Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor

Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 microM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Novel Forms of Coagulation Factor XIa: independence of factor XIa subunits in factor IX activation

Factor XI is the zymogen of a dimeric plasma protease, factor XIa, with two active sites. In solution, and during contact activation in plasma, conversion of factor XI to factor XIa proceeds through an intermediate with one active site (1/2-FXIa). Factor XIa and 1/2-FXIa activate the substrate factor IX, with similar kinetic parameters in purified and plasma systems. During hemostasis, factor IX is activated by factors XIa or VIIa, by cleavage of the peptide bonds after Arg145 and Arg180. Factor VIIa cleaves these bonds sequentially, with accumulation of factor IX alpha, an intermediate cleaved after Arg145. Factor XIa also cleaves factor IX preferentially after Arg145, but little intermediate is detected. It has been postulated that the two factor XIa active sites cleave both factor IX peptide bonds prior to releasing factor IX abeta. To test this, we examined cleavage of factor IX by four single active site factor XIa proteases. Little intermediate formation was detected with 1/2-FXIa, factor XIa with one inhibited active site, or a recombinant factor XIa monomer. However, factor IX alpha accumulated during activation by the factor XIa catalytic domain, demonstrating the importance of the factor XIa heavy chain. Fluorescence titration of active site-labeled factor XIa revealed a binding stoichiometry of 1.9 +/- 0.4 mol of factor IX/mol of factor XIa (Kd = 70 +/- 40 nm). The results indicate that two forms of activated factor XI are generated during coagulation, and that each half of a factor XIa dimer behaves as an independent enzyme with respect to factor IX.     

The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII.

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.     

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

Role of calcium ions and the heavy chain of factor XIa in the activation of human coagulation factor IX

Since optimal rates of factor IX activation by factor XIa require the presence of calcium ions and the heavy chain of the enzyme as well as the active-site-containing light chain, we have studied the effects of calcium ions and the heavy chain on the reaction kinetics. Whereas the amidolytic activities of factor XIa and of its active-site-containing light chain were almost indistinguishable, the two enzymes behaved quite differently when factor IX was the substrate. Factor XIa was 100-fold more potent in the presence of Ca2+ than in its absence. On the contrary, the presence or absence of Ca2+ made very little difference in the case of the isolated light chain of factor XIa. Moreover, the enzymatic activity of the light chain was almost identical with that of intact factor XIa when Ca2+ was absent. Using an optimal concentration of Ca2+, we studied the activation in the presence of various concentrations of two monoclonal antibodies, one (5F4) directed against the light chain of factor XIa and the other (3C1) against its heavy chain. Analysis of 1/V vs. 1/S plots showed that whereas inhibition by 5F4 was noncompetitive, 3C1 neutralized the enzyme in a classical competitive fashion. We conclude that in the calcium-dependent activation of factor IX by factor XIa the heavy chain of the enzyme is involved in the binding of the substrate and this is essential for optimal reaction rates.     

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.     

Activation of human protein C by blood coagulation factor Xa in the presence of anionic phospholipids. Enhancement by sulphated polysaccharides.

The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (20 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca2+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (1:1, w/w) of 75 microM, the apparent Km was 1 microM with a kcat. of 1 min-1. At 25 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-1. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of 2-5 micrograms/ml and 5-10 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca2+ was essential, the dependence being sigmoidal with Hill coefficients ranging from 1.4 to 2.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca2+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like prothrombin fragments F1 or F1+2 could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.     

