Multiparameter Evaluation of the Platelet-Inhibitory Effects of Tyrosine Kinase Inhibitors Used for Cancer Treatment

Figure 1. Affinity profile of common tyrosine kinase inhibitors (TKIs) for the relevant tyrosine kinases implicated in platelet activation, presented as a heatmap of the affinity-based dissociation constants (Kd) [39,40,41] or IC50 values [42] of the indicated TKIs. Values are from binding experiments with the indicated purified kinases, including Src family kinases (SFK), Syk, Tec family kinases (TFK), focal adhesion kinases (FAK), and MEK. Color coding shows the highest affinity (lowest Kd or IC50) in deep red and the lowest affinity (highest Kd or IC50) in white. Data are based in [24].

Figure 2. Dose-dependent effects of TKIs on platelet aggregation induced by collagen. Washed platelets or PRP (250 × 109/L) were incubated with the vehicle (<0.1% DMSO) or TKI for 10 min. Dose–response curves of the inhibition of aggregation induced by collagen Type I (1 µg/mL) are shown for (A) dasatinib, (B) fostamatinib, (C) sunitinib, (D) pazopanib, (E) axitinib, (F) cabozantinib, (G) lapatinib, or (H) vatalanib, measured in either PRP (solid line) or washed platelets (dotted line). Maximum aggregation responses were determined at 8 min; data are shown as means ± SEM (n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3. Effects of eight TKIs on whole-blood thrombus formation under flow on different thrombogenic surfaces. Blood samples were pre-incubated with the vehicle or TKI for 10 min. Samples were then recalcified in the presence of PPACK and perfused for 3.5 min at a wall shear rate of 1000 s−1 over six different coated surfaces. After perfusion, the formed thrombi were stained with AF568-annexin A5, FITC-α-fibrinogen, and AF647-α-CD62P to detect PS exposure, integrin αIIbβ3 activation, and P-selectin expression, respectively. (A) Representative brightfield images of control or fostamatinib-treated blood after flow over collagen Type I, collagen Type III, vWF plus laminin, vWF plus rhodocytin, vWF plus ristocetin, or vWF plus fibrinogen. (B) Subtraction heatmap representing significantly altered effects of parameters P1–P8 (normalized and analyzed as in Figure S2). Note: P1, morphological score of platelet adhesion and thrombus formation; P2, surface area coverage of the adhered platelets; P3, platelet aggregate contraction score; P4, platelet aggregate multilayer score; P5, coverage of multi-layered platelet aggregation; P6, αIIbβ3 activation; P7, P-selectin expression; and P8, PS…

Figure 4. Effects of different TKIs on GPVI-induced intracellular calcium responses. Fura-2-loaded platelets (200 × 109/L) were pre-incubated with vehicle, dasatinib (100 nM), fostamatinib (33 μM), sunitinib (10 μM), pazopanib (10 μM), axitinib (33 μM), cabozantinib (10 μM), lapatinib (33 μM), or vatalanib (10 μM) for 10 min and stimulated with CRP-XL (10 µg/mL) in the presence of 2 mM CaCl2. The histograms show the maximal rises in [Ca2+]i (n = 6). The black dotted line indicates the average of the control values. Data are shown as means ± SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5. Effect of TKIs on populations of activated platelets in response to GPVI stimulation. Washed platelets (25 × 109/L) were incubated with vehicle, fostamatinib (33 µM), cabozantinib (33 µM), or vatalanib (33 µM) for 10 min. Platelet activation was induced by 5 µg/mL of CRP-XL for 15 min and simultaneously labeled for activated integrin αIIbβ3 (PAC1-FITC), α-granule secretion (αTLT1-BV421 and αCD62P-PerCP Cy5.5) and δ-granule/lysosome secretion (αCD63-APC). Separately, PS exposure was induced using 5 µg/mL of CRP-XL, in addition to 4 nM of thrombin for 60 min at 37 °C, labeled by annexin A5-PE. (A) Histograms of integrin αIIbβ3 activation, TLT1, P-selectin, and CD63 expression, as well as PS exposure, are shown as percentages of the control condition. (B) Distribution profiles of the platelet populations after stimulation by CRP-XL. (i) Two-dimensional plots visualizing the platelet fractions per population in the presence of vehicle, fostamatinib, cabozantinib, or vatalanib. (ii) Characterization of CRP-XL-induced platelet populations based on the relative expression levels of activation markers determined by FlowJo analysis using the tSNE and FlowSOM plugins (see Table S2). (C)…

Figure 6. Platelet aggregation and activation induced by different activation pathways in the presence of different TKIs. (A) Washed platelets (250 × 109/L) were incubated with vehicle (control), fostamatinib (33 µM), cabozantinib (33 µM), or vatalanib (33 µM) for 10 min. Platelet aggregation was then induced with 1 µM of 2MeS-ADP, 1 µM of U46619 (thromboxane A2 analogue), 1 µg/mL of rhodocytin, or 4 nM of thrombin. Aggregation responses were measured for 8 min. Histograms show aggregation as a percentage of the control (dotted line). (B,C) Washed platelets (25 × 109/L) were incubated with the vehicle (control) or TKI for 10 min. Platelets were activated by (B) 2 µM of 2MeS-ADP or (C) 4 nM of thrombin for 15 min. Simultaneous labeling was performed for activated integrin αIIbβ3 (PAC1-FITC), α-granule secretion (αTLT1-BV421, αCD62P-PerCP Cy5.5), and δ-granule/lysosome secretion (αCD63-APC). Histograms for integrin αIIbβ3 activation, TLT1, P-selectin, and CD63 expression are given as percentages of the control condition (dotted line). Data are shown as means ± SEM (n = 6); * p < 0.05, ** p < 0.01, and *** p < 0.001.