Experience of a new high-purity factor X concentrate in subjects with hereditary factor X deficiency undergoing surgery.

INTRODUCTION: Maintaining haemostasis in surgery is challenging for hereditary rare bleeding disorders in which multi-coagulation-factor concentrates are the only therapeutic option. Hereditary factor X (FX) deficiency affects 1:500 000 to 1:1 000 000 individuals, and no specific replacement FX concentrate has been available. A high-purity, plasma-derived FX concentrate (pdFX) has been developed for patients with hereditary FX deficiency. AIM: Our objective was to assess the safety and efficacy of pdFX in subjects with FX deficiency undergoing surgery. METHODS: Subjects with hereditary mild-to-severe FX deficiency (basal plasma FX activity [FX:C] <20 IU dL(-1) ) undergoing surgery received pdFX preoperatively to raise FX:C to 70-90 IU dL(-1) and postoperatively to maintain levels >50 IU dL(-1) until the subject was no longer at risk of bleeding due to surgery. Efficacy of pdFX was assessed by blood loss during surgery, requirement for blood transfusion, postoperative bleeding from the surgical or other sites, and changes in haemoglobin levels. Safety was assessed by adverse events (AEs), development of inhibitors, and clinically significant changes in laboratory parameters. RESULTS: Five subjects (aged 14-59 years) underwent seven surgical procedures (four major and three minor). Treatment duration was 1-15 days. For each procedure, pdFX treatment was assessed as "excellent" in preventing bleeding and achieving haemostasis. No blood transfusions were required, no AEs related to pdFX were observed, and no clinically significant trends were found in any laboratory parameters. CONCLUSION: These data demonstrate that pdFX is safe and effective as replacement therapy in five subjects with mild-to-severe FX deficiency undergoing surgery on seven occasions.

Mutagenesis studies toward understanding the mechanism of differential reactivity of factor Xa with the native and heparin-activated antithrombin.

A unique pentasaccharide fragment of high-affinity heparin activates antithrombin (AT) to enhance its rate of complex formation with factor Xa (FXa) by 200-300-fold. Recent results have indicated that the activation of AT is associated with the exposure of a cryptic exosite on the serpin that is an interactive site for FXa in the complex. Previously, we identified Arg(150) on the autolysis loop of FXa as a candidate residue that may specifically interact with the heparin-activated AT. Three other surface loops on FXa including 39, 60, and the sodium-binding 220 loops have been implicated to be critical for the protease interaction with the activated AT. To determine the extent of the contribution of these loops to the specificity of the FXa interaction with activated AT, several loop mutants of the protease were prepared and their reactivity with AT was studied in both the absence and presence of pentasaccharide. Analysis of the inhibition kinetic data suggests that the residues of both 39 and 60 loop make a minor contribution to the recognition of AT in both the native and activated conformation of the serpin. On the other hand, the reactivity of AT with the sodium loop mutants of FXa in the absence of the cofactor was severely impaired. However, the extent of the rate-accelerating effect of pentasaccharide in the AT inhibition of the mutants was not affected. These results suggest that all three loops play a role in the specificity of the FXa-AT interaction; however, neither loop specifically interacts with the activated conformation of the serpin.

Zymogenic and enzymatic properties of the 70-80 loop mutants of factor X/Xa.

