The Elusive Role of the Potential Factor X Cation-binding Exosite-1 in Substrate and Inhibitor Interactions

Abstract

Coagulation factor X (FX) 1 is a vitamin K-dependent zymogen activated into FXa by cleavage at a single bond (Arg 15 -Ile 16 in the chymotrypsin numbering system 2 ) hydrolyzed by either of two complexes: FVIIa bound to tissue factor (TF), which triggers coagulation after injury, or FIXa associated with FVIIIa, which amplifies thrombin production after initiation of coagulation. FXa is the only endogenous prothrombin activator, but substantial thrombin production occurs only within the prothrombinase complex, where FXa binds to FVa in the presence of calcium on an appropriate phospholipid surface. Two inhibitors control FXa activity, viz. the tissue factor pathway inhibitor (TFPI) and antithrombin.Extended binding sites (exosites) play a crucial role in virtually any substrate, cofactor, or inhibitor recognition within the coagulation cascade. Perhaps best characterized is the case of thrombin taking advantage of two exosites for extended interactions in numerous functions (1). Exosite-1 is formed by a set of basic residues comprising loops 34 -40 and 70 -80, which neighbor each other in the folded structure (2); it interacts with fibrinogen, thrombomodulin, platelet activator receptor-1, FV, FVa, heparin cofactor II, and hirudin. Exosite-2, comprising loop 91-102 and helices 165-173 and 233-245 (3), is also formed by a set of basic residues that interact with heparin, with the chondroitin sulfate of thrombomodulin, and within prothrombin with kringle-2 (4). In FVII, the region corresponding to exosite-1 of thrombin is critical for FX activation (5, 6); in FIXa, this region is also important for FX activation in the absence of FVIII and for its inhibition by antithrombin in the absence of heparin (7). In contrast to thrombin, FVIIa, FIXa, and FXa share a negatively charged patch within segment 70 -80, whereas FXa is unique in that both loops 34 -40 and 70 -80 are negatively charged. Thus, the region of FXa corresponding to exosite-1 of thrombin forms a negative cluster, raising the question as to whether it could constitute a functional exosite. FXa has a unique macromolecule substrate, but exosites may also participate in its inhibition and/or in the activation of its zymogen. In fact, a number of studies have established that exosite(s) must be critical in several FXa functions. Electrostatic interactions involving loop 34 -40 of FXa appear to play a significant role in binding to the second Kunitz domain of TFPI (8,9). FXa also appears to interact with a complementary surface on pentasaccharide-activated antithrombin (10), and the region of FXa topologically equivalent to exosite-2 of thrombin seems to be involved in the binding of heparin and FVa (11). Above all, prothrombin recognition by prothrombinase undoubtedly involves one or more exosites on .In a previous study (15), we reported that the catalytic groove of FXa is minimally selective with unconstrained peptides and that FVa has little effect, if any, on this selectivity. These two observations therefore also favor the hypothesis that FXa uses seconda...

Thrombin-activable Factor X Re-establishes an Intrinsic Amplification in Tenase-deficient Plasmas

“…in which S 0 is the initial S2765 concentration, K m , its Michaelis constant for FXa (or activated form of FX FpA ) and I 0 the active concentration of added serpin, estimated as previously described (21).…”

“…Constructions were prepared through a single polymerase chain reaction, and sequence coding for FX FpA was verified by dideoxy chain termination, as previously described (21). Selection of transfected BHK-21 cells and production of FX FpA were also achieved as previously described (19,22,23).…”

“…Observed rates of inhibition (k obs ) were estimated by nonlinear regression analysis of the residual FXa activity with time using a first-order decay equation. Half-life values were calculated by dividing ln(2) by k obs (21). The anti-hemophilic potential of FX FpA was evaluated by its ability to shorten clotting time and increase thrombin formation in normal FVIII-and FIX-deficient plasmas.…”

“…At timed intervals, aliquots were withdrawn, and reactions quenched by diluting samples (1:10, v/v) in Tris, 50 mM; NaCl, 150 mM; pH 8.0 containing 10 mM EDTA, and 100 mM benzamidine. The amount of thrombin was monitored from the rate of 100 M S2238 hydrolysis after further dilution (1:20, v/v) in kinetic buffer without BSA (21).…”

“…Following dilution of 1:10 (v/v) in water, the buffer was exchanged to Tris (50 mM, pH 7.5) containing 500 mM NaCl by anion exchange chromatography onto a Q-Sepharose Fast Flow column (Amersham Biosciences). Calcium concentration was adjusted to 5 mM, and recombinant FX was purified by affinity chromatography as previously described (21 (23). Large quantities of activated FX FpA were prepared by passage through a 1-ml HiTrap N-hydroxysuccinimide-activated column coupled to thrombin (1 mg/ml of gel) according to the manufacturer's instructions.…”