Abstract
The physiological inhibitor of tissue factor (TF).factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI(FL)) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF.FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF.PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF.PSPC) was measured in the presence of preformed complexes of FXa.TFPI(FL) or FXa.TFPI(1-161) (TFPI lacking the third Kunitz domain and C terminus). At TF.PC, FXa.TFPI(FL) and FXa.TFPI(1-161) showed similar rate constants of inhibition (0.07 x 10(8) M(-1) s(-1) and 0.1 x 10(8) M(-1) s(-1), respectively). With phosphatidylserine present, the rate constant of inhibition for FXa.TFPI(FL) increased 3-fold compared with a 9-fold increase in the rate constant for FXa. TFPI(1-161). Incubation of TF.PSPC with FXa.TFPI(FL) in the absence of FVIIa followed by depletion of solution FXa.TFPI(FL) showed that FXa.TFPI(FL) remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa.TFPI(1-161) or at TF.PC membranes. These data suggest that the membrane-bound pool of FXa.TFPI(FL) may be of physiological importance in an on-site regulation of TF.FVIIa activity.
Highlights
The physiological inhibitor of tissue factor (TF)1⁄7factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPIFL) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes
Incubation of TF1⁄7PSPC with FXa1⁄7TFPIFL in the absence of FVIIa followed by depletion of solution FXa1⁄7TFPIFL showed that FXa1⁄7TFPIFL remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa1⁄7TFPI1–161 or at TF1⁄7PC membranes. These data suggest that the membrane-bound pool of FXa1⁄7TFPIFL may be of physiological importance in an on-site regulation of TF1⁄7FVIIa activity
Our study showed that negatively charged phospholipid surfaces act as a safety net in the sense that FXa1⁄7TFPIFL complexes readily bind to the surface
Summary
It remains unclear what the role is of lipid-protein interactions and of the TFPI C terminus when TF is embedded in a macroscopic phospholipid membrane Answering these questions could provide insight in the role of (TF-bearing) cell membranes in the regulation of the TF1⁄7FVIIa-catalyzed initiation of the blood coagulation process. To mimic the processes occurring at TF-bearing cell surfaces as closely as possible, we made use of TF embedded in a macroscopic phospholipid surface to which the reactants were supplied under well defined flow conditions This approach allows a quantitative study of the kinetics of inhibition of TF1⁄7FVIIa-catalyzed FX activation as a function of the phospholipid composition of the membrane and the phospholipid-binding properties of the FXa1⁄7TFPI complex. These phospholipid-bound FXa1⁄7TFPIFL complexes show a potent inhibitory activity against TF1⁄7FVIIa present at the same membrane
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