HIT IC-mediated platelet aggregation in vitro. (A) Human PRP (250 000 platelets/μL) was incubated at 37°C for 15 minutes with aliquots of a mixture of PF4 and heparin (1.5:1 molar ratio) containing 5 μg PF4. The PRP was then incubated at 37°C for an additional 15 minutes with PRT318 at the concentrations indicated. Aggregation was initiated by the addition of the HIT-like antibody KKO (80 μg/mL). Platelet aggregation was assessed under stirring conditions by the increase in light transmission over time. The final percentage aggregation was recorded. In the absence of the PRT318, HIT IC-induced aggregation in human PRP was 50% to 60%. At PRT318 concentrations of more than or equal to 0.3μM, there was complete inhibition. Data are mean ± SEM; n = 3 each group. *P < .007. **P < .0002. (B) Washed ¹⁴C-serotonin-loaded platelets were incubated with PRT318 (1.0μM) or vehicle only before incubation for 1 hour at room temperature with 0.1 or 100 IU /mL heparin and human HIT sera. Positive results are those with > 20% serotonin released at a heparin concentration of 0.1 IU/mL and < 20% with a heparin concentration 100 IU/mL. Data are mean ± SEM; n = 4 for each treatment group. *P < .0001. □ represents control (vehicle only); and ■, PRT treated.