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.

Cellular factor XIII (cFXIII, FXIII-A₂), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.

Bortezomib (BTZ) has demonstrated its efficacy in several hematological disorders and has been associated with thrombocytopenia. There is controversy about the effect of BTZ on human platelets, so we set out to determine its effect on various types of platelet samples. Human platelets were investigated in platelet-rich plasma (PRP) and as gel-filtered platelets (GFPs). Mitochondrial inner membrane potential depolarization and phosphatidylserine (PS) and P-selectin expression levels were studied by flow cytometry, while thrombin generation was measured by a fluorescent method. In PRP, BTZ caused negligible PS expression after 60 min of treatment. However, in GFPs, PS expression was dose- and time-dependently increased in the BTZ-treated groups, as was P-selectin. The percentage of depolarized cells was also higher after BTZ pretreatment at both time points. Peak thrombin and velocity index increased significantly even with the lowest BTZ concentration (p = 0.0019; p = 0.0032) whereas time to peak and start tail parameters decreased (p = 0.0007; p = 0.0034). The difference between PRP and GFP results can be attributed to the presence of plasma proteins in PRP, as the PS-stimulating effect of BTZ could be attenuated by supplementing GFPs with purified human albumin. Overall, BTZ induces a procoagulant platelet phenotype in an experimental setting devoid of plasma proteins.

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective drugs in the treatment of patients with chronic myeloid leukemia. However, based on clinical studies, ponatinib was associated with the development of thrombotic complications. Since endothelial cells (ECs) regulate blood coagulation, their abnormal phenotype may play a role in the development of thrombotic events. We here aimed to investigate the effect of ponatinib on the procoagulant activity of cultured endothelial cells in vitro. Human coronary artery endothelial cells (HCAECs) were incubated with 50, 150, and 1000 nM of ponatinib. Subsequently, phosphatidylserine (PS) exposure and endothelial microvesicles (EMVs) were measured by flow cytometry. In addition, EC- and EMV-dependent thrombin generation was analyzed. To investigate pro-apoptotic effects of ponatinib, the level of Bax and Bcl-xL proteins were studied using Western blot and F3, THBD, and VCAM1 mRNAs were quantified by qPCR. Therapeutic concentrations of ponatinib significantly increased PS expression on ECs and the amount of EMVs which significantly shortened the time parameters of thrombin generation. In addition, these changes were associated with an increased ratio of Bax and Bcl-xL proteins in the presence of the decreased THBD mRNA level. Overall, ponatinib enhances the procoagulant activity of ECs via inducing apoptosis, which may contribute to thrombotic events.

Percutaneous coronary intervention (PCI) is a frequently performed treatment option for recanalization in patients with chronic total occlusion (CTO). As CTO-PCIs are often complicated and challenging for interventionalists, the stressful and damaging nature of the procedure can be remarkable, thus platelets can be easily activated. Our aim was to investigate the effect of CTO-PCI on platelet activation and the expression of selected circulating microRNAs (miR) of platelet and endothelium origin after CTO-PCI. In this study, 50 subjects after CTO-PCI were enrolled. Blood samples were obtained before PCI, at 2 days and 3–6 months after the procedure to measure the degree of platelet activation and the level of plasma miR-223, miR-181b, and miR-126. Patients were divided based on the characteristics of the intervention. Patients with higher Japanese CTO scores and longer duration of PCI showed significantly elevated platelet P-selectin positivity (p = 0.004 and p = 0.013, respectively) 2 days after the procedure compared to pre-PCI and increased concentration of soluble P-selectin 3–6 months after the intervention (higher Japanese CTO score: p = 0.028 and longer duration of PCI: p = 0.023) compared to baseline values. Shorter total stent length caused a significantly lower miR-181b expression at 3–6 months after the intervention (p = 0.031), while no difference was observed in miR-223 and miR-126. One stent thrombosis occurred during the follow-up period. Although these technically challenging CTO-PCIs may cause enhanced platelet activation right after the intervention and long-term endothelial cell dysfunction, these interventions are not associated with more adverse clinical events.