Transient high affinity binding of tissue factor pathway inhibitor-factor Xa complexes to negatively charged phospholipid membranes.

The interaction of tissue factor pathway inhibitor (TFPI), factor Xa, and TFPI-factor Xa complexes with negatively charged phospholipid membranes composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine was studied by ellipsometry. The binding of TFPI alone was negligible; factor Xa bound with moderate affinity, with a dissociation constant Kd = 42 nM. Formation of the TFPI-factor Xa complex drastically enhanced the affinity for phospholipid membranes, Kd = 5 nM, compared to that of either protein alone. TFPI1-161, a TFPI variant lacking the third Kunitz domain and the positively charged C-terminus did not enhance binding affinity of the factor Xa. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data, although upon longer residence at the lipid membrane the desorption rate of TFPI-factor Xa complexes became slower, indicating an increase in affinity with longer residence of the TFPI-factor Xa complexes at the membrane. In contrast, binding of TFPI-factor Xa complexes in the presence of an excess factor Xa was transient; maximal binding is followed by a slow desorption of the complex. Immunoblot analysis revealed that this desorption was accompanied with cleavage of TFPI by membrane-bound factor Xa. Collectively, our results show that phosphatidylserine containing membranes will accumulate tightly bound TFPI-factor Xa complexes, and that uncomplexed, phospholipid-bound, factor Xa, will cause limited proteolysis of TFPI accompanied by simultaneous release of these complexes from the phospholipid membrane.