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Pim kinase regulates TP receptor signaling
megakaryocyte transcriptome database12 identified multi- ple tags for both Pim-1 and Pim-2 kinases and the mRNA transcripts for all three Pim kinases have been identified in the human platelet transcriptome.13,14 Interestingly although triple knockout mice deficient in all three Pim kinase isoforms are viable, they have been shown to have altered hematopoiesis, but there is some dispute as to whether disruption of all three isoforms results in alter- ation of platelet count;10,11 however, platelet counts appear to be unaffected by alteration of Pim-1 expression levels in mice.15,16
Platelets rely on G protein-coupled receptors (GPCR) such as the thromboxane A2 receptor (TPaR), ADP recep- tors (P2Y1 and P2Y12) and the thrombin receptors (PAR1 and PAR4) to mediate platelet activation in response to vessel damage. All platelet GPCR are regulated in some way by receptor cycling/internalization from the platelet surface as well as desensitization.17 Pim-1 kinase has also been shown to have a role in the regulation of GPCR function, through modulation of surface levels of the CXCR4 receptor.18,19 Inhibition of Pim kinase prevents Pim kinase-dependent phosphorylation of CXCR4 at Ser339 and modification of the CXCR4 intracellular C ter- minal domain, resulting in reduced surface expression and signaling. In this study we report the presence of Pim-1 in human and mouse platelets, and reduced throm- bosis in Pim-1 null mice, and following pharmacological inhibition of Pim kinase, but with no associated effect on hemostasis. We describe a novel mechanism of action by which Pim kinase inhibitors negatively regulate TPaR sig- naling.
Methods
Procedures and experiments using human blood were approved by the University of Reading Research Ethics Committee and protocols involving mice were performed according to the National Institutes of Health and Medical College of Wisconsin Institutional Animal Care and Use Committee guidelines and as following procedures approved by the University of Reading Research Ethics Committee.
Platelet isolation, thrombus formation assays, tail bleeding experiments, platelet function tests, aggregometry, granule secretion, flow cytometry, calcium imaging, immunoblotting, image analysis, statistical analyses and materials used are described in the Online Supplementary Methods.
Results
Expression of Pim kinase in human and mouse platelets
Pim kinases are highly expressed in hematopoietic cells.10,11 mRNA transcripts for all three Pim kinases have been identified in the human and mouse platelet tran- scriptomes13,14 and HaemAtlas mRNA expression profiles in hematopoietic cells20 show high expression of Pim-1 in megakaryocytes and moderate expression in platelets (Figure 1, Online Supplementary Figure S1).21 Western blot analysis of platelet lysates identified a protein band of 44 kDa apparent molecular mass in both human and mouse platelet lysates indicating the expression of the larger Pim-1 variant (Pim-1L). A protein band at 32 kDa in mouse platelets also suggested expression of the smaller
Pim-1S. K562 and Jurkat cell lines were included as posi- tive controls8,18,22 (Figure 1B).
Reduced thrombus formation in Pim-1-deficient mice
To determine whether Pim-1 plays a role in the regula- tion of platelet function and thrombosis, we measured the ability of Pim-1-deficient mouse platelets, taken from constitutive Pim-1-deficient mice, to form thrombi on collagen under arterial flow in vitro. Constitutive Pim1–/– mice were as described previously15,16 and global deletion of Pim-1 was confirmed by polymerase chain reaction analysis of genomic DNA (Online Supplementary Figure S2A). Whole blood from Pim-1-/- or Pim-1+/- mice was per- fused over collagen-coated (100 mg/mL) Vena8 biochips for 4 min at an arterial shear rate of 1000 s-1. Thrombus formation was significantly attenuated in blood from Pim-1-/- mice compared to controls, indicating that Pim-1 plays a positive role in the regulation of platelet function and thrombus formation on collagen (Figure 2A). Constitutive Pim-1-/- mice show unaltered platelet counts and no difference in expression levels of major platelet adhesion receptors GPIba, GPIbβ, GPIX, GPV, GPVI and integrins β1 and β3 was observed in Pim-1-/- platelets com- pared to the levels in controls (Online Supplementary Figure S2B). Interestingly, despite the reduced ability to form thrombi, Pim-1-deficient mice showed no alteration in hemostasis as tail bleeding was unaffected compared to that of littermate controls (Figure 2B).
Pim kinase inhibitors reduce thrombus formation but do not disrupt hemostasis
As genetic deletion of Pim-1 in mice resulted in reduced in vitro thrombus formation, we assessed the effects of the Pim kinase inhibitor AZD1208 (100 mM) on thrombus formation in human whole blood. The effect of the Pim kinase inhibitor AZD1208 (100 mM) on thrombus forma- tion on collagen in human whole blood in vitro was also assessed. Human whole blood was pre-incubated with vehicle control or AZD1208 and perfused over collagen- coated (100 mg/mL) Vena8 biochips at either an arterial shear rate (20 dynes/cm3 for 10 min) or a pathological shear rate (135 dynes/cm3 for 5 min). Similarly to what had been observed in Pim-1-deficient mice, a reduction in thrombus formation and stability on collagen under flow in vitro was also observed following AZD1208 treatment in comparison to that seen in vehicle-treated controls under arterial shear conditions (Figure 2C). While throm- bus size and stability appeared reduced at arterial flow rates, the early stages of thrombus formation, including initial adhesion, appeared unaffected by AZD1208 treat- ment. This was further supported by the lack of inhibi- tion of platelet adhesion and spreading on collagen caused by AZD1208 under static conditions (Online Supplementary Figure S3), indicating that the initial adhe- sion to collagen is not affected by Pim kinase inhibition. Interestingly, enhanced inhibition of thrombus formation on collagen was observed following treatment with AZD1208 at pathological shear rates (~80% inhibition) (Figure 2D) in comparison to the inhibition observed at an arterial shear rate (~50% inhibition). In contrast to arterial and pathological shear rates, a slight (but not significant) reduction in thrombus formation was observed in AZD1208-treated platelets compared to vehicle-treated controls under venous shear conditions (Figure 2E). Inhibitor-treated platelets appeared to form ‘woolly’ or
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