Expression, purification and characterization of factor IX derivatives using a novel vector system.

Recent studies have indicated that the loop harboring the S1 specificity site (residues 185-189 in chymotrypsin numbering) of coagulation proteases has several charged residues with important structural and functional roles for the catalytic activity of these proteases. This loop is allosterically linked to the Na(+)-binding site in both factor Xa and thrombin. There are three candidate residues (His-185, Glu-186, and Arg-188) on this loop of factor IXa (fIXa) whose side chains can influence the Na(+) binding and the catalytic function of the protease in the intrinsic Xase complex. In this study, we developed a novel expression/purification vector system, substituted all three residues of factor IX individually with Ala, and expressed the mutant zymogens in mammalian cells. Following activation, all three fIXa mutants exhibited normal activity towards a fIXa-specific chromogenic substrate in the presence of Ca(2+) with no obvious requirement for Na(+) in the reaction. Furthermore, all three mutants interacted with factor VIIIa with near normal affinity and catalyzed the activation of factor X in the intrinsic Xase complex with a normal catalytic efficiency. These results suggest that, unlike thrombin and factor Xa, the charged residues of this loop do not play a functional role in modulating the catalytic function of fIXa in the intrinsic Xase complex.

Down-regulation of factor IXa in the factor Xase complex by protein Z-dependent protease inhibitor.

Protein Z-dependent protease inhibitor (ZPI) is a serpin inhibitor of coagulation factor (F) Xa dependent on protein Z, Ca2+, and phospholipids. In new studies, ZPI inhibited FIXa in the FXase complex. Since this observation could merely represent inhibition of the FXa product whose activity was measured, inhibition of FIXa was investigated five ways. 1) FXase incubation mixtures with/without ZPI/protein Z were diluted in EDTA; FXa activity was measured after reversal of its inhibition. 2) FXase incubation mixtures were immunoblotted for FXa product. 3) FX activation peptide region was 3H-labeled; release of 3H was used to measure FXase activity. 4) Activity was monitored in a FIXa-based clotting assay. 5) FIXa amidolytic activity was measured. In all cases, FIXa was inhibited by subphysiologic levels of ZPI. Unlike inhibition of FXa, inhibition of FIXa did not strictly require protein Z. Low concentrations of FVIIIa increased the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure. Unusual time courses were observed for inhibition of both FIXa in the FXase complex and FXa in the prothrombinase complex. Activity loss stabilized in <100 s at a level dependent on ZPI concentration, suggesting equilibrium interactions rather than typical covalent serpin-protease interactions. Surface plasmon resonance binding experiments revealed binding and dissociation of ZPI/FIXa with Kd (app) of 9-12 nm, similar to the concentration of ZPI needed for 50% inhibition. ZPI may be an unusual physiologic regulator of both the intrinsic FXase and the prothrombinase complexes.

Blood coagulation.

The process of tissue factor initiated blood coagulation is discussed. Reactions of the blood coagulation cascade are propagated by complex enzymes containing a vitamin K-dependent serine protease and an accessory cofactor protein that are assembled on a membrane surface in a calcium-dependent manner. These complexes are 105-109-fold more efficient in proteolyses of their natural substrates than enzymes alone. Based upon data acquired using several in vitro models of blood coagulation, tissue factor initiated thrombin generation can be divided into two phases: an initiation phase and a propagation phase. The initiation phase is characterized by the generation of nanomolar amounts of thrombin, femto- to picomolar amounts of factors VIIa, IXa, Xa, and XIa, partial activation of platelets, and almost quantitative activation of procofactors, factors V and VIII. The duration of this phase is primarily influenced by concentrations of tissue factor and TFPI. The characteristic features of the propagation phase are: almost quantitative prothrombin activation at a high rate, completion of platelet activation, and solid clot formation. This phase is primarily regulated by antithrombin III and the protein C system. Thrombin generation during the propagation phase is remarkably suppressed in the absence of factor VIII and IX (hemophilia A and B, respectively) and at platelet counts <5% of mean plasma concentration. The majority of data accumulated in in vitro models and discussed in this review are in good agreement with the results of in vivo observations.

Modeling human zymogen factor IX.

