The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly

Abstract

Zymogens of the chymotrypsin-like serine protease family are converted to the protease state following insertion of a newly formed, highly conserved N terminus. This transition is accompanied by active site formation and ordering of several surface loops in the catalytic domain. Here we show that disruption of this transition in factor X through mutagenesis (FXa I16L and FXa V17A ) not only alters active site function, but also significantly impairs Na ؉ and factor Va binding. Active site binding was improved in the presence of high NaCl or with saturating amounts of factor Va membranes, suggesting that allosteric linkage exists between these sites. In line with this, irreversible stabilization of FXa I16L with Glu-Gly-Arg-chloromethyl ketone fully rescued FVa binding. Furthermore, the K m for prothrombin conversion with the factor Xa variants assembled into prothrombinase was unaltered, whereas the k cat was modestly reduced (3-to 4-fold). These findings show that intramolecular activation of factor X following the zymogen to protease transition not only drives catalytic site activation but also contributes to the formation of the Na ؉ and factor Va binding sites. This structural plasticity of the catalytic domain plays a key role in the regulation of exosite expression and prothrombinase assembly.Proteolysis of zymogen proteins to their enzymatically active form is a central feature of many physiological processes, including blood coagulation (1). The paradigm for this type of activation mechanism is the zymogen to protease transition in the chymotrypsin-like serine protease family. Bond cleavage at a highly conserved site (Arg 15 -Ile 16 ; the chymotrypsin numbering system is used throughout (2)) unmasks a new N terminus, which becomes an intramolecular ligand for Asp 194 within a hydrophobic environment (3, 4). This transition is associated with a conformational change in the so-called "activation domain" (residues comprising 16 -19, 142-152, 184 -193, and 216 -223) and is responsible for ordering the primary specificity pocket and oxyanion hole (4). These structural changes lead to the maturation of the active serine protease and impart function.The zymogen and the mature protease exist in an equilibrium that typically favors the zymogen; however, this equilibrium can be shifted depending on various conditions. Bode and colleagues (5, 6) Factor X (FX) 3 is also conformationally activated following its conversion from the zymogen to the protease state (15, 16). Factor X is converted to factor Xa (FXa) by either the extrinsic (tissue factor/factor VIIa) or intrinsic (factor VIIIa/factor IXa) tenase enzymatic complexes following cleavage of the Arg 15 -Ile 16 scissile bond, thereby liberating a 52-amino acid activation peptide (17). Factor Xa reversibly associates with its cofactor factor Va (FVa) on an anionic membrane surface in the presence of Ca 2ϩ ions to form prothrombinase, the physiological activator of prothrombin (17). The interaction between membrane-bound FVa and FXa is mediated, at least in par...

Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations

“…For these experiments, pt-FXa was active site-blocked with EGR-CH 2 Cl essentially as described. 23 Sedimentation coefficients and molecular weights were determined by g(s*) analysis. 24 …”

Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro

“…28 Recombinant wt-FX and 2 zymogen-like variants (FX I16L and FX V17A ) were prepared, purified, and characterized as previously described. 5,6 Recombinant and plasma-derived FX were activated with RVV X-CP and purified as described previously. 5,6 Human activated protein C (APC), ATIII, soluble thrombomodulin (sTM), and 2 domain TFPI (residues 13- 33 The concentration of TFPI was determined using M r ϭ 17 400 and E 0.1% 280 nm ϭ 0.45, calculated from the primary structure and by the method of Gill and von Hippel.…”

“…3,4 For FX/FXa, the zymogen to protease transition not only drives catalytic site activation but also contributes to the formation of the FVa binding site. 5,6 Once FXa binds FVa on an anionic membrane surface in the presence of Ca 2ϩ ions to form prothrombinase, the macromolecular enzyme complex rapidly converts prothrombin to thrombin. 1 Although free FXa catalyzes this reaction, prothrombinase is considered the physiologically relevant enzyme.…”

“…21,22 Recently, our laboratory has characterized variants of FXa that could circumvent these associated problems. 6 These FXa derivatives have a mutation at either position 16 or 17 at the N-terminus of the heavy chain (eg, FXa I16L and FXa V17A ). Biochemical studies revealed that these mutations destabilize the normal zymogen to protease conversion process.…”

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

“…Zymogen-like FXa variants were therefore investigated to overcome these limitations. Amino acid substitutions which result in zymogen-like FXa were initially described to impair conversion of FXa into the protease state and to diminish its binding affinity to Na + and factor Va [71]. FX is activated by cleavage at bond Arg194-Ile195 resulting in the formation of a new N-terminus which is inserted into a binding pocket of the heavy chain by forming of a salt bridge between Ile195 and Asp378 [72].…”