Inhibition of Tissue Factor-Factor VIIa-catalyzed Factor X Activation by Factor Xa-Tissue Factor Pathway Inhibitor

Irene Salemink, Jo Franssen, George M. Willems, H. Coenraad Hemker, Theo Lindhout
1999 Journal of Biological Chemistry  
The physiological inhibitor of tissue factor (TF)⅐factor 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 TF⅐FVIIacatalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF⅐PC) or 25% phosphatidylserine and 75% phosphatidylcholine
more » ... was measured in the presence of preformed complexes of FXa⅐TFPI FL or FXa⅐TFPI 1-161 (TFPI lacking the third Kunitz domain and C terminus). At TF⅐PC, FXa⅐TFPI FL and FXa⅐TFPI 1-161 showed similar rate constants of inhibition (0.07 ؋ 10 8 M ؊1 s ؊1 and 0.1 ؋ 10 8 M ؊1 s ؊1 , respectively). With phosphatidylserine present, the rate constant of inhibition for FXa⅐TFPI FL increased 3-fold compared with a 9-fold increase in the rate constant for FXa⅐TFPI 1-161 . Incubation of TF⅐PSPC with FXa⅐TFPI FL in the absence of FVIIa followed by depletion of solution FXa⅐TFPI FL showed that FXa⅐TFPI FL remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa⅐TFPI 1-161 or at TF⅐PC membranes. These data suggest that the membrane-bound pool of FXa⅐TFPI FL may be of physiological importance in an on-site regulation of TF⅐FVIIa activity. Blood coagulation in vivo is initiated when circulating factor VII(a) binds in a calcium-dependent way to its cofactor, tissue factor (TF) 1 (see Refs. 1 and 2 for a review). This complex formation results in enhanced catalytic activity of factor VIIa (FVIIa), which via limited proteolysis, activates factors X (FX) and IX (FIX) (3). TF is a transmembrane glycoprotein, which under normal conditions is expressed only in extravascular tissues (4, 5). The main physiological regulator of TF-induced blood coag-ulation is tissue factor pathway inhibitor (TFPI) (6, 7), a single chain glycoprotein of 42 kDa and a member of the Kunitz family of serine protease inhibitors. TFPI contains an acidic N terminus, followed by three tandemly repeated Kunitz-type inhibition domains, and a basic C-terminal tail (8). Site-directed mutagenesis has revealed that the first Kunitz domain binds to FVIIa and that the second Kunitz domain interacts with the active site of FXa (9). No such functions could be attributed to the third Kunitz domain (10). Yet, various interactions have been ascribed to this domain, e.g. with lipoproteins and heparin, but their importance for the inhibitory function of TFPI is not clear (11, 12). On the other hand, the basic C-terminal region of TFPI (residues 240 -276) has been shown to play a crucial role in the anticoagulant activity of this inhibitor (13, 14). Despite numerous studies, it remains unclear how this basic C terminus modulates the anticoagulant activity of TFPI (15-19). TFPI inhibits the generation of FXa and FIXa by the TF⅐FVIIa complex in a unique, two-step reaction (20). First, TFPI binds Ca 2ϩ independently to FXa, thereby inhibiting the FXa catalytic activity (9). In a second step, the FXa⅐TFPI complex binds in a Ca 2ϩ -dependent way to TF⅐FVIIa. This results in the formation of the quaternary complex TF⅐FVIIa⅐FXa⅐TFPI, in which the proteolytic activity of the TF⅐FVIIa complex is fully neutralized. The effect of TFPI on TF⅐FVIIa activity in the absence of FXa is negligible (21, 22) , implying that the true inhibitor of TF⅐FVIIa activity is the FXa⅐TFPI complex. The rate of complex formation of FXa and TFPI is enhanced by negatively charged phospholipids for full-length TFPI (TFPI FL ) but not for TFPI 1-161 , a truncated variant lacking the third Kunitz domain and the potential phospholipid binding C-terminal tail (16, 23) . Recently (24) , we demonstrated that TFPI FL in complex with FXa has a much higher affinity for anionic phospholipid membranes compared with that of either protein alone. It is well recognized that the binding of blood coagulation enzymes as well as their cofactors and substrates to membranes containing anionic phospholipids may result in an immense increase of the catalytic efficiency of these enzymes. On the other hand, excess binding sites could cause a lowering of both solution concentration and surface density of the reactants (25, 26), resulting in a decrease of reaction rates. Previous studies (17, 22, 23) did not reveal a stimulatory or interfering effect of anionic phospholipids on the complex formation between FXa⅐TFPI and TF⅐FVIIa. These studies, however, were performed in the presence of excess phospholipid vesicles. 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 TF⅐FVIIa-catalyzed initiation of the blood coagulation process.
doi:10.1074/jbc.274.40.28225 pmid:10497177 fatcat:gj5warcgofg5fgrrjqwepxoc5a