The Ca(2+) binding 70-80 loop of factor X (fX) contains one basic (Arg(71)) and three acidic (Glu(74), Glu(76), and Glu(77)) residues whose contributions to the zymogenic and enzymatic properties of the protein have not been evaluated. We prepared four Ala substitution mutants of fX (R71A, E74A, E76A, and E77A) and characterized their activation kinetics by the factor VIIa and factor IXa in both the absence and presence of cofactors. Factor VIIa exhibited normal activity toward E74A and E76A and less than a twofold impaired activity toward R71A and E77A in both the absence and presence of tissue factor. Similarly, factor IXa in the absence of factor VIIIa exhibited normal activity toward both E74A and E76A; however, its activity toward R71A and E77A was impaired approximately two- to threefold. In the presence of factor VIIIa, factor IX activated all mutants with approximately two- to fivefold impaired catalytic efficiency. In contrast to changes in their zymogenic properties, all mutant enzymes exhibited normal affinities for factor Va, and catalyzed the conversion of prothrombin to thrombin with normal catalytic efficiencies. However, further studies revealed that the affinity of mutant enzymes for interaction with metal ions Na(+) and Ca(2+) was impaired. These results suggest that although charged residues of the 70-80 loop play an insignificant role in fX recognition by the factor VIIa-tissue factor complex, they are critical for the substrate recognition by factor IXa in the intrinsic Xase complex. The results further suggest that mutant residues do not play a specific role in the catalytic function of fXa in the prothrombinase complex.

Purification of factor X by hydrophobic interaction chromatography.

Human factor X has been purified to homogeneity by hydrophobic interaction chromatography on phenyl-sepharose. The coagulation protein did not interact with the resin in the presence of 2-3 M NaCl whereas contaminants were retained. This single purification step, in conjunction with classical purification strategies, is a powerful tool in generating high purity factor X and is based on resins which are readily available.

Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography.

A rapid and simple method was developed to separate human vitamin K-dependent plasma proteins from each other, yielding virtually homogeneous pools. The purification technique is based on the single use of hydrophobic interaction chromatography, starting from prothrombin concentrate (PC or DEFIX, also termed factor IX concentrate) as initial material. Phenyl-sepharose HP demonstrated optimal separation by comparing several hydrophobic resins as well as resins used in standard procedures like immobilised heparin and Cibacron blue. Under ideal conditions, factor X could be separated in a single step as well as prothrombin. Factor IX co-eluted with other minor proteins. Focus was given only on these three proteins due to their relative abundance. Complete separation of all proteins present in the starting material was achieved by MonoQ anion-exchange chromatography following the phenyl-sepharose run. The resulting purified material could be demonstrated to be of equal or higher purity than using described methods. This strategy employing hydrophobic interaction chromatography for blood macromolecules could be of immense value for purifying the human vitamin K-dependent proteins and represents a considerable simplification over other purification schemes. It not only involves minimal sample handling but also can be readily up-scaled and is a cost-efficient alternative.

Cloning and recombinant expression of mouse coagulation factor X.

Engineering of recombinant coagulation factor X variants, which can be activated by tumor-associated proteinases may lead to the development of new therapeutic molecules. However, the evaluation of such variants requires an appropriate animal model. Therefore, we isolated the complete coding sequence of mouse coagulation factor X from mouse liver cDNA by polymerase chain reaction. The deduced amino acid sequence codes for a prepro protein of 481 amino acids homologous to factor X sequences from various species. Recombinant mouse factor X was expressed in human embryonic kidney cells and secreted into cell culture supernatant as zymogen, which could be converted to catalytically active factor Xa by Russell's viper venom. Purified recombinant mouse factor X restored coagulation in human factor X deficient plasma, demonstrating that mouse factor X is able to functionally interact with the human blood coagulation system. Recombinant mouse factor X opens the possibility to analyze therapeutically useful variants in the mouse system.

Isolation and characterization of mouse coagulation factor X -- biophysical and enzymological properties.

