Annexin V inhibition of factor IXa-catalyzed factor X activation on human platelets and on negatively-charged phospholipid vesicles.

Annexin V was found to inhibit factor IXa-catalyzed factor X activation on both thrombin-activated human platelets and artificial lipid vesicles containing phosphatidylserine, supporting previous observations of the importance of negatively-charged lipid in potentiating the reaction. Annexin V reduced the Vmax of factor X activation in factor IXa titrations on the platelet surface with an IC50 of 4 nM in the absence of thrombin-activated factor VIII (factor VIIIa), and 4.5 nM in its presence, whereas there was no effect on the EC50,FIXa. This noncompetitive inhibition is consistent with interference of recognition of the factor IXa binding site on the platelet, which was confirmed by equilibrium binding of [125I]-factor IXa to thrombin-activated platelets where, in the absence of factor VIIIa and factor X, annexin V reduced the number of factor IXa binding sites/platelet from 610 to 320, without changing the Kd,app. In the presence of factor VIIIa and factor X, annexin V reduced the number of binding sites, but also raised the Kd,app. Although factor VIIIa improved the affinity of factor IXa for the lipid surface from Kd approximately 60 nM in its absence to Kd 1 nM in its presence, addition of annexin V to factor IXa titrations on lipid vesicles in the presence of factor VIIIa increased the EC50,FIXa with an IC50 of 1.5 nM, without affecting the Vmax. These data provide evidence that factor IXa, although requiring negatively-charged phospholipid for part of its binding site, is accommodated differently on platelets and on artificial vesicles.