Effect of ESH4 and ESH8 on platelet-supported activity of fVIII. (A) Inhibition of 1 unit/mL fVIII activity by ESH4 and ESH8 was evaluated against an fVIII concentration curve in reconstituted platelet-rich plasma. Clotting was initiated by the simultaneous addition of factor XIa, thrombin receptor activation peptides, and Ca⁺⁺. A log-linear plot demonstrates sensitivity to fVIII. ESH4 or ESH8 was incubated with fVIII in the presence of 10% fVIII-deficient plasma for 1 hour prior to mixing with additional fVIII-deficient plasma and initiation of clotting. ESH4 inhibited activity to a greater extent than ESH8. Results are mean ± SEM for triplicates, representative of 3 experiments performed in full and 5 performed with fewer fVIII concentrations. (B) Bar graph comparing residual fVIII activity in various plasma-based activities. aPTT and chromogenic assay results represents mean ± SEM using commercial aPTT and chromogenic reagents. Inhibition was also evaluated in reconstituted plasma and platelet-rich plasma lacking VWF (aPTT [-VWF]), (act platelet [-VWF]). Results are from 4 experiments (aPTT, chromogenic); activated platelets mean ± SD for 3 experiments, activated platelets without VWF (1 experiment). (C) Bar graph comparing residual fVIII activity in the presence of 10 µg/mL ESH8. aPTT without VWF (aPTT [-VWF]).³²  Values on activated platelets are mean ± SEM for 2 experiments and 1 experiment for plasma lacking VWF.