Raising the Active Site of Factor VIIa above the Membrane Surface Reduces Its Procoagulant Activity but Not Factor VII Autoactivation

Abstract

Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membraneanchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k cat . The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex. Tissue factor (TF)3 is the cell surface, type I integral membrane protein that triggers blood clotting in normal hemostasis and many thrombotic diseases (1, 2). TF initiates blood coagulation by binding the plasma serine protease, factor VIIa (fVIIa). The membrane-bound complex of fVIIa and TF can therefore be considered a two-subunit enzyme, with fVIIa as the catalytic subunit and TF as the regulatory subunit. The major substrates for the fVIIa-TF complex are factors X (fX) and IX (fIX), which are converted to active serine proteases by limited proteolysis. Activation of fX and fIX leads to propagation of the clotting cascade and, ultimately, clotted fibrin and activated platelets. Binding of fVIIa to TF embedded in a suitable phospholipid membrane increases its proteolytic activity many thousandfold (3, 4). For optimal fVIIa-TF enzymatic activity, the phospholipid bilayer must contain negatively charged phospholipid, most especially phosphatidylserine (PS) (5, 6). How PS augments the enzymatic activity of fVIIa-TF is not completely understood, but one role is to promote interaction of vitamin K-dependent clotting factors such as fVII, fIX, and fX with phospholipid surfaces (7). These proteins all have an N-terminal domain containing multiple ␥-carboxyglutamate residues (Gla domain) that confers reversible binding to membranes containing PS. Incorporating PS in...

Tissue Factor/Factor VIIa Complex: Role of the Membrane Surface

“…When sTF bound to FVIIa it significantly restricted FVIIa’s motions, resulting in a fairly rigid sTF:FVIIa complex positioned almost perpendicular to the membrane surface [23], in excellent agreement with our previously published fluorescence resonance energy transfer studies [24–26]. Positioning the active site of FVIIa site relative to the membrane may be important in aligning it with membrane-bound substrates (FIX and FX), and in fact we recently showed that perturbing this alignment compromises the ability of TF:FVIIa to activate FX [27]. …”

Activation of the Zymogen to Urokinase-Type Plasminogen Activator Is Associated with Increased Interdomain Flexibility

“…In a similar way, it has been shown that when factor VIIa is captured by tissue factor on the cell surface, the distance of factor VIIa from the cell membrane is critical for the efficient activation of factor X. 36 In pro-uPA, two covalent connections exist between the protease domain and the linker/ATF, that is, the cysteine bridge and the amino acid backbone. In uPA, only one connection exists from the protease domain to the linker/ATF, that is, the cysteine bridge connection.…”

Tissue Factor Residues That Putatively Interact with Membrane Phospholipids

“…Initial rates of FVIIa-catalyzed hydrolysis of Chromozym t-PA substrate (Roche Applied Science, Indianapolis, Indiana, USA) were quantified as described [11]. Reaction conditions were: 5 nM FVIIa, 100 nM membTF, 1 mM Chromozym t-PA in HBSAC (25 mM HEPES pH 7.4, 100 mM NaCl, 5 mM CaCl 2 , 0.1% BSA, 0.1% NaN 3 ) with 0.1% Triton X-100.…”

“…Binding of FVIIa to membrane-anchored TF has multiple effects on enzyme activity: (a) TF allosterically activates FVIIa, independent of the membrane [2]; (b) TF is thought to contribute a substrate-binding site (exosite) to help recognize FIX and FX [3]–[6]; (c) phosphatidylserine (PS) dramatically increases rates of FIX and FX activation by TF/FVIIa [7]; and (d) TF fixes FVIIa’s active site at a set distance above the membrane surface, which may be important for optimal engagement of the scissile bonds of FIX and FX [8]–[11].…”