Mouse factor X was highly purified from plasma using barium ion precipitation and chromatography on anion-exchange and heparin-agarose affinity chromatography columns. Intact and reduced patterns of mouse factor X in SDS-PAGE were similar to those of human factor X. The specific absorption E 1%/1 cm at 280 nm of mouse factor X was found to be 11.2. Content of carbohydrate moieties of mouse factor X, determined to be 10% by weight, differs both quantitatively and quantitatively from that of human factor X, while the gamma-carboxyglutamic acid (Gla) and beta-hydroxy-aspartic acid (beta-OH-Asp) content were essentially the same as for human factor X. The amino-terminal amino acid sequences of the light and heavy chains of mouse factor X separated by SDS-PAGE were ANSFF--FKK and SVALXTSDSE, respectively. Underlined residues are non-identical with those of human factor X. Clotting time-based assays using human factor X-deficient plasma as substrate exhibited the following apparent extents of activation of factor X in mouse plasma, using human plasma as the standard: 195% (intrinsic); 200% (extrinsic); and 190% (RVV-X). Using the purified proteins in the same assay systems, the following apparent activation of mouse factor X was demonstrated, compared with human factorX: 195% (intrinsic); 27% (extrinsic); and 41% (RVV-X). These activity profiles suggest that the human extrinsic coagulation pathway functions less efficiently than the corresponding mouse pathway in the activation of mouse factor X. Furthermore, mouse brain thromboplastin satisfactorily replaced rabbit brain thromboplastin in extrinsic activation of factor X in mouse plasma, but not of human plasma or purified mouse or human factor X, in line with studies by others suggesting that human factor VIIa poorly activates factor X in the presence of mouse tissue factor. While fully RVV-X-activated mouse factor X activated human prothrombin at a rate equal to about 117% of that for human factor X, it hydrolyzed the synthetic substrate, S-2222, at a rate of only about 18% of that for human factor X. These results are expected to be useful in making the mouse suitable for study of the mammalian blood coagulation pathways.

Expression, purification, and characterization of recombinant human factor X.

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the -2 residue (Thr-->Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure-function relationships by mutational analysis.

Contribution of the prothrombin fragment 2 domain to the function of factor Va in the prothrombinase complex.

The prothrombinase complex assembles through reversible interactions between factor Xa, factor Va and acidic phospholipid-containing membranes in the presence of calcium ions. This complex catalyses the conversion of prothrombin to thrombin through two proteolytic steps. We have used prethrombin 2 as a substrate analog for the first cleavage reaction of prothrombin activation (cleavage at Arg323-Ile324) catalyzed by the prothrombinase complex and have also relied on the known ability of prethrombin 2 to interact tightly but reversibly with fragment 2 or fragment 1.2. The kinetics of cleavage at Arg323-Ile324 have been assessed with these substrate analogs to investigate the contribution of cofactor-substrate interactions mediated by the fragment 2 domain to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex. Initial velocity measurements indicated that the rate of prethrombin 2 cleavage by the factor Xa-PCPS binary complex was increased by a factor of approximately 1300 upon the addition of saturating concentrations of factor Va to assemble prothrombinase. Although the measured initial velocity was higher when either fragment 2 or fragment 1.2 was present, the factor Va-dependent enhancement in initial rate (2600- and 1500-fold) was comparable in each case. Steady state kinetic constants were obtained using prethrombin 2, prethrombin 2 plus fragment 2, and prethrombin 2 plus fragment 1.2 as substrates. For each substrate, the addition of saturating concentrations of factor Va to the Xa-PCPS binary complex led to increases in catalytic efficiency of between 1000 and 9000-fold. The kcat/Km for prethrombin 2 cleavage by prothrombinase was essentially identical to that obtained for prethrombin 2 saturated with fragment 2. Thus, comparable accelerating effects of factor Va are observed independent of the presence of the fragment 2 domain in the substrate. The results indicate that interactions between factor Va and the substrate mediated by the fragment 2 domain do not contribute in a dominant way to the ability of factor Va to enhance the catalytic efficiency of factor Xa within the prothrombinase complex.

Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex.

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1-52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase gamma (ARHgamma), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1-49 and a mixture of Xdes 1-34 and Xdes 1-43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1-49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1-49 of the activation peptide or with carbohydrate structures attached to these residues.

Review of conformation-specific affinity purification methods for plasma vitamin K-dependent proteins.