Kinetics of coagulation factor X activation by platelet-bound factor IXa

Thrombin-activated human platelets, in the presence of factors VIIIa and X, have specific, high-affinity (Kd approximately 0.5 nM), saturable binding sites for factor IXa that are involved in factor X activation [Ahmad, S.S., Rawala-Sheikh, R., & Walsh, P.N. (1989) J. Biol. Chem. 264, 3244-3251]. To determine the functional consequences of factor IXa binding to platelets, a detailed kinetic analysis of the effects of platelets, phospholipids, and factor VIII on factor IXa catalyzed factor X activation was done. In the absence of platelets, phospholipids, or factor VIII, the Michaelis constant (Km = 81 microM) was greater than 500-fold higher than the factor X concentration in human plasma. Unactivated platelets and thrombin-activated factor VIII, alone or in combination, had no effect on the kinetic parameters, whereas thrombin-activated platelets caused a major decrease in Km (0.39 microM) with no significant effect on kcat (0.052 min-1) and allowed factor VIIIa to decrease the Km further to a concentration (0.16 microM) near that of factor X in plasma and to increase the kcat 24,000-fold to 1240 min-1. Sonicated mixed phosphatidylserine/phosphatidylcholine vesicles (25/75, mol/mol) had kinetic effects similar to those of activated platelets. When factor IXa binding to thrombin-activated platelets and rates of factor X activation were measured simultaneously at saturating concentrations of factor X and factor VIIIa, the kcat was independent of factor IXa concentration, and the mean kcat value was 2391 min-1. The increase in catalytic efficiency (kcat/Km) in the presence of thrombin-activated platelets and factor VIIIa was (17.4 x 10(6))-fold.     

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.     

The evaluation of complex-dependent alterations in human factor VIIa.

Factor VIIa is a plasma glycoprotein which, when bound to the integral membrane glycoprotein tissue factor, forms an enzymatic complex that is essential for normal hemostasis. We have developed a fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide) which can be used to directly measure the enzymatic activity of factor VIIa in the presence and absence of tissue factor and phospholipid. The sensitivity of this substrate allows for detection of factor VIIa at concentrations below 10(-9) M. The kinetics of substrate hydrolysis by factor VIIa were evaluated and it was observed that the binding of factor VIIa to tissue factor increases the catalytic efficiency (kcat/Km) of factor VIIa substrate hydrolysis greater than 100-fold. The increase in enzymatic efficiency of factor VIIa, when complexed to tissue factor, is mediated primarily by an increase in kcat. These data suggest that tissue factor induces an alteration in the catalytic site of factor VIIa, which allows for more efficient hydrolysis of the small fluorescent substrate. Measurements conducted using various phospholipids and detergents demonstrated that the increase in catalytic efficiency of factor VIIa, when complexed to tissue factor, is independent of the supporting surface. The differential rate of substrate hydrolysis when factor VIIa is complexed to tissue factor was used to estimate the binding of factor VIIa to tissue factor. From these data an apparent dissociation constant for factor VIIa binding to tissue factor was calculated to be between 1.1 and 2.1 nM with a binding stoichiometry of 1.04:1 (factor VIIa:tissue factor). When the reactivity of this small fluorescent substrate toward single-chain factor VII was investigated, both in the presence and absence of tissue factor, no substrate hydrolysis was observed.     

A moderate form of hemophilia B is caused by a novel mutation in the protease domain of factor IXVancouver

A genomic phage library was constructed using lymphocyte DNA from a patient with cross-reacting material-positive, moderately severe hemophilia B. The library was screened by using a full-length factor IX cDNA as a hybridization probe. DNA sequence analysis of the factor IX exons and intron/exon junctions revealed a single point mutation at nucleotide 31,311 of the gene. This mutation occurs in the protease domain of factor IXa and changes the codon for isoleucine 397 (ATA) to a threonine codon (ACA). The resulting abnormal protein has been named factor IXVancouver. Factor IXVancouver was isolated from the patient's plasma by barium citrate adsorption, affinity chromatography on a Ca2+-dependent antibody bound to agarose, and anion-exchange chromatography. On gel electrophoresis, the purified protein exhibited a normal molecular weight and a normal pattern of activation cleavages with bovine factor XIa. Kinetic studies on the purified protein indicated that the Km of factor IXaVancouver for human factor X was 3.4 times higher than that of normal factor IXa. The kcat of factor IXaVancouver was 12.5% of the kcat of normal factor IXa. Structural models of the protease domain of human factor IXa and of factor IXaVancouver were constructed, based on the homology of factor IXa with related serine proteases of known structure. The factor IXaVancouver model suggests that hydrogen bonding between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385 reduces the ability of factor IXaVancouver to bind factor X in a configuration favoring catalysis.     

