Investigating the Roles of Platelet PAR4 in Hemostasis, Thrombosis and Viral Infection Using a Newly Generated PAR4 Floxed Mouse

Introduction: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes and lung epithelial cells, and activated by multiple ligands including thrombin and cathepsin G. Importantly, PAR4 is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and protection from thrombosis which are attributed to loss of platelet PAR4, but this has not been specifically demonstrated in mice. Additionally, global PAR4 deficiency increases mortality after influenza A virus infection, but the cell type/s responsible for the enhanced mortality have not been determined. Here, we describe the generation of PAR4 floxed (PAR4 ^fl/fl) mice that can be used to delete PAR4 in a cell type-specific manner, and examine the effect of megakaryocyte/platelet-specific deletion of PAR4 on hemostasis, thrombosis and viral infection using PAR4 ^fl/fl;PF4Cre ⁺ mice.

Methods: PAR4global knockout (PAR4 ^-/-), MK/platelet-specific knockout (PAR4 ^fl/fl;PF4Cre ⁺) and appropriate littermate control mice were used for experiments. Platelet function was determined by light transmission aggregometry and flow cytometry. Hemostasis was assessed in the saphenous vein laser injury model. Platelet plug formation was visualized by intravital microscopy following saphenous vein laser ablation (~50 μm diameter injury), followed by 2 subsequent ablations to reinjure the same site unless on-going bleeding was occurring. Mice were treated with ibrutinib (12.5 mg/kg) to inhibit GPVI signaling, or dabigatran etexilate (chow containing 10 mg/g) or recombinant hirudin (50 mg/kg) to inhibit thrombin activity. Thrombosis was assessed in the carotid artery FeCl ₃ model. The carotid artery was exposed and 8% FeCl ₃ applied for 3 mins. Blood flow was observed for 30 mins and occlusion was defined as no blood flow for 2 mins. To study susceptibility to viral infection, mice were challenged intranasally with a mouse-adapted H1N1 influenza A virus (H1N1 IAV PR8; 0.02 hemagglutination assay units), which induces mortality in 20% of WT mice. Mortality was defined as body weight loss greater than 25%, which required euthanasia.

Results: As expected, PAR4 ^fl/fl;PF4Cre ⁺ platelets were unresponsive to thrombin or PAR4-specific stimulation, while the response to other agonists was retained. In the saphenous vein laser injury hemostasis model, PAR4 ^fl/fl;PF4Cre ⁺ mice were able to rapidly form a hemostatic platelet plug, but the majority of plugs (7/8) were unstable and re-opened after several minutes, leading to severely prolonged total bleeding times. We observed similar findings in global PAR4 ^-/- mice with 8/12 plugs re-opening. To investigate the mechanism mediating initial platelet plug formation, we inhibited GPVI signaling in PAR4 ^fl/fl;PF4Cre ⁺ mice using the Btk inhibitor ibrutinib. Ibrutinib administration shortened time to plug re-opening in PAR4 ^fl/fl;PF4Cre ⁺ mice but plugs were still able to form, which is likely mediated by GPIbα/VWF. We observed a similar phenotype to PAR4 ^fl/fl;PF4Cre ⁺ mice in mice treated with the direct thrombin inhibitor hirudin, suggesting thrombin is the primary activator of PAR4 during hemostatic plug formation. In the FeCl ₃-induced carotid artery thrombosis model, both PAR4 ^fl/fl;PF4Cre ⁺ and PAR4 ^-/- mice were significantly protected compared to controls. Finally, when challenged with the mouse-adapted H1N1 IAV PR8, PAR4 ^fl/fl;PF4Cre ⁺ mice demonstrated similar body weight loss and survival as littermate controls.

Conclusions: Our results in mice demonstrate that 1) platelet PAR4 is not required for initial hemostatic plug formation but is necessary for maintaining hemostatic plug stability, 2) loss of platelet PAR4 protects from arterial thrombosis, and 3) platelet PAR4 does not alter the course of H1N1 IAV infection, at least at the virus dose used in this study. In summary, we generated a novel mouse line carrying a floxed PAR4 allele which can be used to investigate cell-specific roles of PAR4 in disease.