There are seven known vitamin K-dependent proteins in blood. These proteins require calcium ion for expressing their full biological activities. Calcium ion also induces conformational changes in this class of proteins. Taking advantage of the ligand induced conformational changes, a number of unique approaches of affinity chromatography have been developed. These methodologies have been very useful tools for both the purification and for understanding the structure-function relationships of this class of proteins. One method is the use of metal ion dependent immunoaffinity chromatography. The antigen can be dissociated from the antibodies with either the removal or addition of calcium ion under physiological conditions. The other method is pseudoaffinity chromatography. This method uses conventional ion-exchange or hydrophobic resin and manipulates the mobilities of the proteins on these resins by the presence or absence of calcium ions. Researchers working with other calcium binding proteins or other proteins that are known to undergo ligand induced conformational changes may benefit from the experience of these unique conformation-specific affinity chromatography approaches.

Structural integrity of the gamma-carboxyglutamic acid domain of human blood coagulation factor IXa Is required for its binding to cofactor VIIIa.

This report describes the analysis of a novel mutant human factor IX protein from a patient with hemophilia B (factor IX activity <1%; factor IX antigen 45%). Enzymatic amplification of all eight exons of the factor IX gene followed by direct sequence analysis reveals a single nucleotide change (a guanine --> adenine transition) in exon 2 at nucleotide 6409 which results in a glycine --> arginine substitution at amino acid 12 in the gamma-carboxyglutamic acid rich (Gla) domain of the mature protein. Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband. The purified protein is indistinguishable from normal factor IX by polyacrylamide gel electrophoresis. Characterization of the variant in purified component assays reveals that it is activated normally by its physiologic activator factor XIa, but its phospholipid-dependent activation by the factor VIIa-tissue factor complex is diminished. In the presence of phospholipid and 5 mM Ca2+, the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intrinsic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX, factor X, is not observed. The determination of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant factor IX for the cofactor factor VIIIa is 172-fold lower than normal. Competition studies using active site-inactivated factor IXas in the intrinsic tenase complex confirm that the defect in the variant protein is in its binding to factor VIIIa. We conclude that the structural integrity of the Gla domain of human factor IX is critical for the normal binding of factor IXa to factor VIIIa in the intrinsic tenase complex. In addition, a glycine at amino acid 12 is necessary for normal activation of factor IX by the factor VIIa-tissue factor complex.

Binding properties of the coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis.

The binding properties of the coagulation factor IX/factor X-binding anticoagulant protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis (habu snake) were investigated with an enzyme-linked immunosorbent assay. The half-maximal binding and maximal binding of IX/X-bp to both factors IX and X were observed at concentrations of Ca2+ ions of 0.4 mM and 1 mM, respectively. Concentration of IX/X-bp at half-maximal binding to solid-phase bovine factor IX and solid-phase bovine factor X were 0.4 +/- 0.1 nM and 1.1 +/- 0.4 nM, respectively, in the presence of 1 mM Ca2+ ions. The kinetics of binding activity of IX/X-bp to bovine factors IXa and Xa and to human factors IX and X resembled those of the binding to bovine factors IX and X. IX/X-bp did not bind to solid-phase coagulation factors other than factor IX/IXa and factor X/Xa, for example, prothrombin, factor VII, protein C, and protein Z, under the conditions of the experiment. To localize the binding sites of IX/X-bp on the coagulation factors, the ability of IX/X-bp to bind to various fragments derived from factors IX and X was examined. The binding of IX/X-bp to solid-phase factor IX was inhibited by a peptide containing the 4-carboxyglutamic acid (Gla) domain derived from factor IXa beta' (residues 1-42) in the liquid phase, but the binding was not inhibited by Gla-domainless factor IXa beta'. Half-maximal binding of IX/X-bp to solid-phase Gla-domain peptide of factor IX occurred at 9.2 +/- 1.9 nM. Factor X was partially reduced and the S-carboxymethylated light and heavy chains of factor X were prepared. IX/X-bp bound to the S-carboxymethylated light chain of factor X but not to the heavy chain. The binding of IX/X-bp to solid-phase factor X was inhibited by the Gla-domain peptide of factor X (residues 1-44) but not by Gla-domainless factor X. IX/X-bp bound to PCGFX, a recombinant human protein C whose Gla-domain region (residues 1-43) had been replaced by residues 1-43 of human factor X. The affinity of binding was about one tenth of that to intact human factor X. IX/X-bp was unable to bind at all to human protein C. These data indicate that IX/X-bp is a protein that binds to the Gla-domain regions of factors IX and X in the presence of Ca2+ ions.

