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The synergy of adenosine and ibrutinib on platelet aggregation in chronic lymphocytic leukemia

Hagger, Madison Barbara Allerton (2021) The synergy of adenosine and ibrutinib on platelet aggregation in chronic lymphocytic leukemia. Honours thesis, Murdoch University.

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Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults caused by a gradual clonal expansion of B-cells in the bone marrow. Bruton’s Tyrosine Kinase (BTK) plays a role in the signalling of immune cells such as B-cells and platelets and has been implicated in the survival pathways of cancer. The BTK inhibitors, ibrutinib and acalabrutinib, are used to treat CLL; however, a significant bleeding risk has been observed in patients taking these inhibitors that is not seen in people with BTK-mutation (X-lined agammaglobulinemia (XLA)). CLL patients using ibrutinib and acalabrutinib have platelet dysfunction characterised by impaired response to collagen-related peptide (CRP; a specific glycoprotein VI (GPVI) agonist) and collagen. A disease factor may potentiate the antiplatelet effects of BTK inhibitors to cause significant bleeding issues in CLL patients. The CLL microenvironment is characterised by high levels of CD73-generated adenosine, which plays a role in suppressing immune responses such as platelet aggregation. Thus, it is hypothesized that adenosine amplifies the effect of ibrutinib and acalabrutinib to inhibit platelet function, which would explain the associated increased bleeding risk.

Plasma was collected from stable, untreated CLL patients (n = 24) and age-matched healthy controls (n = 10). Using ELISA and the Malachite green assay, soluble CD73 levels and activity were determined. For ex vivo platelet activation studies, blood was collected from healthy volunteers into sodium citrate for whole blood assays (n=2), or ACD anticoagulant (n=6) for washed platelet tests. Flow cytometry, light transmission aggregometry, and Western blotting were used to delineate the impact of adenosine on the antiplatelet activities of ibrutinib and acalabrutinib.

Soluble CD73 was detected in the plasma of CLL patients, and its activity was confirmed by measuring phosphate generation from AMP using the Malachite green assay and a selective CD73 inhibitor. In whole blood platelet activation assays, adenosine potentiated the antiplatelet effect of ibrutinib and acalabrutinib. In washed platelets, using clinically relevant concentrations, ibrutinib and acalabrutinib inhibited CRP (P < 0.0001) but not collagen-induced platelet aggregation as single agents (P > 0.05). When combined with the adenosine derivative, 2-Hexynyladenosine-5'-N-ethylcarboxamide (HENECA), both inhibitors managed to reduce collagen-induced platelet aggregation, although Ibrutinib’s effect was stronger (P < 0.0001). The combination of HENECA with ibrutinib or acalabrutinib broadens the antiplatelet activity of these inhibitors as evidenced by changes in collagen-mediated phosphorylation of the downstream kinases: ERK, AKT, VASP, BTK and SYK.

The results of this study provide initial evidence that increased bleeding risk present in the CLL microenvironment might be caused by the synergy between the BTK inhibitors and high levels of adenosine. Furthermore, the synergistic effect was more pronounced with ibrutinib rather than acalabrutinib. Future studies should focus on examining the association between plasma adenosine levels, CD73 and platelet activation in CLL patients.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): Medical, Molecular and Forensic Sciences
Supervisor(s): Metharom, Pat and Elaskalani, Omar
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