Tyrosine kinase inhibitors modulate platelet function in vitro, ex vivo and in vivo

The introduction of Bcr-Abl in chronic myeloid leukaemia has led to quickly developing targeted tyrosine kinase inhibitors (TKIs), imatinib which specifically interrupt the abnormal protein tyrosine kinase pathways of the Bcr-Abl fusion protein. Subsequently, imatinib resistance due to BCR-Abl kinase mutations led to the development and approval of more effective second generation TKIs, nilotinib and dasatinib. Nilotinib and dasatinib have shown a greater efficacious on leukaemic cells with rapid and depth of haematological and molecular remission rates. However, cumulative data have reported an excess of serious vascular complications including arterial thrombosis in patients receiving nilotinib in comparison with other TKIs, with resultant interest in delineating the pathophysiology and implications for rationale cardiovascular risk modification. The adherence of platelets under specific circumstances of high flow shear rates in stenotic blood arteries increases the responsiveness of platelet reactivity, which results in enhanced in vivo arterial thrombosis. In the context of the vasculature, the role of platelets in pro-atherothrombotic mechanisms is now well recognised and implicates pro-inflammatory adhesive molecules in the mediation of the interactions of platelets and endothelium at site of ruptured atherosclerosis plaques.

We demonstrated that dasatinib and imatinib, but not nilotinib, inhibited ADP, CRP, and collagen-induced platelet aggregation. Nilotinib also potentiated PAR-1-mediated alpha granule release. Accordingly, pretreatment of C57BL/6 mice with nilotinib (25 and 50 mg/kg), but not imatinib (25 and 50 mg/kg) or dasatinib (2.5 and 5 mg/kg), significantly increased both platelet thrombus growth on type I collagen under ex vivo but not in vitro arterial flow conditions. Intravital microscopy of vascular injury of mesenteric arterioles and carotid artery induced by ferric chloride confirmed a significant increase in in vivo thrombus formation and stability over time in mice treated with nilotinib but not with imatinib or dasatinib. In addition, the effect of nilotinib was independent of platelet glycoprotein surface expression. Nilotinib selectively potentiated alpha granule exocytosis in humans and mice, but it did not induce spontaneous conversion of the major platelet integrin αIIbβ3 into its active conformation for binding to its ligand, fibrinogen, and mediation of platelet aggregation. The thesis data confirmed that whole blood from nilotinib-treated CML patients showed increased platelet adhesion under flow and increased plasma levels of soluble P-selectin and CD40L, as well as enhanced in vivo thrombin generation. These results indicate that chronic treatment of CML patients with nilotinib selectively induces both endothelial and platelet activation and that these perturbations could predispose them to the risk of formation of pathological blood clots in vivo. The studies presented in this thesis have provided the first evidence of potentiation of platelet and/or endothelium activation, adhesive molecule secretion and increased thrombin generation following nilotinib treatment. The results suggested that the pro-thrombotic phenotypes of nilotinib therapy required the presence of endothelial cells to prime the platelets in order to trigger potentiation of in vivo arterial thrombus formation. Ultimately, the results from this thesis indicated that nilotinib may modulate atherothrombosis by multiple off targeting mechanisms that affect the physiological profiles of platelets, endothelial cells and leukocytes.