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A.J. Unsworth et al.
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Figure 1. Expression of Pim kinase in human and mouse platelets. (A) HaemAtlas analysis of Pim kinase mRNA expression levels. Pim kinase mRNA levels were quantified in human megakaryocytes and a range of blood cells by analysis of gene array data. Megakaryocytes (MK), human erythroblasts (EB), human umbilical vein endothelial cells (HUVEC), monocytes (CD14), granulocytes (CD66), mature B cells (CD19), natural killer cells (CD56), cytotoxic T cells (CD*) and helper T cells (CD4); 10+ (lighter colors) was deemed high expression. (B) Human and mouse washed platelets (three preparations) were lysed in SDS PAGE Laemmli sample buffer, separated on SDS PAGE gels and transferred to PVDF membranes before immunoblotting (IB) with anti-Pim-1 antibody. K562 and Jurkat cell lysates were included as positive controls. Actin was included as a loading control. Representative blots are shown.
‘loose’ aggregates compared to the aggregates formed in vehicle-treated controls, but no difference in fluorescence intensity of platelets adhered was observed. These find- ings indicate that Pim kinase inhibition does not signifi- cantly alter platelet adhesion and thrombus formation at low shear rates compared to the effects observed at high- er shear rates. To examine whether Pim kinase inhibition could regulate thrombosis in vivo, we performed intravital microscopy following ferric-chloride-induced injury in mice pretreated with AZD1208 (100 μM) or vehicle con- trol (Figure 2F). As with in vitro thrombus formation, treat- ment with AZD1208 resulted in a dramatic attenuation in the ability of platelets to form thrombi in vivo, at the site of ferric-chloride-induced injury with significantly pro- longed occlusion times observed in AZD1208-treated mice (1463±37 s) compared to those in vehicle-treated controls (697±72 s). This supports a role for Pim kinase in the positive regulation of platelet function, and the anti- platelet properties of Pim kinase inhibitors. Interestingly, despite the dramatic attenuation in the ability of platelets to form thrombi in vivo, as with genetic deletion, pharma- cological inhibition of Pim kinase was not associated with altered hemostasis. Tail bleeding assays performed in mice indicated that treatment with AZD1208 (100 mM) did not cause any significant increase in bleeding (Figure 2G), suggesting that despite the observed inhibitory effect on thrombus formation, normal hemostasis is not compromised following inhibition or genetic deletion of Pim kinase.
Pim kinase inhibitors reduce platelet aggregation
To determine how Pim kinase plays a role in the regu- lation of platelet function, human washed platelets were pretreated for 10 min with a range of concentrations of the pan-Pim kinase inhibitor AZD1208 before stimula- tion with platelet agonists. As shown in Figure 3A-E pre- treatment of platelets with AZD1208 inhibited aggrega- tion stimulated by collagen (1 mg/mL) or the thrombox- ane A2 (TxA2) mimetic U46619 (0.3 mM). A slight inhibi- tion was also observed in CRP-XL stimulated platelets (0.3 mg/mL). In contrast no inhibition of thrombin- (0.03 U/mL) or ADP- (10 mM) induced platelet aggregation was observed following treatment with increasing concentra-
tions of AZD1208 (up to 10 mM). Treatment with four other structurally unrelated Pim kinase inhibitors, PIM- 447 (LGH-447), SGI-1776, SMI-4a and CX6258, inhibited platelet aggregation stimulated by either collagen or U46619 but not thrombin, recapitulating findings with AZD1208 and supporting a Pim kinase-dependent mode of action (Online Supplementary Figure S4).
Pim kinases have been shown previously to play impor- tant roles in cell survival as Pim-2 is known to phospho- rylate and inactivate Bcl-2-associated death promoter (BAD).23 Inhibition of Pim kinase enables activation of BAD and initiation of apoptosis.5 If Pim kinase inhibitors activate apoptosis in platelets, this could cause the observed reduction in aggregation. To investigate whether Pim kinase inhibition triggers apoptosis in platelets, phosphatidylserine exposure (a marker of mem- brane flippage) was determined by measuring annexin V binding by flow cytometry following treatment for 2 h with AZD1208 or the BCL-2 inhibitor ABT-263, an acti- vator of apoptosis in platelets (Online Supplementary Figure S5A). ABT-263 treatment caused an increase in annexin V binding but no difference was observed in AZD1208- treated platelets compared to vehicle-treated controls over the same incubation time. Furthermore, caspase cleavage did not occur in platelets following 2 h of treat- ment with Pim kinase inhibitors: AZD1208 (10 mM), SGI- 1776 (10 mM), SMI-4a (30 mM) or CX6258 (10 mM), but did occur following treatment with the BCL-2 inhibitor ABT-263 (Online Supplementary Figure S5B). Together these observations suggest that Pim kinase inhibition does not initiate platelet apoptosis.
Pim kinase inhibitors reduce thromboxane A2-mediated a-granule secretion and integrin activation
Alpha granule secretion and activation of integrin aIIbβ3 are critical events in platelet activation and aggregation. We investigated the effects of AZD1208 (10 μM) on a- granule secretion and aIIbβ3 activation by measuring sur- face P-selectin exposure and fibrinogen binding, respec- tively. AZD1208 (10 mM) inhibited a-granule secretion and integrin activation evoked by collagen, CRP-XL and U46619 compared to the effects in vehicle-treated con-
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haematologica | 2021; 106(7)