Modern theoretical techniques are employed to provide complete three dimensional structure for the zymogen and activated forms of human coagulation factors IX and IXa. These structures are fully calcium bound and equilibrated in an electrically neutral aqueous environment. The relationship of structure to mutational data is examined. We find that a substantial relative orientational change of the catalytic domain occurs on activation. Also, we find that the electrostatistically dipolar nature of the catalytic domain is substantially modified upon activation, with cleavage of the negatively charged activation peptide leaving behind a largely hydrophobic face in factor IXa. While the backbone atoms of the catalytic residues have little relative movement, nearby loops are found that do move. The presence or absence of these changes likely defines specificity.

Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation.

Tissue factor is the major initiator of coagulation. Both factor IX and factor X are activated by the complex of factor VIIa and tissue factor (VIIa/TF). The goal of this study was to determine the specific roles of factors IXa and Xa in initiating coagulation. We used a model system of in vitro coagulation initiated by VIIa/TF and that included unactivated platelets and plasma concentrations of factors II, V, VIII, IX, and X, tissue factor pathway inhibitor, and antithrombin III. In some cases, factor IX and/or factor X were activated by tissue factor-bearing monocytes, but in some experiments, picomolar concentrations of preactivated factor IX or factor X were used to initiate the reactions. Timed samples were assayed for both platelet activation and thrombin activity. Factor Xa was 10 times more potent than factor IXa in initiating platelet activation, but factor IXa was much more effective in promoting thrombin generation than was factor Xa. In the presence of VIIa/TF, factor X was required for both platelet activation and thrombin generation, while factor IX was only required for thrombin generation. We conclude that VIIa/TF-activated factors IXa and Xa have distinct physiologic roles. The main role of factor Xa that is initially activated by VIIa/TF is to activate platelets by generating an initial, small amount of thrombin in the vicinity of platelets. Factor IXa, on the other hand, enhances thrombin generation by providing factor Xa on the platelet surface, leading to prothrombinase formation. Only tiny amounts of factors IX and X need to be activated by VIIa/TF to perform these distinct functions. Our experiments show that initiation of coagulation is highly dependent on activation of small amounts of factors IXa and Xa in proximity to platelet surfaces and that these factors play distinct roles in subsequent events, leading to an explosion of thrombin generation. Furthermore, the specific roles of factors IXa and Xa generated by VIIa/TF are not necessarily reflected by the kinetics of factor IXa and Xa generation.

Activation of bovine factor IX by the reaction product of bovine factor VII and human tissue factor.

A study was carried out on the alternate activation of factor IX (FIX) by bovine FVII and human tissue factor (TF) rather than by activated factor XI (FXIa). The reaction product of bovine FVII and human TF functioned as a FIX activator in the assay system used. Published studies suggest that in the presence of Ca2+ ions, the complex of human FVII-TF readily activates both human FIX and human FX, and at low TF concentrations, FIX appears to be the preferred substrate for the reaction product of FVII and TF. This may explain the discrepancy between the mild bleeding of hereditary FXI deficiency and the severe bleeding of hereditary FIX deficiency. The results obtained with bovine FVII and a crude human TF preparation confirm that at low TF concentrations, bovine FIX is the preferred substrate rather than FX. At higher TF concentrations, bovine FX was rapidly activated.

Extrinsic activation of human blood coagulation factors IX and X.

We studied activation of human coagulation factors IX and X by factor VIIa in the presence of calcium ions, phospholipid (phosphatidylserine/phosphatidylcholine, 50/50, mol/mol) and purified tissue factor apoprotein. Activation of factor IX and factor X was found to occur without a measurable lag-phase and hence initial rates of factor IXa and factor Xa formation could be determined. Like previously observed for the activation of factor X, the activation of factor IX was saturable with respect to factor VIIa, tissue factor apoprotein and phospholipid. The results suggested that in the presence of a Ca2+ ions the same ternary complex of factor VIIa-tissue factor apoprotein-phospholipid is responsible for the activation of factor IX and factor X. Both the apparent Km of 22 nM-factor IX and the apparent Kcat of 28 min-1 were about 3-fold lower than the corresponding parameters of factor X activation by this complex. Hence, the catalytic efficiency (Kcat/Km) of factor IX and factor X activation was about equal. However, the two substrates inhibited the activation of each other by competition for the same catalytic sites. The apparent Kinh of factor IX for inhibition of extrinsic factor X activation is 30 nM. The apparent Kinh of factor X for inhibition of extrinsic factor IX activation is 116 nM. From these kinetic data it was calculated that at plasma concentration of factors IX and X, the rate of extrinsic factor IX activation would be half the rate of factor X activation.(ABSTRACT TRUNCATED AT 250 WORDS)