Activated factor X and thrombin formation triggered by tissue factor on endothelial cell matrix in a flow model: effect of the tissue factor pathway inhibitor.

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma-TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.

Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for omega-aminocarboxylic acids.

The kringle 2 (E161T/C162S/EEE[K2HPg/C169S]TT) and the kringle 3 (TYQ[K3HPg]DS) domains of human plasminogen (HPg) were expressed in Escherichia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein. A coagulation factor Xa (FXa)-sensitive cleavage site was introduced to remove the N-terminal histidine tag. In r-K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter-kringle disulfide bridge between kringles 2 and 3. Recombinant proteins were isolated by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non-denaturing and oxidising environment. The free thiol group in position 297 in r-K3 was selectively alkylated with iodoacetamide. The hexahistidine tail was successfully removed with FXa. The N-terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data. The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments. r-K2 exhibits weak binding to lysine-Bio-Gel. The weak binding affinity of r-K2 for omega-aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6-aminohexanoic acid (NH2C5COOH) indicating a Kd of approximately 401 microM. In contrast, r-K3 seems to be devoid of a binding affinity for omega-aminocarboxylic acids. Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH2C5COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > kringle 3.

Expression and localization of tissue factor-based procoagulant activity (PCA) in pigeon monocyte-derived macrophages.

Monocyte-derived macrophages, focal to initiation and progression of atherosclerosis, have been implicated in thrombotic complication of this disease. In the present study we demonstrated tissue factor based procoagulant activity in cultured macrophages from the White Carneau pigeon following endotoxin (1-2 micrograms/ml) stimulation. This macrophage procoagulant activity paralleled activity obtained with pigeon brain homogenate. We used Enzyme-Linked Coagulation Assay (ELCA), an ultrasensitive microtiter plate assay, to measure procoagulant activity in these cells. Through the use of clotting factors purified from pigeon plasma, procoagulant activity could be detected with as few as 1-3 cells. Tissue factor antigen, detected through the use of immunogold labelling in conjunction with a polyclonal antibody which was highly specific to human tissue factor, was distributed uniformly over the plasma membrane of the endotoxin-stimulated cells. These studies suggest that this procoagulant activity might play an important role in the pathobiology of atherosclerosis in White Carneau pigeons by initiating fibrin polymerization and thus leading to thrombotic complications of the disease.

Specific molecular interaction sites on factor VII involved in factor X activation.

Factor VII, a serine-protease zymogen, and tissue factor, the cellular receptor/coenzyme, are the protein components of the macromolecular complex which initiates the extrinsic pathway of the coagulation cascade. Previous studies were directed to the identification of functional sites on factor VII which mediate factor X activation, employing a series of potentially inhibitory synthetic peptides representing the primary structure of factor VII and antibodies to selected peptides. The involvement of at least four high-affinity interactive regions [factor VII (44-50), (196-229), (285-305) and (376-396) peptides] on the surface of factor VII was clearly demonstrated. The minimal sequences for the expression of inhibitory activity of these four molecular recognition domains on factor VII were identified using short and overlapping peptides. The short factor VII-(206-218)-peptide (most inhibitory peptide in the sequence 196-229 on factor VII) inhibited the binding of factor VII to the tissue-factor-expressing J82 cell line. Furthermore, radiolabeled [Tyr201] factor VII-(199-221)-peptide, with a tyrosine substituted for the normal tryptophan residue, was specifically bound to J82 cells, and also the binding of the radiolabeled peptide to this cell line was specifically inhibited by a monoclonal antibody to tissue factor, confirming that the interaction site for tissue factor on factor VII is present within the peptide sequence 196-229. Kinetic analyses suggested that the regions represented by factor VII-(285-305)- and factor VII-(376-396)-peptides are involved in factor X recognition and the chemical cross-linking of the radiolabeled peptides resulted in specific binding to factor X, confirming that these two regions on factor VII represent the substrate-recognition site. Furthermore, these radiolabeled peptides specifically interact with the heavy chain of factor X, suggesting that the complementary binding region for the substrate-recognition site on factor VII are present on the heavy chain of factor X.

