Inhibition of factor IXa and factor Xa by antithrombin III/heparin during factor X activation

We investigated the kinetics of the inhibitory action of antithrombin III and antithrombin III plus heparin during the activation of factor X by factor IXa. Generation and inactivation curves were fitted to a three-parameter two-exponentional model to determine the pseudo first-order rate constants of inhibition of factor IXa and factor Xa by antithrombin III/heparin. In the absence of heparin, the second-order rate constant of inhibition of factor Xa generated by factor IXa was 2.5-fold lower than the rate constant of inhibition of exogenous factor Xa. It appeared that phospholipid-bound factor X protected factor Xa from inactivation by antithrombin III. It is, as yet, unclear whether an active site or a nonactive site interaction between factor Xa and factor X at the phospholipid surface is involved. The inactivation of factor IXa by antithrombin III was found to be very slow and was not affected by phospholipid, calcium, and/or factor X. With unfractionated heparin above 40 ng/ml and antithrombin III at 200 nM, the apparent second-order rate constant of inhibition of exogenous and generated factor Xa were the same. Thus, in this case phospholipid-bound factor X did not protect factor Xa from inhibition. In the presence of synthetic pentasaccharide heparin, however, phospholipid-bound factor X reduced the rate constant about 5-fold. Pentasaccharide had no effect on the factor IXa/antithrombin III reaction. Unfractionated heparin (1 micrograms/ml) stimulated the antithrombin III-dependent inhibition of factor IXa during factor X activation 400-fold. In the absence of reaction components this stimulated was 65-fold. We established that calcium stimulated the heparin-dependent inhibition of factor IXa.     

The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin . antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide . inhibitor complex are 2.4 x 10(8) M-1 min-1 and 4.0 x 10(6) M-1 min-1, respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 x 10(5) M-1 min-1 and 4.0 x 10(4) M-1 min-1, respectively. Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin c antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparin . antithrombin complex are 3.0 x 10(8) M-1 min-1 and 1.7 x 10(9) M-1 min-1, respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor IXa or mucopolysaccharide-thrombin interaction products by the heparin . antithrombin complex are 2.0 x 10(7) M-1 min-1 and 3.0 x 10(8) M-1 min-1, respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 x 10(4) M-1 min-1 and 4.25 x 10(5) M-1 min-1, respectively. Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Kinetic comparison of bovine blood coagulation factors IXa alpha and IXa beta toward bovine factor X

The Vmax/Km (microM -1 min -1.) for bovine factor X activation by bovine factor IXa alpha, in the presence of sufficient [Ca2+] to saturate the initial reaction rate, was 0.007. When factor IXa beta was substituted for factor IXa alpha in this reaction, the Vmax/Km decreased to 0.001, suggesting that factor IXa alpha was a more potent catalyst under these conditions. When phospholipid (PL) vesicles (egg phosphatidylcholine/bovine brain phosphatidylserine, 4:1 w/w) were added to these same systems, at levels sufficient to saturate their effects, little change in the Vmax/Km occurred when factor IXa alpha was the enzyme. However, when factor IXa beta was employed, the Vmax/Km dramatically increased to 0.023, demonstrating that factor IXa beta responded to PL addition to a much greater extent than did factor IXa alpha. Upon addition of thrombin-activated factor VIII (factor VIIIa,t), at a suboptimal level, to the above systems, the Vmax/Km for factor X activation by factor IXa alpha/Ca2+/PL/factor VIIIa,t was increased to 1.0, whereas this parameter for factor X activation by factor IXa beta/Ca2+/PL/factor VIIIa,t under the same conditions was found to be 27.3. During these studies, it was discovered that the factor X which became activated to factor Xa during the course of reaction participated in several feedback reactions: activation of factor X, activation of factor VIII, and conversion of factor IXa alpha to factor IXa beta. All feedback reactions, which are capable of complicating the kinetic interpretation, were inhibited by performing the studies in a system which contained a rapid factor Xa inhibitor, Glu-Gly-Arg-CH2Cl, thus allowing kinetic constants to be accurately determined. The results show that while factor IXa alpha is a more efficient enzyme than factor IXa beta toward factor X activation in the absence of cofactors, the response of factor IXa beta to the reaction cofactors, PL and factor VIIIa,t, is much greater than that of factor IXa alpha.     

Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.     

Interaction of factor Xa with heparin does not contribute to the inhibition of factor Xa by antithrombin III-heparin

Factor Xa modified by reductive methylation (greater than 92%) loses the capacity to bind heparin as determined both by gel chromatography and by sedimentation equilibrium ultracentrifugation. The kinetic properties of methylated factor Xa differ, with respect to KM and Vmax for a synthetic tripeptide substrate and for antithrombin III inhibition rate constants, from those of the unmodified enzyme. The 10,000-fold rate enhancement elicited by the addition of heparin to the antithrombin III inhibition reaction, however, is the same. The observed second-order rate constants (k"obs) for antithrombin III inhibition of factor Xa and methylated factor Xa are 3000 and 340 M-1 s-1, respectively, whereas k"obs values for the inhibition of factor Xa or methylated factor Xa with antithrombin III-heparin are 4 X 10(7) and 3 X 10(6) M-1 s-1, respectively. These findings provide direct evidence that the interaction of factor Xa with heparin is not involved in the heparin-enhanced inhibition of this enzyme.     

Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by approximately 100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of approximately 1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.     

Mechanism of catalysis of inhibition of factor IXa by antithrombin in the presence of heparin or pentasaccharide

Because of the homology between factor IXa and factor Xa (f.IXa and f.Xa, respectively), and the critical upstream position of f.IXa in the coagulation cascade, the contribution of the heparin-derived pentasaccharide to antithrombin-mediated inhibition of f.IXa was investigated. Pentasaccharide promotes inhibition of both f.IXa and f.Xa generated in recalcified plasma. This result demonstrates that antithrombin is the predominant inhibitor of f.IXa in plasma, and that the activity of antithrombin is promoted by pentasaccharide. Kinetic experiments reveal that pentasaccharide increases the rates of antithrombin-mediated inhibition of both f.IXa and f.Xa by 2 orders of magnitude. These findings indicate that pentasaccharide-induced conformational changes in antithrombin enhance its capacity to inhibit both f.IXa and f.Xa. In the presence of Ca2+, full-length heparin produces an additional approximately 10-fold increase in the rates of inhibition of both enzymes, consistent with a template role of heparin. Heparin binding to f.Xa was previously shown to be promoted in the presence of Ca2+. Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca2+ compared with its absence. Thus, Ca2+ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa. Furthermore, our data suggest that the efficacy of pentasaccharide for prevention and treatment of thrombotic disorders may reflect its action at two sites in the coagulation system.     

Molecular interactions of the intrinsic activation complex of coagulation: binding of native and activated human factors IX and X to defined phospholipid vesicles

The assembly of proteins of the intrinsic activation complex has been partially elucidated. In the present study we examine the association of gamma-carboxylated serine proteinase zymogens factors IX and X, and their proteolytically activated counterparts factors IXa and Xa to unilamellar lipid vesicles of defined composition using three types of physical measurement. Utilizing relative light scatter to estimate the dissociation constants for binding in the presence of calcium ions, it appears that factor IXa (0.93 +/- 0.37 microM) may preferentially associate with phospholipids relative to factor IX (0.35 +/- 0.08 microM). In contrast, factor X (0.34 +/- 0.14 microM), the substrate for factor IXa, appears to bind to phospholipid with a higher affinity than factor Xa (0.58 +/- 0.13 microM). These observations are compatible with the hypothesized dynamics where the forward 'traffic' is facilitated by favoring the association of factor IXa with factor X. The dissociation constants were estimated by molecular exclusion chromatography (1.1 - 2.5 microM) and do not reflect these relative and ordered differences in association with lipid vesicles. Quasi-elastic light scatter analyses indicate that each protein appears to saturate the same vesicle surface, consistent with competition for similar surface lipids, although the molecular shell formed by factor Xa (36 A) is smaller, suggesting that it has a different packing on the phospholipid surface than the other proteins (64-79 A). The pattern of preferential affinities for phospholipid is consistent with a kinetically functional forward traffic through the reaction precursors to products, and suggests that these preferential affinities may assist in the ordering of the four proteins in the intrinsic activation complex.     

