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. 2019 Oct 31;20(21):5430.
doi: 10.3390/ijms20215430.

Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets

Ildikó Beke Debreceni  1 , Gabriella Mezei  2 , Péter Batár  3 , Árpád Illés  4 , János Kappelmayer  5
Affiliations

Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets

Ildikó Beke Debreceni et al. Int J Mol Sci. .

Abstract

Tyrosine kinase inhibitors (TKI) such as the BCR-ABL inhibitor dasatinib and nilotinib are highly effective therapies for chronic myeloid leukemia (CML). However, several lines of evidence suggest that dasatinib can induce bleeding which may be due to impaired collagen-induced platelet adhesion, aggregation, and secretion. Sarcoma family kinases (SFK) play central role in the GPVI-induced signaling pathway. We aimed to investigate whether and how dasatinib can modulate SFK-mediated platelet procoagulant activity in a purified system and in dasatinib/nilotinib treated CML patients. In platelet rich plasmas of healthy volunteers, dasatinib dose-dependently reduced convulxin-induced phosphatidylserine exposure and attenuated thrombin formation. Similarly to these changes, integrin activation and clot retraction were also significantly inhibited by 100 nM dasatinib. Platelets isolated from dasatinib treated patients showed a significantly lower phosphatidylserine expression upon convulxin activation compared to premedication levels. In these samples, thrombin generation was significantly slower, and the quantity of formed thrombin was less compared to the trough sample. Western blot analyses showed decreased phosphorylation levels of the C-terminal tail and the activation loop of SFKs upon dasatinib administration. Taken together, these results suggest that dasatinib inhibits the formation of procoagulant platelets via the GPVI receptor by inhibiting phosphorylation of SFKs.

Keywords: bleeding; dasatinib therapy; glycoprotein VI; platelet activation; thrombin formation.

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Conflict of interest statement

The authors state that they have no conflict of interest.

Figures

Figure 1
Figure 1
Dasatinib inhibits convulxin-induced procoagulant phosphatidylserine (PS) exposure and thrombin generation in vitro. Platelet rich plasma (PRP) from healthy donors were incubated with 10 or 100 nM dasatinib, 5000 nM nilotinib or vehicle (0.2% DMSO) for 10 min. Platelets, pretreated with vehicle (without Tyrosine kinase inhibitors (TKI)) or TKI, subsequently they were stimulated with 12.5 ng/mL convulxin for 15 min. Exposure of PS was determined by FITC-labeled annexin V (A). The histogram shows percentages of platelets binding FITC-annexin V. Representative thrombin generation curves of TKI pretreated and convulxin stimulated PRPs (B). Lagtime (C), time to peak (D), peak thrombin (E), and Endogenous Thrombin Potential (ETP) (F) parameters were evaluated. The results are the mean ± SEM of five different experiments.
Figure 2
Figure 2
Inhibitory effect of dasatinib on convulxin-induced integrin αIIbβ3 activation and clot retraction. Platelets in control PRPs were pretreated with dasatinib/nilotinib or vehicle (without TKI) and subsequently were stimulated with convulxin. The active conformation of the integrin αIIbβ3 was detected by FITC-labeled PAC1 monoclonal antibody and analyzed by flow cytometry. The percentages of platelets binding FITC-PAC1 are shown (A). Clot retraction of platelets was elicited by high calcium concentration in TKI pretreated and convulxin-activated PRPs (B). Results were expressed as extruded percent of the original volume. The results are the mean ± SEM of 5 different experiments. Panel C shows a photo series to present the clot retraction phenomenon.
Figure 3
Figure 3
Treatment of chronic myeloid leukemia (CML) patients with dasatinib suppresses PS exposure and generation of thrombin in PRP. Blood was collected from dasatinib treated CML patients (n = 5) before and at 1 h after witnessed drug administration. Platelets of patients were stimulated with 12.5 ng/mL convulxin for 15 min in PRP. PS exposure (A) and thrombin generation were investigated. Panel (B) shows representative overlay thrombin generation curves. Time parameters of thrombin generation, lagtime (C); time to peak (D); and quantity of generated thrombin, peak (E) and ETP (F), were evaluated.
Figure 4
Figure 4
Nilotinib treatment does not affect PS expression and thrombin generation in PRP of CML patients. Samples were from nilotinib treated CML patients (n = 5) before and at 1 h after witnessed drug administration. Platelets of patients were stimulated with convulxin for 15 min in PRP. PS exposure (A) and thrombin generation were investigated. Panel (B) shows representative overlay thrombin generation curves. Time parameters of thrombin generation, lagtime (C); time to peak (D); and quantity of generated thrombin, peak (E) and ETP (F), were evaluated.
Figure 5
Figure 5
Integrin αIIbβ3 activation and clot retraction in PRPs of TKI treated CML patients. Blood was collected from dasatinib (n = 5) or nilotinib (n = 5) treated CML patients before and at 1 h after witnessed drug administration. Platelets of patients were stimulated with convulxin in PRP. The percent of active conformation of integrin αIIbβ3 is shown by FITC-PAC1 binding in the cases of dasatinib or nilotinib treated patients respectively (Panels A and B). Panels C and D show results of clot retraction in PRPs of dasatinib or nilotinib treated patients.
Figure 6
Figure 6
Dasatinib inhibits the phosphorylation of C-terminal tail and activation loop of Sarcoma family kinases (SFK) in CML patients. Lysates of platelets from dasatinib (n = 3) or nilotinib (n = 3) treated patients were examined with the indicated antibodies against the inhibitory (A–C) and the full activatory (D–F) phosphorylation of Lyn, Fyn, and Src kinases. The quantity of phosphorylated SFKs in platelet lysates before drug administration (0) was regarded as 100 %. Columns show the mean and SEM.
Figure 7
Figure 7
Phosphorylation status of SFK after dasatinib treatment upon convulxin activation. Gel filtrated human platelets from a healthy donor were incubated with 10 or 100 nM dasatinib or vehicle (0.2% DMSO) for 10 min. After incubation, platelets were left non activated or were activated with 12.5 ng/mL convulxin for 15 min. Platelet lysates were immunoblotted with phosphospecific antibodies against the inhibitory (A) and the full activatory (B) phosphorylation of Lyn, Fyn, and Src kinases.
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