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

Abstract

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 TF⅐FVIIa-catalyzed 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 (TF⅐PSPC) was measured in the presence of preformed complexes of FXa⅐TFPI FL 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 coagulation 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)(16)(17)(18)(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 ...

von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets

“…Recombinant human tissue factor was a kind gift of Dr Y. Nemerson (Mount Sinai School of Medicine, New York, NY, USA) and was relipidated as described before [13]. Human α‐thrombin was prepared as previously described [14].…”

Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor

“…Human FXa was obtained from Enzyme Research Laboratories (South Bend, IN). TFPI was kindly provided by T. Lindhout (Cardiovascular Research Institute Maastricht) (40). Full-length TFPI was produced in Escherichia coli, and the truncated variant of TFPI (amino acid residues 1-161) was expressed in Sacharomyces cerevisiae.…”

Regulation of Platelet Factor Va-dependent Thrombin Generation by Activated Protein C at the Surface of Collagen-adherent Platelets

“…The Rotating Disc Device-Rotating disc experiments were performed in a device described previously (15). Briefly, a circular glass coverslip with a diameter of 20 mm (Menzel Glä ser, Braunschweig, Germany), was rotated at 63 rad/s at the bottom of a cylindrical reaction vessel containing reactants in 3 ml of buffer A.…”