Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Figures

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  Figure 1. Sustained Syk phosphorylation in thrombin-stimulated platelet is integrin-dependent. Washed platelets at 5 × 108/mL were stimulated with Horm collagen (30 µg/mL), thrombin (1 U/mL), TRAP (10 µM) or fibrinogen (200 µg/mL) in the presence or absence of eptifibatide (9 µM). Activation was stopped at stated time after addition of the agonist. (A) Representative blot of Syk tyrosine phosphorylation from 6 donors. (Ai) Whole cell lysates were immunoprecipitated with anti-Syk mAb and probed with anti-phospho-tyrosine mAb 4G10; (Aii) Percentage of Syk tyrosine phosphorylation, based on Western blots, from 6 donors (mean ± s.d.); (Aiii) Aggregation trace of platelets stimulated with thrombin (1 U/mL), representative of 3 donors, and (Aiv) graph showing aggregation as % light transmission (mean ± s.d.) after 50 min of agonist stimulation. (B) Representative Western blot (Bi), and calculated percentage of Syk (Bii), PLCγ2 (Biii) and LAT (Biv) phosphorylation in response to thrombin (Thromb) from 5 donors (mean ± s.d.). Horm collagen (Col) was used as positive control. (C) Representative blot (Ci); mean ± s.d. of Syk (Cii), PLCγ2 (Ciii) and LAT (Civ) phosphorylation in response to collagen…

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  Figure 2. Inhibition of Syk causes loss of contraction and platelet adhesion on collagen and plaque material. Recalcified blood from 6 donors was perfused for 7 min on Horm collagen and plaque at room temperature at a shear rate of 1000 s−1. (A,B) Representative images of aggregates formed on collagen (A) and plaque (B), taken during post-perfusion at times 0 and 10 min with vehicle or with PRT-060318 (n = 6). Scale bar = 100 µm. (C,D) Disaggregation on collagen (C) or on plaque (D) was monitored by taking brightfield images every 60 sec and measured using morphological (Ci,Di), contraction (Cii,Dii) and multilayer scores (Ciii,Diii) [16]. Aggregates were post-perfused with rinse buffer ± vehicle (DMSO) or the Syk inhibitor PRT-060318 (10 µM). Data are shown as mean ± s.e.m. * p < 0.05, ** p < 0.005.

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  Figure 3. Platelet activation imaging of thrombi postperfused under condition of Syk inhibition. Recalcified blood samples labelled with APC-αCD41a mAb (purple), FITC-fibrinogen (green) and PE-αGPVI mAb (red) was perfused for 7 min over immobilised collagen or plaque material at room temperature and arterial shear rate (1000 s−1). Post-perfusion for 3 min was with rinse buffer containing vehicle (DMSO) or Syk inhibitor PRT-060318 (10 µM). (A) Representative microscopic images, showing remaining aggregates on collagen (Ai) or plaque (Aii) after 3 min of post-perfusion with vehicle or PRT-060318 (n = 4 donors). (B) Graphs showing scores of thrombus morphology (Bi), thrombus contraction (Bii) and thrombus multilayer (Biii) [16]. (C) Graphs of fluorescence intensity (arbitrary units, a.u.) of platelet aggregates on collagen (Ci) or plaque material (Cii) fixed after 3 min of post-perfusion. Scale bar = 50 µm. Data are shown as mean ± s.d., * p < 0.05, ** p < 0.005, *** p < 0.0005, one-tailed Student’s paired t-test.

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  Figure 4. Inhibition of Syk causes the same extent of disaggregation observed with antagonists of adenosine diphosphate (ADP) and thromboxane A2 (TxA2). Recalcified blood was perfused for 7 min on Horm collagen at 37 °C at arterial shear (1000 s−1), and post-perfused for 10 min with rinse buffer with vehicle (DMSO) or inhibitor of Syk, Src or Btk (PRT-060318 10 µM, dasatinib 10 µM or ibrutinib 7 µM, respectively). (A) Representative images of aggregates at 0, 5, and 10 min of post-perfusion with vehicle, PRT-060318 (n = 5) or dasatinib (n = 6). (B–D) Graphs showing extent of disaggregation, as measured from scores of thrombus morphology, contraction and multilayer,16 at every 60 s (Bi,Ci,D) or at 10 min (Bii,Cii) of post-perfusion with either vehicle, PRT-060318 (PRT) (Bi,ii), dasatinib (Ci,ii) or ibrutinib (D), alone or combined with indomethacin (Indo, 10 µM) and ticagrelor (Tica, 10 µM). Scale bar = 50 µm. Data are shown as mean ± s.e.m. * p < 0.05, ** p < 0.005, *** p < 0.0005, one-tailed paired Student’s t-test.

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  Figure 5. Blockage of GPVI with the anti-GPVI nanobody 21 (Nb21) causes minor thrombus instability. Recalcified blood was perfused for 7 min on Horm collagen at 37 °C at arterial shear (1000 s−1), and successively post-perfused for 10 min with rinse buffer and blocking anti-GPVI nanobody Nb21 (500 nM). (A) Representative images after perfusion of blood from 6 donors, taken during post-perfusion at time 0, 5 and 10 min. (B) Graphs showing extent of disaggregation, monitored from recorded brightfield images every min, and measured as morphological, contraction and multilayer scores [16]. Scale bar = 50 µm. Data are shown as mean ± s.e.m. * p < 0.05, ** p < 0.005 one-tailed Student’s paired t-test.