Skip to main content
. 2020 May 28;106(7):1968–1978. doi: 10.3324/haematol.2019.223529

Figure 2.

Figure 2.

Genetic deletion and pharmacological inhibition of Pim-1 kinase attenuates thrombus formation on collagen, but does not cause bleeding. (A) DiOC6 loaded mouse whole blood from Pim1+/- (black) or Pim1-/- (red) mice was perfused through collagen-coated (100 μg/mL) Vena8Biochips at a shear rate of 1500 s-1. (i) Representative images taken at the end of recording are shown. (ii) Thrombus formation was determined after 4 min by comparing the percentage area covered. (B) Tail bleeding in Pim1+/- or Pim1-/- mice represented as time to cessation of bleeding (s). (C-E) DiOC6 loaded human whole blood was pretreated with vehicle (black) or 100 μM AZD1208 (red) for 10 min before perfusion through collagen-coated (100 μg/mL) Vena8Biochips at (C) an arterial shear rate of 20 dyn/cm2, (D) a pathological shear rate of 135 dyn/cm2, or (E) a venous shear rate of 4.5 dyn/cm2. The concentration of 100 μM AZD1208 was chosen because of the reduced bioavailability of AZD1208 in plasma. Thrombus formation was determined after 5 min (pathological shear) or 10 min (arterial and venous shear) by comparing percentage of maximum vehicle-treated fluorescence intensity, which measures both surface area coverage and thrombus size, in the vehicle and treated samples. (i) Representative images taken at the end of recording are shown. (ii) Data expressed as percentage of maximum vehicle-treated fluorescence intensity. (F) Thrombus formation was determined in vivo following ferric chloride-induced injury in mice pretreated for 10 mins with vehicle or 100 μM AZD1208. DiOC6 was used to enable visualization of platelets. (i) representative images taken at 0, 300, 600 and 900 s. (ii) Data expressed as time to occlusion (s). (G) Tail bleeding determined as time to cessation of bleeding (s) in mice pretreated with vehicle or 100 μM AZD1208 for 10 min. Results are mean ± standard error of mean for n≥3, *P≤0.05, **P≤0.01 ****P≤0.001 in comparison to vehicle control; where normalized data are shown statistics were performed prior to normalization.