Functional difference between intrinsic and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages

Activation of coagulation factor X via the intrinsic pathway requires the assembly of factors IXa and VIII on lipid membranes. It is known that the platelet expresses membrane sites for assembly of factors IXa/VIII and promotes efficient factor X activation. We now show that human blood monocytes, but not lymphocytes or polymorphonuclear leukocytes, also express appropriate sites for factors IXa/VIII assembly. The maximal rate of factor X activation by factors IXa (0.75 nM) and VIII (1 unit/ml) assembled on monocytes is similar to the maximal rate on platelets. This rate, adjusted per micromole of lipid phosphorus, is 1636 +/- 358 nM factor Xa/min on monocyte, and 1569 +/- 54 nM factor Xa/min on platelets. At physiologic concentrations of factors X and VIII, the activation rate increases with factor IXa concentration asymptotically approaching a maximum. Half-maximal rate is achieved with 1.0 +/- 0.16 nM factor IXa. Monocytes and macrophages, but not platelets, can express membrane tissue factor and thus promote simultaneous assembly of two distinct factor X-activating protease complexes. In these studies, blood monocytes and alveolar macrophages are used as membrane sources in kinetic experiments comparing factor X activation by intrinsic (factor IXa/VIII) versus extrinsic (factor VII/tissue factor) protease complexes. At plasma concentration of factors VIII and VII, apparent Km on the monocyte is 14.6 +/- 1.4 nM for intrinsic and 117.0 +/- 10.1 nM for extrinsic activation. The apparent Km on alveolar macrophages is 12.1 +/- 1.9 and 90.6 +/- 10.2 nM for intrinsic and extrinsic activation, respectively. Maximal rates on monocytes at saturating concentration of factors IXa, VIII, and VII are 48.0 +/- 11.2 nM factor Xa/min, for intrinsic activation, and 16.5 +/- 5.5 nM factor Xa/min, for extrinsic activation. These data show that the monocyte/macrophage is the only blood-derived cell type with membrane sites for both intrinsic and extrinsic pathway assembly. We have exploited this characteristic of the monocyte/macrophage membrane to demonstrate that factor X activation by the intrinsic pathway protease is more efficient than activation via the extrinsic pathway protease complex.     

Thrombin generation and inactivation in the presence of antithrombin III and heparin

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the generation and inhibition of activated coagulation factor X in pure systems and in human plasma

The overall generation and inhibition of human factor Xa have been studied in pure systems and plasma to determine the kinetic characteristics of inhibition during factor Xa generation. Generation curves were measured amidolytically in a pure system containing factor X and antithrombin, which was activated with the factor X-activating enzyme of Russell's viper venom (RVV-X). The measured change in factor Xa level with time was fitted to a 3-parameter 2-exponential model to determine apparent first-order rates of inhibition. With antithrombin at 4.5 microM, the inhibition rate constant thus obtained was very close to the known rate of inhibition of exogenous enzyme. Factor Xa generation curves were also analyzed in plasma; however, to reduce interference in the assay of thrombin, congenitally prothrombin-deficient plasma was used containing 0.5 microM D-Phe-Pro-Arg-chloromethylketone. In plasma, factor Xa generated in the presence of phospholipid and Ca2+ ions by RVV-X, factor IXa, or tissue factor was inhibited more slowly than exogenous enzyme. The reduction was particularly severe with tissue factor activation, where the rate was 0.04-0.06 min-1. This protection by tissue factor was also observed in pure systems and apparently required factor VII.     

