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A.J. Unsworth et al.
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Figure 3. AZD1208 inhibits platelet aggregation. (A-E, i and ii) Washed human platelets were pretreated with increasing concentrations of AZD1208 (0.1, 1, 10 μM) prior to stimulation with (A) collagen (1 mg/mL), (B) CRP-XL (0.3 mg/mL), (C) U46619 (0.3 mM), (D) thrombin (0.03 U/mL) or (E) ADP (10 mM) and aggregation moni- tored using optical light transmission aggregometry. (i) Representative traces and (ii) quantified data are shown. (A-E, iii) Human washed platelets were pretreated with 10 mM AZD1208 (red) or vehicle (black) prior to stimulation with (A) collagen (0.01-10 mg/mL), (B) CRP-XL (1-3 μg/mL), (C) U46619 (3 nM - 3 mM), (D) thrombin (0.01-1 U/mL) or (E) ADP (0.1-100 μM) and aggregation was monitored after 5 min using an optical light transmission plate-based aggregometry assay; quantified data are shown. Results are mean ± standard error mean for n≥3, *P≤0.05 ***P≤0.005 in comparison to vehicle control; where normalized data are shown statis- tics were performed prior to normalization.
TPaR.-dependent mechanism. The inhibition observed in collagen-stimulated platelets is likely due to the loss of TxA2 signaling, indicating that Pim kinase has a positive regulatory role in the regulation of TPaR signaling. Similar results were also observed following treatment with structurally unrelated Pim kinase inhibitors SGI- 1776, PIM-447 (LGH447), SMI-4a and CX6258 in the presence or absence of indomethacin (Online Supplementary Figure S4D-G). In further support of this, we also observed that AZD1208-mediated inhibition of thrombus formation on collagen under flow occurred via a TPaR-dependent mechanism, as indomethacin (10 mM) did not cause further inhibition of thrombus formation when combined with AZD1208 treatment (Figure 4C). In support of the effects of AZD1208 being mediated via Pim kinase, and a role for Pim kinase in the regulation of platelet TPaR signaling, platelets from Pim1-/- mice (red) displayed reduced fibrinogen binding, compared to con- trols (black), following stimulation with U46619 (10 mM) or CRP-XL (10 mg/mL) but not following stimulation with thrombin (0.01 U/mL) (Figure 4D).
Pim kinase inhibitors inhibit platelet function independently of COX1
The synthesis of TxA2 in platelets is dependent on acti- vation of COX1 following platelet stimulation. To deter- mine whether the inhibitory effects of Pim kinase inhibitors were due to altered COX activity, thrombox- ane B2 (TxB2) levels (a marker of TxA2 release) were determined following stimulation of platelets with
arachidonic acid (a direct substrate for COX1 and the TxA2 synthesis pathway) or collagen and compared to the levels following treatment with indomethacin, a COX1 inhibitor, in the presence of the TPaR antagonist GR32191 to remove secondary TPaR feedback mecha- nisms. While indomethacin caused almost complete ablation of both collagen- and arachidonic acid-induced platelet TxB2 generation, AZD1208 did not cause any significant alterations in TxB2 generation indicating that it does not regulate COX1 activity (Figure 5A). In further support of AZD1208 mediating its inhibitory actions via inhibition of TPaR receptor signaling and not inhibition of cyclo-oxygenase activity, while AZD1208 causes inhibition of U46619-mediated platelet aggregation, concentrations of indomethacin that cause ablation of platelet responses to arachidonic acid are unable to inhibit platelet activation by the TPaR agonist U46619 (Figure 5A).
Pim kinase inhibitors reduce signaling events downstream of TPaR
The TPaR receptor is coupled to both Gq and Ga13 pro- teins. Gq couples the TPaR to phospholipase C which in turn regulates calcium mobilization and the activation of protein kinase C (PKC), key mediators of granule secre- tion and activation of integrin aIIbβ3. Ga13 regulates the Rho/Rho-kinase signaling pathway which regulates the phosphorylation of myosin IIa and is important for the regulation of cytoskeletal rearrangements and platelet shapechange.
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haematologica | 2021; 106(7)