Figure 2. Impact of intravenous PTL060.

A and B, Two color immunofluorescence images of cross sections through aorta harvested at 6 hours post‐intravenous injection of 10 μg/g PTL060 (A) or equimolar (5 μg/g) chemically modified hirulog analogue HLL (B) stained with isotype control or anti‐HLL antibody as indicated. Blue ‐DAPI. (Please note Sections examined at all other time points showed less evidence of binding by PTL060). C through E, Flow cytometric assessment of binding to erythrocytes (C), CD11b+ leukocytes (D) and platelets (gated on CD41+) (E) obtained from mice given either saline control, chemically modified hirulog analogue HLL (2.5 μg/g), or PTL060 (5 μg/g). Graphs show percentage of population binding anti‐HLL antibody (left column) and the geometric mean of the fluorescence intensity of binding (right column). Samples were taken from mice at the time points post‐injection as indicated. n=3 per group. F and G, Thrombin clotting times (seconds±SEM) in plasma. Blood was collected into citrated tubes at the times specified under terminal anesthesia before spinning at 15 000g for 10 minutes to separate out cellular components and plasma. Thrombin times performed by adding 25 U (F) or 50 U (G) thrombin to 100 μL of plasma and recording time for a fibrin clot to form. Mice (n=3 per group) injected with PTL060 (5 μg/g—filled squares) or equimolar dose of chemically modified hirulog analogue HLL (2.5 μg/g—circles). Plasma from mice treated with PTL060 was centrifuged for a further 20 minutes at 10 000g, to remove any membrane bound PTL060, before repeating assessments (open squares). H, Graph depicting tail bleeding times in minutes±SEM at various times after intravenous injection of control phosphate buffered saline (open circles), PTL060 10 μg/g (squares) or equimolar (5 μg/g) chemically modified hirulog analogue HLL (closed circles). N=6 per group. Mouse euthanized at 20 minutes if tail still bleeding.