10.  Inhibition of factor VIII activation by thrombin

Factor VIII is converted to factor VIIIa by thrombin during the aPTT reaction, and factor VIIIa is required for efficient activation of factor X by factor IXa.  Diminished activation of factor VIII, therefore, can prolong the factor IXa clotting time.  The graph below shows that monoclonal Fab fragments inhibited thrombin-induced activation of factor VIII by ~80% when assayed in the presence of von Willebrand factor, which binds and stabilizes factor VIII.  The half-maximal effect occurred at ~30 nM Fab.

Factor VIII (1 nM) was incubated with 0.2 nM thrombin and various concentrations of monoclonal Fab in 0.15 M NaCl, 20 mM Hepes, 5 mM CaCl2, 1 mg/ml poly(ethyleneglycol) 8000, pH 7.4, at room temperature in the presence of 200 nM von Willebrand factor. After 5 min, the mixture was diluted in the same buffer to 0.8 nM factor VIII in 2.5 nM factor IXa, 20 µM PCPS, and 200 nM factor X. The initial rate of factor X activation was measured using a chromogenic substrate for factor Xa.  Under the conditions of the assay, the initial rate of factor X activation was linearly proportional to the concentration of activated factor VIII.