Factor VIIa

FVIIaFVIIa

IntroductionIntroduction

Factor VIIa (FVIIa)is a single chain trypsin-like serine protease [1](EC 3.4.21.21) of 406 residues. The FVII[2] zymogen is a glycoprotein consisting of an amino-terminal (N-linked) γ-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like (EGF1 and EGF2) domains, a short linker peptide, and a carboxy terminal serine protease domain (Figure 1). The active form, FVIIa, is generated by a specific cleavage of a peptide bond between Arg-152 and Ile-153 at the end of the linker peptide by either factor Xa (FXa) or thrombin (IIa). This cleavage generates an N-terminal light chain of 152 residues linked to a heavy chain of 254 residues by a disulfide bridge . Following cleavage the newly formed N-terminal inserts itself into a cavity, or the activation pocket, forming a salt bridge with Asp343 (Asp194 trypsin numbering).Formation of this salt bridge allows for the maturation of FVIIa to its active form.

FVIIa mechanismFVIIa mechanism

GeneralGeneral

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 107-fold6. There are four distinct steps that are required for the full activity of the TF-FVIIa complex (Scheme 1): 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. . (Add fig 9 from ref 6).

Structural changes in FVIIa activationStructural changes in FVIIa activation

Cation interactionCation interaction

The Gla domain binds seven Ca++ 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. Binding of Ca++ induces an increase in the α-helical content of that region. Ca++ binding in the protease domain, mediated by Glu210 and Glu220, produces subtle local changes presumably important for TF binding12

TF interactionTF interaction

The binding epitope of TF to FVIIa is a stripe running along the whole length of the TF protein. 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 (check). 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.

BACE1 (β-site of APP cleaving enzyme) also called β-Secretase and memapsin-2 is a 52 kD class I transmembrane aspartic acid protease that cleaves the Amyloid Precursor Protein (APP) in a rate limiting step that contributes to the accumulation of β-amyloid plaques in Alzheimer’s disease (AD). A subsequent cleavage by γ-secretase generates a 40 or 42 amino acid β-amyloid peptide. These peptides can form Aβ plaques that may have deleterious effects on neuronal function and contribute to pathologies of AD. Under normal conditions, BACE1 activity generates a monomeric and soluble Aβ peptide that may play a physiological role in decreasing excitotoxicity and neurotransmission at glutamatergic synapses. Additionally, α-secretase and γ-secretase cleave APP to generate p3 and the carboxy terminal fragment AICD in a non-amyloidogenic pathway. In AD, amyloidogenic pathways become preferential over non-amyloidogenic and Aβ plaques appear under increased levels of BACE1 catalytic activity.

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