Factor VIIa: Difference between revisions

FVIIa alone shows very little proteolytic activity and only becomes fully active when complexed to its obligatory cofactor, tissue factor (TF) and cations, mainly Ca++. TF, located in the vessel wall, is exposed to circulating FVIIa upon injury or some type of stimulus and forms a TF-FVIIa complex. A unique property of TF-FVIIa among other coagulation enzyme complexes is that phospholipids are not an obligate requirement for the assembly of the complex. However, the activity of the complex towards its substrates (FIX and FX) requires a lipid surface which is provided by the membrane-anchored TF. The TF-phospholipid complex enhances the efficiency (kcat/Km) of FVIIa-catalyzed reactions by the 10^7-fold. There are four distinct steps that are required for the full activity of the TF-FVIIa complex: 1) proteolytic activation of single-chained FVII to two-chain disulfide bridged FVIIa 2) binding of Ca++ 3) interaction of TF with FVIIa 4) acidic-membrane association and proper orientation of substrate<ref>PMID:1537862</ref><ref>PMID:18640965</ref>. .

===Structural changes in FVIIa activation===

The Gla domain binds four Ca++ ions and three Mg++ ions arranged in a linear fashion. Ca++ induced changes in the Gla domain are responsible for major structural rearrangements in that region that facilitate binding of FVIIa to membrane<ref>PMID:8844844</ref>. Binding of Ca++ induces an increase in the α-helical content of that region. The EGF1 domain contains one Ca++ binding site believed to be important for TF binding. The protease domain binds bidns one Ca++, one Na+ and two Zn++ ions. The Zn++ ions have been shown to inhibit the activity of FVIIa specifically by reducing its affinity for TF. Ca++ binding in the protease domain, mediated by Glu210 and Glu220, produces subtle local changes presumably important for TF binding. The Na+ binding site is located in a hydrophobic cavity responsible for TF binding<ref>PMID: 16757484 </ref>.  

====TF interaction====

 

The Gla domain of FVIIa binds to the C-domain of TF. The interaction is mainly hydrophobic termed the “hydrophobic stack”. EGF1 binds a single Ca++ ion and packs into a groove formed by the two modules of TF. This interface is the largest and  contributes the most energetically in binding of the cofactor to the protease domain13. EGF2 and the catalytic domain interact with the N-domain of TF. Two “lock and key” interactions are observed. One is the side chain of Phe50 of TF which is trapped in the pocket formed by the end of EGF2 domain of FVIIa. Second, the side chain Met306 of FVIIa is enclosed by TF residues Arg74, Phe76, Glu92, Leu94. This Met306 residue is responsible for the thermodynamic coupling to the active site and is unique to FVIIa. Mutation of this residue results in the failure of TF to decrease the dissociation rate of the enzyme from the cofactor. Cofactor interactions, specifically through Met306 lead to subtle changes which then influence the position of Asp331??. Asp331 is the specificity-determining residue in the binding pocket. Constraint and stabilization promote formation of a hydrogen bond between the amide of Arg315 (170C) and carbonyl of Gly372 (223). When bound to TF the activation region of FVIIa contains a large number of hydrogen bonds between the main chain and side chain atoms. At least 11 water molecules are identified in the catalytic domain region. An interesting observation is that the center of this region contains water, in contrast to the hydrophobic interactions in the other two interface regions. These hydrophilic interactions may be more efficient at mediating TF affinity and substantial conformational changes to induce activity of FVIIa. Therefore TF and cations are obligatory cofactors in the allosteric regulation of FVIIa activity by stabilizing the disordered, felxible FVIIa and restraining the enzyme for catalytic activity.

 

There are three steps serine proteases take to hydrolyze an amide bond: 1)activation of amide bonds by the interaction of the general acid with the carbonyl oxygen of the substrtate amide bond which disrupts resonance stabilization 2) activation of water by general base 3)activation of amines by protonation before expulsion. Serine proteases hydrolyze amide bonds with rates of 10^10–fold higher than the uncatalyzed reactions.   

In FVIIa Ser344(195) of the catalytic triad is activated by a His193(57), or the general base, which itself is stabilized by a hydrogen bond to Asp242(102). These reactions result in a formation of a tetrahedral intermediate and the oxyanion hole. The oxyanion hole is stabilized by interactions with main chain NHs. The activated Ser344 then attacks the scissile bond of the substrate. The general base His193 transfers the abstracterd proton from Ser to the amine leaving group, the tetrahedral intermediate (transition state) collapses and an acylenzyme intermediate is formed releasing the product. The general base His193 abstracts a proton from water as it attacks the acylenzyme to again form a tetrahedral intermediate. His193 then acts as an acid and protonates Ser344 releasing the product acid and regenerates the enzyme. This reaction is largely possible by having a His193 with a pKa ~7 necessary for deprotonation15, a hydrogen bonding network or “the charge relay system” activating Ser344 for nucleophilic attack, stabilization of the negatively charged oxyanion of the tetrahedral intermediate by the main chain NHs of Ser344 and Gly342(193). The kinetics are described in three steps: 1) binding of enzyme to substrate (k+1, k-1), 2) acylation of enzyme (k2), and 3) deacylation (k3)