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A.J. Unsworth et al.
44 kDa and 32 kDa variants of Pim-1 kinase in human and mouse platelets. While Pim-2 and Pim-3 were not identified in our assays, expression of either paralog in human and mouse platelets cannot be ruled out. Comparison of thrombus formation on collagen under flow in vitro established that platelets from Pim-1-defi- cient mice showed significant attenuation in comparison to that in wild-type controls, highlighting a role for Pim- 1 kinase in the regulation of platelet function and throm- bus formation under arterial flow on collagen. Despite the reduction in thrombotic potential, deletion of Pim-1 was not associated with altered hemostasis, indicating that drugs targeting Pim kinase activity could offer an antithrombotic therapeutic strategy that is not associated with the increased bleeding risk usually observed with other antiplatelet agents.
In support of the antithrombotic potential of Pim kinase inhibitors we determined that several structurally different Pim kinase inhibitors, AZD1208, PIM-447, SGI- 1776, SMI-4a and CX6258, which are pan-Pim kinase inhibitors that target all three Pim kinases, all caused inhi- bition of platelet functional responses, including aggrega- tion in response to GPVI agonists collagen and CRP-XL, CXCR4 ligand SDF-1a (CXCL12) and TxA2 mimetic U46619, but not to other GPCR agonists including throm- bin, TRAP6 and ADP, with an inhibitory profile similar to that observed by Lordkipanidzé et al. in a patient with a mutation in the TP receptor.27,37 Furthermore inhibition of collagen-induced platelet responses was found to be due to an inhibition of TPaR signaling, as the level of platelet aggregation observed in the presence of indomethacin or the TPaR antagonist GR32191 was not further decreased following treatment with AZD1208 (10 mM). Similarly to the reduced thrombus formation observed in whole blood from Pim-1-deficient mice, AZD1208 caused signif- icant attenuation of thrombus formation on collagen under flow in vitro at both arterial and pathological shear rates and a dramatic inhibition of thrombus formation in vivo (Figure 2). Interestingly, however, no effect on platelet adhesion and thrombus formation at venous flow rates was observed following treatment with AZD1208; this, combined with the lack of effect on bleeding in mice following damage to the tail vein, suggests that although Pim kinase inhibition or deficiency reduces thrombus for- mation under high arterial shear, this does not alter thrombus formation or hemostasis at venous or low shear. This absence of effect is likely due to the lack of inhibition of PAR or P2Y receptor-mediated platelet acti- vation by Pim kinase inhibitors. Previous studies have shown that while GPVI deficiency or inhibition has vary- ing effects in tail bleeding assays,38,39 PAR or P2Y12 defi- ciency or P2Y1 inhibition results in significant increases in tail bleeding and alteration of hemostasis in mice.39-41 As initial adhesion to collagen is unaffected following treat- ment with AZD1208, maintenance of PAR and P2Y receptor responses may compensate for the lack of TP receptor signaling, allowing for normal hemostasis. The lack of bleeding effect following treatment with AZD1208 is consistent with the lack of reported bleed- ing-related adverse effects in patients in a recent phase I clinical study investigating the efficacy of AZD1208 in solid and hematologic cancers.24 This provides further evi- dence that Pim kinase inhibitors may not be associated with drug-induced platelet dysfunction-related bleeding events and points to future use of Pim kinase inhibitors as
a possible treatment strategy for individuals with increased risk of cardiovascular disease and atherosclero- sis, conditions associated with pathological shear rates.
Investigation into how Pim kinases elicit their inhibito- ry effects on TPaR signaling, revealed that AZD1208 inhibited TPaR downstream signaling events, pointing to upstream regulation of TPaR signaling most likely via direct regulation of the TPaR. It has previously been described that Pim-1 kinase regulates CXCR4 activity in Jurkat and chronic lymphocytic leukemia cells via regula- tion of surface expression levels of the receptor.18,19 Inhibition or deletion of Pim kinase reduces surface expression levels of CXCR4 and inhibits CXCL12/SDF-1 signaling.29,30 Similarly, in our study we identified reduced surface expression levels of TPaR in platelets following treatment with AZD1208 compared to the levels in vehi- cle-treated controls.
The TPaR signaling pathway is a key target to reduce cardiovascular disease-related thrombotic events and inflammation in patients. Despite widespread use, cur- rently available GPCR-targeted therapies are associated with variable outcomes and adverse side effects in patients. Aspirin is the ‘gold standard’ antiplatelet agent for the prevention of arterial thrombosis. Aspirin targets platelet TPaR signaling via an indirect route, by inhibiting COX1, the enzyme that controls synthesis of TxA2. Aspirin, however, has dose-limiting off-target effects on COX2, an enzyme involved in synthesis of endogenous inhibitors of platelet function that can increase the risk of thrombosis when repressed. Aspirin is also less effective in patients suffering from diabetes, hypertension and obesity, and is associated with an increased risk of severe bleeding42 with one recent meta-analyses suggesting that in low-risk individuals the harms of aspirin outweigh the cardiovascular benefits.43-45 Our findings suggest that Pim kinases may be a safer target to control thrombosis.
The mechanism underlying the regulation of TPaR sig- naling by Pim-1 kinase requires further investigation. In other cell types the 44 kDa variant of Pim-1 kinase, Pim- 1L is localized to the plasma membrane and associated with the phosphorylation and regulation of several mem- brane proteins, while the smaller 32 kDa variant Pim-1S predominantly localizes to the cytosol and nucleus.7,46 One possibility is that Pim kinase phosphorylates the TPαR. controlling its surface expression levels, similar to the mechanism observed for CXCR4 in chronic lympho- cytic leukemia cells. Sequence alignments of the Pim kinase substrate recognition sequence have identified four putative Pim kinase phosphorylation sites within the TPaR sequence, including one within the first intracellu- lar loop, a region that has previously been shown to be associated with TPaR surface expression and receptor function.27 Pim kinase inhibition may therefore disrupt phosphorylation of the receptor and reduce surface expression via increasing its internalization or preventing the dynamic process of receptor recycling to the surface. It is also possible that Pim kinase does not phosphorylate the receptor directly and instead orchestrates interactions with other proteins that regulate receptor surface expres- sion.18,19
This work identifies a novel, Pim kinase-dependent reg- ulatory mechanism for the TPaR and represents a new targeting strategy that is independent of COX1 inhibition or direct antagonism of the TPaR that, while reducing thrombosis, does not increase the risk of bleeding.
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haematologica | 2021; 106(7)