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PubMed2001

Factor IXa:factor VIIIa interaction. helix 330-338 of factor ixa interacts with residues 558-565 and spatially adjacent regions of the a2 subunit of factor VIIIa.

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca(2+) and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K(d(A2)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 100 nm, dEGR-IXa(R333Q) approximately 1.8 micrometer, and dEGR-IXa(helixVII) >10 micrometer. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit. Apparent K(d(peptide)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 4 micrometer, and dEGR-IXa(R333Q) approximately 62 micrometer. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.

Amino Acid Substitution↗
PubMed1981

Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates.

The subsite specificities of bovine factor IXa, factor Xa, factor XIa, factor XIIa, thrombin, plasma kallikrein, and trypsin were mapped with amino acid, dipeptide, and longer peptide thioester substrates. Each substrate contained a P1 Arg residue. The P1' residues included thiol residues which are analogues of valine, leucine, and isoleucine, respectively, and the P2 residue included 12 representative amino acid residues. Longer substrates with the sequence at the antithrombin III reactive site and at the zymogen activation site of various coagulation factors were also studied. The enzymatic hydrolysis of the thioesters was measured in the presence of 4,4'-dithiodipyridine which provides a very sensitive assay for the free thiol. The thioesters were excellent substrates for the coagulation factors studied, and the kcat/Km values for the best thioester substrates were higher than those previously reported for most of these enzymes. Thrombin and plasma kallikrein were the most active of the coagulation factors toward the thioester substrates. The best substrate for thrombin was Z-Gly-Arg-SCH2C6H5, although substrates containing proline in the P2 position were also quite effective. Some of the better substrates for plasma kallikrein had a P2 Phe or Trp residue. Factor IXa was the least reactive of the coagulation factors and hydrolyzed only four of the dipeptide thioesters. Substrates with bulky hydrophobic groups such as Phe or Trp in the P2 position were the most reactive with factor IXa. Factor Xa hydrolyzed all the thioester substrates tested, the most reactive being Z-Gly-Arg-SCH2C6H5. This is consistent with the fact that glycine and arginine are present in the P2 and P1 positions, respectively, of the factor Xa sensitive bonds in prothrombin which is the physiological substrate for factor Xa. Bovine factor XIa showed the least amount of specificity of the various coagulation factors and was quite reactive toward all of the thioester substrates. The most sensitive substrate for this enzyme was also Z-Gly-Arg-SCH2C6H5. Factor XIIa preferred the dipeptide with a P2 Phe, although the simpler thioester Z-Arg-SCH2CH(CH3)2 was more reactive. Trypsin hydrolyzed all of the thioester substrates at a high rate and showed little substrate specificity. With all enzymes studied, extension of the thioester substrate beyond P2 or the P1' thiol leaving group did not lead to an improvement in hydrolysis. Due to their high kcat/Km values and the ease of detecting the thiol leaving group, thioester substrates should be extremely useful for future studies of coagulation proteases.

Amino Acids, Sulfur↗
PubMed1985

Assembly of the intrinsic factor X activating complex--interactions between factor IXa, factor VIIIa and phospholipid.

The activation of blood coagulation factor X by factor IXa is strongly stimulated by the non-enzymatic cofactors phospholipid, Ca2+ and activated factor VIII. In this paper we present a method by which we were able to determine binding affinities of factor IXa for phospholipids (either in the absence or presence of factor VIIIa) from kinetic measurements of factor X activation. It is shown that rates of factor X activation in the presence of phospholipids can be saturated with an excess factor VIIIa at limiting amounts of factor IXa and vice versa. Our data indicate that the enzymatic unit in the intrinsic factor X activator is a 1:1 stoichiometrical complex of factor IXa and factor VIIIa bound to phospholipid. Titrations with factor IXa at fixed concentrations of phospholipid and factor X show that the apparent dissociation constant of factor IXa for phospholipid is lowered from 10(-6) M to 10(-8) M by the presence of factor VIIIa. We conclude, that in analogy with the role of factor Va in prothrombin activation, phospholipid-bound factor VIIIa functions as a high-affinity binding site ("receptor") for factor IXa in the intrinsic factor X activating complex. Therefore, factor VIIIa increases the observed Vmax of factor X activation by 1) enhancing the kcat of the reaction and 2) increasing the amount of phospholipid-bound factor IXa that participates in factor X activation.