Inhibition of serine proteinases by p-carbethoxyphenyl esters of epsilon-guanidino- and epsilon-amino caproic acid: thermodynamic and molecular modeling study

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of epsilon-guanidino-caproic acid methanesulphonate (epsilon-GCA-CEP) on the catalytic properties of human LYS77-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine alpha-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine beta-trypsin (EC 3.4.21.4), porcine pancreatic beta-kallikrein-B (EC 3.4.21.35), human urinary kallikrein (EC 3.4.21.35) and the Mr 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M; T = 21 +/- 0.5 degrees C), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl epsilon-amino-caproate hydro chloride (epsilon-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (Ki) for epsilon-GCA-CEP and epsilon-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) epsilon-GCA-CEP interacts with bovine factor Xa and bovine alpha-thrombin with an higher affinity than that observed for epsilon-ACA-CEP binding; (ii) both inhibitors associate to bovine beta-trypsin with the same affinity; and (iii) epsilon-ACA-CEP inhibits human Lys77-plasmin and the Mr 54,000 species of human urokinase with an higher affinity than that reported for epsilon-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of epsilon-ACA-CEP for ancrod, crotalase, porcine pancreatic beta-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by epsilon-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of epsilon-GCA-CEP and epsilon-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).     

Allosteric activation of antithrombin critically depends upon hinge region extension

Antithrombin (AT) inhibits most of the serine proteases generated in the blood coagulation cascade, but its principal targets are factors IXa, Xa, and thrombin. Heparin binding to AT, via a specific pentasaccharide sequence, alters the conformation of AT in a way that promotes efficient inhibition of factors IXa and Xa, but not of thrombin. The conformational change most likely to be relevant to protease recognition is the expulsion of the N-terminal portion of the reactive center loop (hinge region) from the main beta-sheet A. Here we investigate the hypothesis that the exosites on the surface of AT are accessible for interaction with a protease only when the hinge region is fully extended, as seen in the related Michaelis complex between heparin cofactor II and thrombin. We engineered a disulfide bond between residues 222 on strand 3A and 381 in the reactive center loop to prevent the extension of the hinge region upon pentasaccharide binding. The disulfide bond did not significantly alter the ability of the variant to bind to heparin or to inhibit thrombin. Although the basal rate of factor Xa inhibition was not affected, that of factor IXa inhibition was reduced to the limit of detection. In addition, the disulfide bond completely abrogated the pentasaccharide accelerated inhibition of factors Xa and IXa. We conclude that AT hinge region extension is the activating conformational change for inhibition of factors IXa and Xa, and propose models for the progressive and activated AT Michaelis complexes with thrombin, factor Xa, and factor IXa.     

Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism

The kinetics of the activation of human prothrombin catalyzed by human prothrombinase was studied using the fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Prothrombinase proteolytically activates prothrombin to alpha-thrombin by cleavages at Arg273-Thr274 (bond A) and Arg322-Ile323 (bond B). The differential fluorescence properties of DAPA complexed with the intermediates and products of human prothrombin activation were exploited to study the kinetics of the individual bond cleavages in the zymogen. When the catalyst was composed of prothrombinase (human factor Xa, human factor Va, synthetic phospholipid vesicles, and calcium ion), initial velocity studies of alpha-thrombin formation indicated that the kinetic constants for the cleavage of bonds A or B were similar to the constants that were obtained for the overall reaction (bonds A + B). The progress of the reaction was also monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results indicated that the activation of human prothrombin catalyzed by prothrombinase proceeded exclusively via the formation of meizothrombin (bond B-cleaved) as an intermediate. Kinetic studies of the cofactor dependence of the rates of cleavage of the individual bonds indicated that, in the absence of the cofactor, cleavage at bond B would constitute the rate-limiting step in prothrombin activation. Progress curves for prothrombin activation catalyzed by prothrombinase and monitored using the fluorophore DAPA were typified by the appearance of a transient maximum, indicating the formation of meizothrombin as an intermediate. When factor Xa alone was the catalyst, progress curves were characterized by an initial burst phase, suggesting the rapid production of prethrombin 2 (bond A-cleaved) followed by its slow conversion to alpha-thrombin. Gel electrophoresis followed by autoradiography was used to confirm these results. Collectively, the results indicate that the activation of human prothrombin via the formation of meizothrombin as an intermediate is a consequence of the association of the cofactor, human factor Va, with the enzyme, human factor Xa, on the phospholipid surface.     

