PF801 DASATINIB ATTENUATES THROMBIN GENERATION AND CLOT RETRACTION OF CONVULXIN ACTIVATED HUMAN PLATELETS

Abstract

Background:Tyrosine kinase inhibitors (TKI) are a very effective group of drugs that considerably prolong survival of patients with chronic myeloid leukemia. Several lines of evidence suggest that dasatinib an inhibitor of BCR‐ABL may induce bleeding due to its effect on platelets. Previously it was shown that dasatinib induced impaired platelet adhesion, aggregation and secretion. In our in vitro studies we investigated the effect of dasatinib on the procoagulant activity and on clot retraction of human platelets.Aims:We hypothesized that dasatinib treatment may impair various platelet functions in activated platelets.Methods:Platelet‐rich plasmas (PRPs) were prepared from citrate‐anticoagulated blood samples of healthy volunteers using low speed centrifugation. Subsequently PRPs were incubated with or without dasatinib, then platelets were activated by a GPVI agonist, the snake venom convulxin. Phosphatidylserine (PS) expression of activated platelets was examined by flow cytometry. The thrombin generation test (TGT) of PRPs was performed in the presence of 1pM Tissue factor (PRP reagent) by a fluorimetric assay. Fluorescence was detected by a Fluoroskan Ascent fluorimeter (Thermo Fischer Scientific), and the thrombin generation curves were analyzed by the Thrombinoscope software (Thrombinoscope BV). The activated form of GPIIb/IIIa was detected by measuring PAC1 expression by flow cytometry. Clot retraction of platelets was elicited by high calcium concentration in PRP and results were expressed as retracted percent of the original volume and was followed up to 1 hour. Sarcoma Family Kinases (SFK) play role in platelet activation, thus their phosphorylation levels were examined from dasatinib pretreated platelet lysates by Western‐blot.Results:Convulxin resulted in a dose‐dependent increase of platelet PS expression peaking at 51% when 12.5 ng/mL convulxin was used. The lower end of the therapeutic range of dasatinib (10 nM) showed no effect on the rate of this activation marker while the high therapeutic dasatinib concentration (100 nM) considerably prevented this activating effect. PS expressed on the platelet surface may serve as an anchor for the multiprotein complexes of the coagulation cascade. Thus, we investigated the effect of dasatinib on thrombin generation in PRP and found that 100 nM dasatinib attenuated thrombin formation as it reduced the peak thrombin values from 322 ± 31 nM thrombin to 204 ± 62 nM. Similarly, PAC1 expression on convulxin activated platelets was significantly prevented by preincubation with 100 nM dasatinib (8% versus 81%). Clot retraction was 80% in non activated samples and this was decreased to 53% upon convulxin activation that was completely prevented by dasatinib preincubation. We found that dasatinib pretreatment downregulated the phosphorylation levels of Fyn, Lyn and Src kinases in a concentration dependent manner.Summary/Conclusion:Dasatinib is an effective TKI that alters platelet reactions to activation via the collagen receptor GPVI. This effect may be mediated via its effect on phosphorylation status of SFK and can contribute to bleeding symptoms observed during dasatinib treatment.