Blood Coagulation Factors↗
PubMed1981

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.

Antithrombin III↗
PubMed1982

The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa, and thrombin.

Reactions between near equimolar amounts of antithrombin and Factors IXa or Xa resulted in the formation of a free proteolytically modified, two-chain form of the inhibitor, in addition to the inactive antithrombin-protease complexes. The modified inhibitor produced by either enzyme was electrophoretically identical with that formed in the reaction with thrombin. As in the latter reaction, the formation of the modified antithrombin by Factor Xa was increased in the presence of heparin, while only small amounts were produced by Factor IXa both in the absence and presence of the polysaccharide. NH2-terminal sequence analyses of the isolated modified inhibitor formed by Factor Xa showed that a single Arg-Ser bond in the COOH-terminal end of the inhibitor had been cleaved. This cleavage site is identical with that identified in free thrombin-modified antithrombin. The purified antithrombin-Factor IXa and antithrombin-Factor Xa complexes were dissociated by ammonia or hydroxylamine into free enzyme and a modified two-chain form of the inhibitor. Electrophoresis studies and NH2-terminal sequence analyses showed that the modified antithrombin obtained from either complex was identical with that produced in free form by the two enzymes and also with the modified inhibitor that is released from the antithrombin-thrombin complex. The fact that identical results were obtained for the reactions between antithrombin and three enzymes with different specificities strongly suggests that the observed Arg-Ser cleavage site is the active site of antithrombin.

Amino Acid Sequence↗
PubMed1985

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.

Blood Coagulation↗
PubMed1999

The second epidermal growth factor-like domain of human factor IXa mediates factor IXa binding to platelets and assembly of the factor X activating complex.

Factor IXa binding to the activated platelet surface is required for efficient catalysis of factor X activation. Platelets possess a specific binding site for factor IXa, occupancy of which has been correlated with rates of factor X activation. However, the specific regions of the factor IXa molecule that are critical to this interaction have not yet been fully elucidated. To assess the importance of the second epidermal growth factor (EGF2) domain of factor IXa for platelet binding and catalysis, a chimeric protein (factor IXa(Xegf2)) was created by replacement of the EGF2 domain of factor IX with that of factor X. Competition binding experiments showed 2 different binding sites on activated platelets (approximately 250 each/platelet): (1) a specific factor IXa binding site requiring the intact EGF2 domain; and (2) a shared factor IX/IXa binding site mediated by residues G(4)-Q(11) within the Gla domain. In kinetic studies, the decreased V(max) of factor IXa(Xegf2) activation of factor X on the platelet surface (V(max) 2. 90 +/- 0.37 pM/min) versus normal factor IXa (37.6 +/- 0.15 pM/min) was due to its decreased affinity for the platelet surface (K(d) 64.7 +/- 3.9 nM) versus normal factor IXa (K(d) 1.21 +/- 0.07 nM), resulting in less bound enzyme (functional complex) under experimental conditions. The hypothesis that the binding defects of factor IXa(Xegf2) are the cause of the kinetic perturbations is further supported by the normal k(cat) of bound factor IXa(Xegf2) (1701 min(-)(1)) indicating (1) an intact catalytic site and (2) the normal behavior of bound factor IXa(Xegf2). The EGF2 domain is not a cofactor binding site since the mutant shows a normal rate enhancement upon the addition of cofactor. Thus, the intact EGF2 domain of factor IXa is critical for the formation of the factor X activating complex on the surface of activated platelets.

Animals↗
PubMed1994

Factor IXa protects factor VIIIa from activated protein C. Factor IXa inhibits activated protein C-catalyzed cleavage of factor VIIIa at Arg562.

