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.

Annexin A5↗

PubMed1996

The role of electrostatic interactions in the assembly of the factor X activating complex on both activated platelets and negatively-charged phospholipid vesicles.

Factor X was activated by factor IXa on the surface of either activated platelets or artificial lipid vesicles in the presence of different NaCl concentrations. The Vmax of reactions using platelets was optimal at physiologic [NaCl] both in the absence and in the presence of factor VIIIa. In contrast, the Vmax of reactions using vesicles decreased with increasing [NaCl] in the absence of factor VIIIa, and increased with increasing [NaCl] when cofactor was present. In the absence of factor VIIIa, the EC50FIXa' although stable to changes in [NaCl] in platelet-supported reactions, was found to increase significantly as [NaCl] increased in vesicle-supported reactions and correlated with the decreased Vmax. Thus, in contrast to platelet-supported reactions, enzyme interaction with negatively-charged vesicles was highly dependent upon electrostatic interactions. In the presence of factor VIIIa, the EC50FIXa of vesicle-supported reactions decreased with increasing [NaCl], indicating that interactions between FIXa and FVIIIa can increase enzyme affinity when fewer ionic interactions are favored. The EC50FVIIIa was insensitive to changes in [NaCl] on both surfaces. The K(m)app derived from platelet-supported titrations of factor X was lowest just above physiological [NaCl], whereas on vesicles K(m)app was minimal at the lowest [NaCl] tested. Thus, the direct interaction of factor X and factor IXa with the artificial lipid surface is highly dependent upon ionic interactions with the negatively-charged polar heads of phospholipids. However, the interaction of factor IXa and factor X with the activated platelet surface must rely both on electrostatic interactions with lipid and on other interactions provided by surface proteins.

Animals↗

PubMed1995