29399905