The factor IX gamma-carboxyglutamic acid (Gla) domain is involved in interactions between factor IX and factor XIa

During hemostasis, factor IX is activated to factor IXabeta by factor VIIa and factor XIa. The glutamic acid-rich gamma-carboxyglutamic acid (Gla) domain of factor IX is involved in phospholipid binding and is required for activation by factor VIIa. In contrast, activation by factor XIa is not phospholipid-dependent, raising questions about the importance of the Gla for this reaction. We examined binding of factors IX and IXabeta to factor XIa by surface plasmon resonance. Plasma factors IX and IXabeta bind to factor XIa with K(d) values of 120 +/- 11 nm and 110 +/- 8 nm, respectively. Recombinant factor IX bound to factor XIa with a K(d) of 107 nm, whereas factor IX with a factor VII Gla domain (rFIX/VII-Gla) and factor IX expressed in the presence of warfarin (rFIX-desgamma) did not bind. An anti-factor IX Gla monoclonal antibody was a potent inhibitor of factor IX binding to factor XIa (K(i) 34 nm) and activation by factor XIa (K(i) 33 nm). In activated partial thromboplastin time clotting assays, the specific activities of plasma and recombinant factor IX were comparable (200 and 150 units/mg), whereas rFIX/VII-Gla activity was low (<2 units/mg). In contrast, recombinant factor IXabeta and activated rFIX/VIIa-Gla had similar activities (80 and 60% of plasma factor IXabeta), indicating that both proteases activate factor X and that the poor activity of zymogen rFIX/VII-Gla was caused by a specific defect in activation by factor XIa. The data demonstrate that factor XIa binds with comparable affinity to factors IX and IXabeta and that the interactions are dependent on the factor IX Gla domain.     

Assembly and expression of an intrinsic factor IX activator complex on the surface of cultured human endothelial cells.

Endothelial cells expose specific receptors for blood clotting factors and, upon perturbation, can initiate and propagate the reactions of the extrinsic pathway of blood coagulation leading to fibrin formation on the cell surface. The existence of an intrinsic mechanism of Factor IX activation on cultured human umbilical vein cells (HUVECs) was investigated by studies of the interaction between HUVECs and two proteins of the contact activation system, the cofactor high molecular weight kininogen (H-kininogen) and the zymogen Factor XI. In the presence of zinc ions (10-300 microM), 125I-labeled H-kininogen bound to HUVECs in a time-dependent, reversible, and saturable manner, with calcium ions exerting an inhibitory effect on the zinc-dependent binding. Analysis of the binding data by the LIGAND computer program indicated that HUVECs, in the presence of 2 mM CaCl2 and 100 microM ZnCl2 at 37 degrees C, bound 1.14 x 10(7) H-kininogen molecules per cell with an apparent dissociation constant of 55 nM. HUVEC-bound H-kininogen functions as the cell surface receptor for both 125I-labeled Factor XI and 125I-labeled Factor XIa, since HUVECs cultured in contact factor-depleted serum do not detectably bind either the zymogen or the enzyme in the absence of H-kininogen and zinc ions. In the presence of saturating concentrations of H-kininogen, 2 mM CaCl2 and 100 microM ZnCl2, the binding of 125I-labeled Factor XI and Factor XIa to HUVECs was time-dependent, reversible, and saturable, with apparent dissociation constants of 4.5 and 1.5 nM, respectively. HUVEC-bound complexes of H-kininogen and Factor XI generated Factor XIa activity only after the addition of purified Factor XIIa, and cell-bound Factor XIa in turn activated Factor IX, as documented by a 3H-labeled activation peptide release assay for 3H-Factor IX activation. The results indicate that cultured HUVECs provide a surface for the assembly and expression of an intrinsic Factor IX activator complex that may participate in the initiation of blood coagulation at sites of vascular injury.     

Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397

Factor IXLong Beach has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IXwild type and the two mutants (factor IXVal and factor IXThr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IXVal and factor IXwild type had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IXThr had only 5% clotting activity compared with normal factor IX, a slightly lower Km and significantly reduced kcat, using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXawild type-factor X model and the factor IXaThr-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond.     

Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.     

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.     

Tissue factor (coagulation factor III) inhibition by apolipoprotein A-II

Apolipoprotein A-II (apoA-II) has been shown to inhibit tissue factor participation in the activation of coagulation factor X by factor VIIa. The magnitude of inhibition was dependent on the concentration of the enzyme (factor VIIa) and substrate (factor X) present in the reaction. With factor VIIa at 0.86 nM, 0.41 microM apoA-II inhibited factor X activation as much as 50% at 200 nM factor X, with inhibition decreasing to 39% at 3 nM factor X. When factor X was held constant at 100 nM, 0.41 microM apoA-II inhibited its activation by 80% when factor VIIa was present at 26.7 pM, but the inhibition decreased to 47% when factor VIIa was increased to 1.75 nM. Kinetically, increasing apoA-II decreased the reaction Vmax. ApoA-II produced little effect on the apparent Km, but the apparent K1/2 for factor VIIa in the reaction increased as apoA-II concentration increased. In the presence of 0.75 pM bovine tissue factor, reconstituted with 4.31 microM phosphatidylserine-phosphatidylcholine (30:70, w/w) vesicles, and in the absence of apoA-II, the apparent Km was near 7 nM factor X when factor VIIa was present at 0.86 nM. Under the same conditions with factor X at 100 nM, the apparent K1/2 was near 56 pM factor VIIa. As apoA-II was added to 0.41 microM, the apparent K1/2 increased to about 200 pM factor VIIa. The aggregate results support a model in which apoA-II inhibits tissue factor potentiation of factor VIIa activity. Because the apparent K1/2 increases when apoA-II is added, the factor VIIa can apparently protect tissue factor from the effects of apoA-II. Thus, apoA-II appears to inhibit factor X activation by preventing the appropriate association of tissue factor with factor VIIa.     

The factor IX phospholipid-binding site is required for calcium-dependent activation of factor IX by factor XIa

To determine the functional role of the metal-dependent conformational changes in Factor IX, two populations of conformation-specific anti-Factor IX antibodies were prepared. Anti-Factor IX X Mg(II) antibodies bind to Factor IX in the presence of Mg(II) and other metal ions, but not in the absence of metal ions. Anti-Factor IX X Ca(II)-specific antibodies bind to Factor IX in the presence of Ca(II) and Sr(II), but not in the presence of Mn(II), Mg(II), and Ba(II). In the presence of a metal ion that induces the conformational transition recognized by the anti-Factor IX X Mg(II) antibodies, the concentrations of CaCl2 and SrCl2 needed for the half-maximal binding of the anti-Factor IX X Ca(II)-specific antibodies to Factor IX were reduced 3- and 20-fold, respectively. Factor IX binding to phospholipid vesicles was inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but was not inhibited by the Fab fragments of the anti-Factor IX X Mg(II) antibodies. Factor XIa activation of Factor IX was also inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but not by the anti-Factor IX X Mg(II) antibodies. These results support the hypothesis that Factor IX undergoes two metal-dependent conformational transitions: FIX----FIX'----FIX*. The first transition (FIX----FIX') is metal-dependent but cation-nonselective; the second transition (FIX'----FIX*) is metal-selective for Ca(II) or Sr(II). The second transition results in the expression of conformational determinants necessary for membrane binding and the Ca(II)-dependent activation of Factor IX by Factor XIa. These results suggest chemical similarity between a surface of a domain of Factor XIa and phospholipid vesicles, both of which interact with Factor IX in the presence of Ca(II).