Factor VIIIa is inactivated by both factor IXa and activated protein C. The latter protease rapidly attacked a site at Arg562 (A2 subunit), whereas both proteases slowly cleaved factor VIIIa at Arg336 (A1 subunit). Cofactor inactivation catalyzed by activated protein C was 8-fold faster than that catalyzed by factor IXa. Simultaneous reaction of factor VIIIa with the two enzymes resulted in a rate of inactivation intermediate to that observed for the individual proteases. Under these conditions, the activated protein C-catalyzed cleavage at Arg562 was inhibited such that cofactor inactivation resulted primarily from cleavage at Arg336. Substitution of factor IXa modified in its active site with 6-(dimethylamino)-2-naphthalenesulfonyl-glutamylglycylarginyl++ + chloromethyl ketone (DEGR-IXa) for the native enzyme yielded a similar rate of activated protein C-catalyzed cleavage at the A1 site, whereas cleavage at the A2 site was virtually eliminated. However, the inclusion of protein S resulted in a marked increase in cleavage at the A2 site that correlated with an increased rate of cofactor inactivation. Active site-modified activated protein C inhibited the factor IXa-dependent enhancement of factor VIIIa reconstitution from isolated subunits. In addition, the factor VIIIa-dependent fluorescence enhancement of DEGR-activated protein C was inhibited by EGR-IXa. These results indicate that factor IXa can reduce the rate of activated protein C-catalyzed cofactor inactivation by selectively blocking cleavage at the A2 domainal site, an effect reversed by protein S. One mechanism consistent with the reciprocal inhibitory effects of the proteases is that activated protein C and factor IXa occupy overlapping sites on the cofactor. Thus, factor IXa may protect factor VIIIa by preventing activated protein C binding.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Factor X bound to the surface of activated human platelets is preferentially activated by platelet-bound factor IXa.

Factor X is a zymogen in the blood coagulation system which is activated by the serine protease, factor IXa, in a reaction that is promoted by the presence of stimulated platelets. We have shown previously that platelets possess a binding site for factor IXa, the occupancy of which is correlated with the rate of factor X activation (Ahmad et al., 1989b,c). Similarly, we have described a different binding site on the surface of activated platelets to which the substrate for this reaction, factor X, can bind (see the accompanying paper). This "zymogen binding site" is of moderate affinity and is relatively nonspecific; apparently shared to some degree by factor X and other vitamin K-dependent proteins, most notably prothrombin. We have found that prothrombin fragment 1 not only is able to displace factor X from this platelet binding site but also possesses the ability to inhibit the platelet-dependent activation of factor X. We have developed two mathematical models for the activation of factor X by platelet-bound factor IXa. The first model assumes that factor X is activated in a manner that is totally unrelated to the presumptive zymogen binding site, whereas the second model requires factor X to first bind to this site before it may interact with platelet-bound factor IXa and become activated. Within the context of each of these models, we have evaluated three mechanisms by which prothrombin fragment 1 may inhibit factor X activation. The data presented herein are most consistent with the precept that platelet-bound factor X is activated by platelet-bound factor IXa (kcat approximately 0.0011 s-1) in an explicitly two-dimensional reaction (Km.2D approximately 230 molecules per platelet). Prothrombin fragment 1 is believed to disrupt this reaction by competing with factor X for the zymogen binding site (Ki approximately 470 nM) and, to a lesser degree, by displacing factor IXa from its binding site (Ki approximately 7 microM). These findings suggest that platelet-bound zymogen factor X represents a kinetically important pool of substrate that is preferentially activated on the surface of activated platelets.

Binding Sites↗
PubMed1998

The A2 subunit of factor VIIIa modulates the active site of factor IXa.

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Isolated subunits of factor VIIIa were examined for their ability to accelerate the factor IXa-catalyzed activation of factor X. The A2 subunit enhanced the kcat for this conversion by 100-fold whereas the Km for factor X was unaffected. The apparent Kd for the interaction of A2 subunit with factor IXa was approximately 300 nM. Similar results were obtained using purified A2 expressed as the isolated domain in Chinese hamster ovary cells, although this material was less stable than the factor VIIIa-derived material. Isolated A1 and A3-C1-C2 subunits showed no effect on the rate of factor X conversion. A2 subunit increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa (Deltar = 0.015) and markedly increased anisotropy in the presence of factor X (Deltar = 0.057), suggesting that it contributes to the orientation of the factor IXa active site and its relation to substrate. A synthetic peptide to A2 residues 558-565 inhibited the A2-dependent enhancement of factor X activation with an IC50 = 40 microM, a value similar to its Ki for inhibition of the intrinsic factor Xase (105 microM). These results indicate that the isolated A2 subunit modulates the active site of factor IXa and identifies a functional role for this subunit in factor VIIIa.

Animals↗
PubMed2001

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.