The influence of factor Va on the active site of factor Xa.

The interaction of factor Xa with factor Va on the membrane surface results in a 3,000-fold increase in the kcat for the activation of prothrombin catalyzed by factor Xa. The reaction between the transition state irreversible inhibitor dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) and factor Xa was characterized and employed to evaluate changes in the active site of factor Xa resulting from its interaction with factor Va, which may account for the increased catalytic efficiency of the enzyme complex. Inhibition studies indicated a two-step inhibition reaction involving a reversible binding step (Ki = 1.13 microM) and an irreversible alkylation step (ki = 0.65 s-1). The interaction between factors Va and Xa in solution or on membranes resulted in a small decrease in the overall second-order rate constant (ki/Ki) for the inhibition reaction. The incorporation of DEGRck into the active site of factor Xa results in a large change in the fluorescence intensity of the dansyl moiety. The fluorescence change was employed to study the reaction between enzyme and inhibitor directly by stopped-flow fluorescence measurements. The fluorescence traces were biphasic, indicating that the association of DEGRck with factor Xa and the subsequent covalent modification of the active site could be resolved because of differences in fluorescence intensities of the intermediate and product. This interpretation was verified by rapid chemical quench experiments. The reaction between DEGRck and factor Xa was characterized by a second-order association rate constant of 8.38 +/- 0.28 x 10(5) M-1.s-1 and an intrinsic rate constant for the alkylation step of 1.67 +/- 0.25 s-1. The rate constant for the alkylation step was unchanged in the presence of factor Va and membranes, whereas the association rate constant was modestly decreased by approximately 50%. The decrease in the association rate constant did not result from the partitioning of factor Xa to the membrane surface and could therefore be ascribed to an effect of factor Va on the protease. The data suggest that the interaction between factors Va and Xa on the membrane surface does not detectably alter the catalytic residues but may result in changes in the binding or accessibility of substrate to extended macromolecular recognition sites on the protease.

Importance of factor Xa in determining the procoagulant activity of whole-blood clots.

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.

Analysis of the functions of the first epidermal growth factor-like domain of factor X.

Upon activation, factor X participates in the prothrombin activation complex. Similar to 4-carboxyglutamic acid (Gla)-domainless protein C, the Gla-domainless factor X (GDFX) contains a high affinity Ca(2+)-binding site critical for the function of these molecules. In the case of protein C, we recently demonstrated that the high affinity Ca(2+)-binding site critical for activation is outside the first epidermal growth factor (EGF) homology domain. To examine if this is also true for factor X, we have expressed in human 293 cells a deletion mutant of factor X (E2FX) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of factor X. Direct binding studies by equilibrium dialysis indicate that E2FX contains a single Ca(2+)-binding site with a dissociation constant (Kd) of 154 +/- 48 microM. The functional properties of E2FX are equivalent or improved over those of GDFX. For instance, the factor X coagulant protein of Russell's viper venom activates E2FX three times faster than recombinant GDFX. Kinetic analysis of prothrombin activation in the absence of membranes indicates that activated GDFX and E2FX bind to factor Va with equal affinity (Kd = 4.1 microM). The Ca2+ concentration required for half-maximal prothrombin activation rates in the above activation system shifted from 721 +/- 113 microM for activated GDFX to 193 +/- 64 microM for activated E2FX. GDFX and E2FX activation rates with the soluble tissue factor-factor VIIa complex were identical as was the Ca2+ dependence of the reaction. We conclude that E2FX retains a high affinity Ca(2+)-binding site and that the first EGF homology domain does not appear to have a positive functional role in the GDFX molecule. However, Ca2+ occupancy of the Ca(2+)-binding site in the first EGF domain of intact factor X may be essential for optimal prothrombin activation.