Effect of SLK on platelet oxidative fragmentation and dissolution of ex vivo collagen-induced platelet aggregates. (A) Effect of SLK on platelet fragmentation and (B) platelet DCFH oxidation. C indicates control IgG; and Pt, patient IgG. Black, gray, horizontal, vertical, and white bars represent serial dilutions of Ab, starting at 12.5 μg /mL. Data are representative of 2 different experiments. (C) Effect of prestimulated platelet PAR-1 on induction of platelet fragmentation with SLK. Gel-filtered platelets were treated with the thrombin PAR-1 agonist TFLLRN (200μM) for 30 minutes at 37°C and then followed with various SLK concentrations (5-40 μg/mL) for 4 hours. Gray and black bars represent without and with TFLLRN (T) prestimulation, respectively. Note increased sensitivity of activated platelets to SLK at low SLK concentration. (D) Collagen platelet aggregate. Platelet aggregates were prepared by incubating gel-filtered platelets with 1 μg/mL collagen at 37°C for 1 hour in Tyrode buffer with intermittent shaking. A11 or SLK or Ctl scFv (13CG2) was incubated with aggregates at different time points at 37°C. The numbers of platelet/aggregates were then counted. Data and SEM are given for 3 separate experiments at 0.85μM reagent in which each time point represents 5 measurements.