Binding Sites↗
PubMed1999

Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa.

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.

Antibodies, Monoclonal↗
PubMed1990

Heparin-stimulated inhibition of factor IXa generation and factor IXa neutralization in plasma.

Generation and inhibition of activated factor IXa was studied in factor XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine). Interference of other (activated) clotting factors with the factor IXa activity measurements could be avoided by using a highly specific and sensitive bioassay. Factor IXa generation curves were analyzed according to a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed factor IXa formation and pseudo first order kinetics of inhibition of factor XIa and factor IXa. In the absence of heparin, factor IXa activity in plasma reached final levels that were found to increase with increasing amounts of factor XIa used to activate the plasma. When the model was fitted to this set of factor IXa generation curves, the analysis yielded a rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of factor IXa activity was observed (the estimated rate constant of inhibition of factor IXa was 0). Thus, in the absence of heparin, the final level of factor IXa in plasma is only dependent on the initial factor XIa concentration. While neutralization of in situ generated factor IXa in normal plasma was negligible, unfractionated heparin dramatically enhanced the rate of inactivation of factor IXa (apparent second order rate constant of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic pentasaccharide heparin, the smallest heparin chain capable of binding antithrombin III, stimulated the inhibition of in situ generated factor IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per microgram pentasaccharide/mL). We found that free calcium ions were absolutely required to observe an unfractionated heparin and pentasaccharide-stimulated neutralization of factor IXa activity. Factor XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not affected by unfractionated heparin or pentasaccharide in the range of heparin concentrations studied.

Activation Analysis↗
PubMed1989

Platelet receptor occupancy with factor IXa promotes factor X activation.

To investigate the activated platelet surface as a locus for factor X activation, the functional consequences of factor IXa binding to platelets were studied. The concentration of factor IXa required for half-maximal rates of factor X activation in the presence of factor VIIIa and thrombin-activated platelets was 0.53 nM, which is close to the Kd (0.56 nM) for factor IXa binding to platelets under identical conditions, determined from equilibrium binding studies. In direct comparative experiments, there was a close correspondence between equilibrium binding of factor IXa to thrombin-activated platelets in the presence of factor VIIIa and kinetic determinations of factor X activation rates. Analysis by polyacrylamide gel electrophoresis revealed that 125I-labeled factor IXa bound to platelets was structurally intact and did not form covalent complexes with platelet proteins. Factor IXa active site-inhibited by 5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycylarginyl chloromethyl ketone was shown to be a competitive inhibitor of factor IXa binding in the absence (Ki = 2.3 nM) and presence (Ki = 0.43 nM) of factor VIIIa and factor X and of factor X activation (Ki = 0.4 nM) by factor IXa in the presence of factor VIIIa, indicating that the generation of factor Xa is not required for factor IXa binding and that factor IXa bound to activated platelets in the presence of factor VIIIa is closely coupled with rates of factor X activation. We conclude that factor IXa bound tightly to a platelet receptor in the presence of factor VIIIa is the enzyme active in factor X activation.

Blood Platelets↗
PubMed1988

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.

Algorithms↗
PubMed1995

Localization of factor IXa and factor VIIIa interactive sites.

The contribution of the catalytic and noncatalytic domains of factor IXa to the interaction with its cofactor, factor VIIIa, was evaluated. Two proteolytic fragments of factor IXa, lacking some or all of the serine protease domain, failed to mimic the ability of factor IXa to enhance the reconstitution of factor VIIIa from isolated A1/A3-C1-C2 dimer and A2 subunit. Both fragments, however, inhibited this factor IXa-dependent activity. Selective thermal denaturation of the factor IXa serine protease domain eliminated its effect on factor VIIIa reconstitution. Modification of factor IXa with dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-IXa) stabilized this domain, and heat-treated DEGR-IXa retained its ability to enhance factor VIIIa reconstitution. These results indicate the importance of the serine protease domain as well as structures residing in the factor IXa light chain (gamma-carboxyglutamic acid and/or epidermal growth factor domains) for cofactor stabilizing activity. In the presence of phospholipid, the A1/A3-C1-C2 dimer produced a saturable increase in the fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa (Fl-FFR-IXa). This effect was inhibited by a factor IXa fragment comprised of the gamma-carboxyglutamic acid and epidermal growth factor domains. The difference in Fl-FFR-IXa anisotropy in the presence of A1/A3-C1-C2 dimer (delta r = 0.043) compared with factor VIIIa (delta r = 0.069) represented the contribution of the A2 subunit, A peptide corresponding to factor VIII A2 domain residues 558-565 decreased the factor VIIIa dependent-anisotropy of Fl-FFR-IXa to a value similar to that observed with the A1/A3-C1-C2 dimer. These results support a model of multiple interactive sites in the association of the enzyme-cofactor complex and localize sites for the A1/A3-C1-C2 dimer and the A2 subunit to the factor IXa light chain and serine protease domain, respectively.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Interaction of factor IXa with factor VIIIa. Effects of protease domain Ca2+ binding site, proteolysis in the autolysis loop, phospholipid, and factor X.

