Many natural enzymes need the assistance of protein cofactors to catalyze chemical reactions at a physiologically relevant speed and several of the enzymes that make up for the coagulation cascade are no exception in this regard. Notably, activated factors VII, IX and X display relatively poor enzymatic activity towards their respective macromolecular substrates. The reason for their low proteolytic activity originates from a number of structural restrictions. For instance, not all enzymes are capable to efficiently fold their new amino-terminus into the active site pocket, leaving the catalytic triad immature. Furthermore, serine protease activation is often associated with a reduced plasticity of the protease domain, which improves their proteolytic activity. Nevertheless, some enzymes still require additional stabilization to reduce flexibility of their protease domain. Protein cofactors are designed to optimize the proteolytic activity of such serine proteases, and can improve the catalytic efficiency of these enzymes by one-thousand to one-million fold. The allosteric changes induced by these protein cofactors are specific to each cofactor/enzyme pair. When focusing on the cofactor role of Factor VIIIa (FVIIIa; which stimulates the catalytic activity of factor IXa; FIXa), several aspects are of importance. First, FVIIIa has high affinity for phosphatidylserine-containing phospholipid-membranes, favoring formation of the FVIIIa/FIXa complex at the membrane surface. Being assembled at the membrane surface limits their movements to two dimensions, and enforces the affinity between both proteins. Second, the interactions between FVIIIa and FIXa involve an extended protein surface, which includes interactions between the FVIIIa light chain and FIXa light chain as well as between the FVIIIa A2 domain and the FIXa protease domain. Due to this extended interactive surface, the complex mimics a staked tree, in which FVIIIa orients the FIXa active site at the appropriate distance from the membrane surface. Moreover, binding of the FVIIIa A2 domain to FIXa surface loops reduces flexibility of the protease domain, and it is likely that allosteric changes induced by the A2-domain optimize the conformation of the active site region. Finally, FVIIIa provides also a binding site for the substrate FX. This not only allows FVIIa to function as a molecular bridge between enzyme and substrate, but also helps to align the FX activation peptide with the FIXa active site. This multistep process by which FVIII acts as a cofactor for FIXa may help us to understand how other non-FVIII molecules can be used to stimulate FIXa activity. Several molecular entities have been reported that are enhancing FIXa activity, including short synthetic peptides, monoclonal antibodies and, perhaps best known at this moment, bispecific antibodies that bind both FIXa and FX. Given the complex molecular structure that FVIIIa has and needs to stimulate FIXa activity, it is of interest to reflect on how this translates to the non-FVIII molecules in terms of regulation and potential cofactor activity. Differences in regulation and activity are of particular relevance for laboratory monitoring of these molecules and in the therapeutic setting. Knowing these limitations will help us to optimize the therapeutic application of non-FVIII molecules.
Disclosures
Lenting: Spark Therapeutics: Honoraria; Catalyst Biosciences: Honoraria; Sobi: Honoraria; Shire/Takeda: Honoraria; NovoNordisk: Honoraria; Biotest: Honoraria; LFB: Honoraria; Roche: Honoraria; laelaps therapeutics: Equity Ownership.
Mechanism of the CBM35 domain in assisting catalysis by Ape1, a Campylobacter jejuni O-acetyl esterase