Modulation of human prothrombin activation on phospholipid vesicles and platelets using monoclonal antibodies to prothrombin fragment 2

Prothrombin contains two kringle domains that are removed during activation to the blood clotting enzyme alpha-thrombin. By analogy with other kringle-containing proteins the prothrombin kringles may play a role in the protein-protein interactions necessary for prothrombin activation. Four monoclonal antibodies to prothrombin kringle 2 have been produced against human prothrombin, and a fifth monoclonal antibody was produced against a synthetic peptide consisting of amino acid residues 216-231 of kringle 2. Each antibody was tested for its ability to block prothrombin activation by factor Xa. In the presence of phosphatidylcholine/phosphatidylserine vesicles and factor Va, two of the antibodies, alpha HII-3 and alpha HII-4, inhibited prothrombin activation at a 90 and 50% level, respectively. Two other monoclonal antibodies (alpha HII-6 and alpha HII-7) and the antipeptide antibody (alpha HII-5) had no effect on prothrombin activation. When factor Xa was the catalyst alone, antibody alpha HII-3 lost the ability to inhibit prothrombin activation whereas antibody alpha HII-4 again partially inhibited the reaction. When human platelets were the reaction surface, the patterns of inhibition by the anti-fragment 2 antibodies were identical to that observed with phospholipid vesicles. These data suggest a role for prothrombin fragment 2 in activation, possibly by mediating the interaction of substrate prothrombin with factor Xa or factor Va on the phospholipid surface.     

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.     

Localization of an antithrombin exosite that promotes rapid inhibition of factors Xa and IXa dependent on heparin activation of the serpin

We have previously shown that exosites in antithrombin outside the P6-P3' reactive loop region become available upon heparin activation to promote rapid inhibition of the target proteases, factor Xa and factor IXa. To identify these exosites, we prepared six antithrombin-alpha 1-proteinase inhibitor chimeras in which antithrombin residues 224-286 and 310-322 that circumscribe a region surrounding the reactive loop on the inhibitor surface were replaced in 10-16-residue segments with the homologous segments of alpha1-proteinase inhibitor. All chimeras bound heparin with a high affinity similar to wild-type, underwent heparin-induced fluorescence changes indicative of normal conformational activation, and were able to form SDS-stable complexes with thrombin, factor Xa, and factor IXa and inhibit these proteases with stoichiometries minimally altered from those of wild-type antithrombin. With only one exception, conformational activation of the chimeras with a heparin pentasaccharide resulted in normal approximately 100-300-fold enhancements in reactivity with factor Xa and factor IXa. The exception was the chimera in which residues 246-258 were replaced, corresponding to strand 3 of beta-sheet C, which showed little or no enhancement of its reactivity with these proteases following pentasaccharide activation. By contrast, all chimeras including the strand 3C chimera showed essentially wild-type reactivities with thrombin after pentasaccharide activation as well as normal full-length heparin enhancements in reactivity with all proteases due to heparin bridging. These findings suggest that antithrombin exosites responsible for enhancing the rates of factor Xa and factor IXa inhibition in the conformationally activated inhibitor lie in strand 3 of beta-sheet C of the serpin.     

The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway

Prolixin-S is a salivary anticoagulant of the blood-sucking insect, Rhodnius prolixus, and known as an inhibitor of the intrinsic Xase. We report here its inhibitory mechanisms with additional important anticoagulation activities. We found prolixin-S specifically bound to factor IX/IXa in the presence of Ca(2+) ions. Light scattering and surface plasmon resonance studies showed that prolixin-S interfered with factor IX/IXa binding to the phospholipid membrane, indicating that prolixin-S inhibit Xase activity of factor IXa by interference with its Xase complex formation. Furthermore, reconstitution experiments showed that prolixin-S binding to factor IX strongly inhibited factor IXa generation by factor XIa. We also found that prolixin-S inhibited factor IXa generation by factor VIIa-tissue factor complex and factor IXalpha generation by factor Xa. These results suggest that prolixin-S inhibits both intrinsic and extrinsic coagulations by sequential inhibition of all coagulation pathways in which factor IX participates. It was also suggested that prolixin-S may bind to factor IX/IXa by recognizing conformational change of the Gla domain induced by Ca(2+) binding.