We previously identified a high affinity Ca2+ binding site in the protease domain of factor IXa involving Glu235 (Glu70 in chymotrypsinogen numbering; hereafter, the numbers in brackets refer to the chymotrypsin equivalents) and Glu245[80] as putative ligands. To delineate the function of this Ca2+ binding site, we expressed IXwild type (IXWT), IXE235K, and IXE245V in 293 kidney cells and compared their properties with those of factor IX isolated from normal plasma (IXNP); each protein had the same Mr and gamma-carboxyglutamic acid content. Activation of each factor IX protein by factor VIIa.Ca2+.tissue factor was normal as analyzed by sodium dodecyl sulfate-gel electrophoresis. The coagulant activity of IXaWT was approximately 93%, of IXaE235K was approximately 27%, and of IXaE245V was approximately 4% compared with that of IXaNP. In contrast, activation by factor XIa.Ca2+ led to proteolysis at Arg318-Ser319[150-151] in the protease domain autolysis loop of IXaE245V with a concomitant loss of coagulant activity; this proteolysis was moderate in IXaE235K and minimal in IXaWT or IXaNP. Interaction of each activated mutant with an active site probe, p-aminobenzamidine, was also examined; the Kd of interaction in the absence and presence (in parentheses) of Ca2+ was: IXaNP or IXaWT 230 microM (78 microM), IXaE235K 150 microM (145 microM), IXaE245V 225 microM (240 microM), and autolysis loop cleaved IXaE245V 330 microM (350 microM). Next, we evaluated the apparent Kd (Kd,app) of interaction of each activated mutant with factor VIIIa. We first investigated the EC50 of interaction of IXaNP as well as of IXaWT with factor VIIIa in the presence and absence of phospholipid (PL) and varying concentrations of factor X. At each factor X concentration and constant factor VIIIa, EC50 was the free IXaNP or IXaWT concentration that yielded a half-maximal rate of factor Xa generation. EC50 values for IXaNP and IXaWT were similar and are as follows: PL-minus/X-minus (extrapolated), 2.8 microM; PL-minus/X-saturating, 0.25 microM; PLplus/X-minus, 1.6 nM; and PL-plus/X-saturating, 0.09 nM. Further, Kd,app of binding of active site-blocked factor IXa to factor VIIIa was calculated from its ability to inhibit IXaWT in the Tenase assay. Kd,app values in the absence and presence (in parentheses) of PL were: IXaNP or IXaWT, 0. 19 microM (0.07 nM); IXaE235K, 0.68 microM (0.26 nM); IXaE245V, 2.5 microM (1.35 nM); and autolysis loop-cleaved IXaE245V, 15.6 microM (14.3 nM). We conclude that (a) PL increases the apparent affinity of factor IXa for factor VIIIa approximately 2,000-fold, and the substrate, factor X, increases this affinity approximately 10-15-fold; (b) the protease domain Ca2+ binding site increases this affinity approximately 15-fold, and lysine at position 235 only partly substitutes for Ca2+; (c) Ca2+ binding to the protease domain increases the S1 reactivity approximately 3-fold and prevents proteolysis in the autolysis loop; and (d) proteolysis in the autolysis loop leads to a loss of catalytic efficiency with retention of S1 binding site and a further approximately 8-fold reduction in affinity of factor IXa for factor VIIIa.

1-Carboxyglutamic Acid↗
PubMed1995

Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin.

The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII-depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors-independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors-independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.

Amino Acid Sequence↗