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The physiological inhibitor of tissue factor (TF)zfactor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPIFL) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TFzFVIIacatalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TFzPC) or 25% phosphatidylserine and 75% phosphatidylcholine (TFzPSPC) was measured in the presence of preformed complexes of FXazTFPIFL or FXazTFPI1‐161 (TFPI lacking the third Kunitz domain and C terminus). At TFzPC, FXazTFPIFL and FXazTFPI1‐161 showed similar rate constants of inhibition (0.07 3 10 8 M 21 s 21 and 0.1 3 10 8 M 21 s 21 , respectively). With phosphatidylserine present, the rate constant of inhibition for FXazTFPIFL increased 3-fold compared with a 9-fold increase in the rate constant for FXazTFPI1‐161. Incubation of TFzPSPC with FXazTFPIFL in the absence of FVIIa followed by depletion of solution FXazTFPIFL showed that FXazTFPIFL remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXazTFPI1‐161 or at TFzPC membranes. These data suggest that the membrane-bound pool of FXazTFPIFL may be of physiological importance in an on-site regulation of TFzFVIIa activity.

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−fa...

The structure and function of biological membranes can be expected to be sensitive to the extent of hydrophobic matching between the length of the membrane-spanning part of intrinsic membrane proteins and the hydrophobic thickness of the lipid bilayer. To gain insight into the consequences of hydrophobic mismatch on a molecular level, we have carried out systematic studies on well-defined peptidehpid complexes, using artifical transmembrane peptides, anchored to the membrane by tryptophan residues. It is shown that hydrophobic mismatch can result in a dramatic change in lipid organization, and that the presence of interfacially localized aromatic amino acid residues is important for determining the exact consequences of hydrophobic mismatch.

SummaryTissue factor : factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa : TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa : TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa : TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Arg199-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa : TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa : TFPI towards TF : factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF : factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

The basic C-terminus of Tissue Factor Pathway Inhibitor (TFPI) appears to be essential for its anticoagulant activity when tested in a diluted thromboplastin prothrombin time assay. Although the data reported so far have increased our knowledge about the C-terminus as a major binding site for heparin, lipoproteins and phospholipids, it is still unclear how this region of TFPI plays a role in its anticoagulant mode of action. We earlier reported that in the presence of phospholipid the rate of association of factor Xa with full length TFPI (FL-TFPI) is about 10-fold faster than with C-terminus truncated TFPI. This in turn makes that, in vitro, full length TFPI is a more potent inhibitor of tissue factor – factor VIla catalyzed factor X activation than truncated TFPI. Binding studies, utilizing an ellipsometer, revealed that in contrast to the complex of C-terminus truncated TFPI (TFPI1-161) and factor Xa, the FL-TFPI.factor Xa complex has a high affinity for negatively charged phospholipids. However, when examined in a tubular flow reactor containing tissue factor embedded in a phospholipid bilayer composed of 25 mol% phosphatidyl-serine/75 mol% phosphatidylcholine, no differences in the potency of FL-TFPI and TFPI 1–161 to inhibit factor X activation were found. The two variants of TFPI did show an interesting difference though. We found that the quaternary complex of TF.factor VIla.FL-TFPI.factor Xa was much more stable than the complex containing TFPI1-161. This difference could not be attributed to their different phospholipid-binding properties since the same difference in stability was found on membranes that contained only DOPC.

The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin, The rate constant of association of prothrombinase with full-length TFPI was 2.1 x 10(7) M(-1). s(-1) and 0.05 x 10(7) M(-1). s(-1) for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65 x 10(-4) s(-1) in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.