Elimination of P1 Arginine 393 Interaction with Underlying Glutamic Acid 255 Partially Activates Antithrombin III for Thrombin Inhibition but Not Factor Xa Inhibition

Abstract

The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu255 on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg393–Glu255 hydrogen bond and ionic interactions were eliminated by converting Glu255 to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu255-mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wild-type parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000- and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although “approximation” is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all. The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu255 on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg393–Glu255 hydrogen bond and ionic interactions were eliminated by converting Glu255 to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu255-mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wild-type parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000- and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although “approximation” is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all. antithrombin III full-length high affinity heparin heparin pentasaccharide factor Xa observed pseudo-first-order rate constant apparent second-order rate constant second-order association rate constant dissociation equilibrium constant fluorophore-assisted carbohydrate electrophoresis stoichiometry of inhibition ionic strength polyethylene glycol Antithrombin III (ATIII)1 is a key inhibitor of blood coagulation enzymes and belongs to the serpin family of protease inhibitors whose members employ a common inhibition mechanism. However, the rate at which ATIII inhibits its target enzymes is abnormally low for a serpin, and activation by heparin or vascular wall heparan sulfate proteoglycans (HSPGs) is necessary for it to achieve the high inhibition rates characteristic of other serpins. The physiological importance of ATIII-heparin/HSPG interactions is supported by the occurrence of thrombosis in individuals carrying ATIII molecules with defects in heparin binding or activation, and by the clinical use of pharmaceutical heparin as an anticoagulant in patients with hereditary and acquired thrombosis. Because of the extensive use of heparin as a pharmaceutical, the mechanism for heparin activation of ATIII anticoagulant activity has been extensively investigated. These studies show that dramatically increased rates of thrombin and factor Xa inhibition can be achieved upon binding of heparin. Heparin binding to antithrombin is a two-step process consisting of an initial low affinity interaction that induces a large scale protein conformational change, which leads to high affinity binding and ATIII activation (1Olson S. Shore J. J. Biol. Chem. 1982; 257: 14891-14895Abstract Full Text PDF PubMed Google Scholar). The rate enhancement for the inhibition of thrombin results from an approximation (bridging) mechanism in which long, high affinity heparin chains (>18 sugars) facilitate a >1000-fold increased rate of association between thrombin and ATIII (2Olson S. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar). In contrast, a specific anticoagulantly active pentasaccharide sequence of heparin is sufficient for accelerating the rate of factor Xa inhibition by ATIII. Binding of this pentasaccharide causes a protein conformational change and is associated with a 270-fold increased rate of factor Xa inhibition and a 1.7-fold increase in rate of inhibition of thrombin (3Olson S.T. Bjork I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar). As a part of the effort to understand how heparin activates antithrombin III, it is important to determine why its basal rate of target enzyme inhibition is depressed compared with other serpins. The low rates of association between ATIII and its target enzymes have been attributed to problems with accessibility of its P1 residue, arginine 393. On the basis of comparisons of x-ray structures for native antithrombin (4Schreuder H. deBoer B. Dijkema R. Mulders J. Theunissen H. Grootenhuis P. Hol W. Struct. Biol. 1994; 1: 48-54Crossref PubMed Scopus (267) Google Scholar, 5Carrell R. Stein P. Fermi G. Wardell M. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and native α-1 antitrypsin (6Elliott P. Lomas D. Carrell R. Abrahams J. Nat. Struct. Biol. 1996; 3: 676-681Crossref PubMed Scopus (239) Google Scholar), two factors were proposed to constrain the reactive loop and to limit accessibility of the ATIII P1 residue in the native molecule. One constraint involves shortening of the reactive loop and an associated decrease in loop mobility and flexibility due to incorporation of its amino-terminal side (including the P14 residue) into β-sheet A. The structure of an antithrombin III-pentasaccharide co-crystal (7Jin L. Abrahams J. Skinner R. Petitou M. Pike R. Carrell R. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (629) Google Scholar) and studies of a P14 serine-to-tryptophan substitution mutant (8Huntington J. Olson S. Fan B. Gettins P. Biochemistry. 1996; 35: 8495-8503Crossref PubMed Scopus (126) Google Scholar) show that upon occupation of the pentasaccharide binding site, the P14 residue is expelled from the sheet A, lengthening the reactive loop. Expulsion of the P14 residue by pentasaccharide binding, P14 modification with fluorescein (9Huntington J. Gettins P. Biochemistry. 1998; 37: 3272-3277Crossref PubMed Scopus (45) Google Scholar), or P14 substitution with glutamic acid (10Futamura A. Gettins P. J. Biol. Chem. 2000; 275: 4092-4098Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) is associated with a 200–300-fold increase in the rate of association with factor Xa and a much smaller 2-fold increase in the rate of reaction with thrombin. Therefore, the P14 constraint on reactive loop length and mobility plays a significant role in depressing the native ATIII rate of factor Xa inhibition but makes a negligible contribution to the low rate of thrombin inhibition. Native ATIII recognition by target proteinases is additionally thought to be affected by a second reactive loop constraint, which is also specific to antithrombin compared with other serpins. The second constraint directly involves the arginine 393 P1 residue and causes it to face inwards toward the body of the inhibitor rather than outwards toward the solvent in native ATIII (Fig. 1). As observed in crystal structures of native ATIII (4Schreuder H. deBoer B. Dijkema R. Mulders J. Theunissen H. Grootenhuis P. Hol W. Struct. Biol. 1994; 1: 48-54Crossref PubMed Scopus (267) Google Scholar, 5Carrell R. Stein P. Fermi G. Wardell M. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), this constraint involves ionic and hydrogen-bonding interactions between the P1 (Arg393) and P6′ (Arg399) arginines of the reactive loop polypeptide and a glutamic acid residue (Glu255), which projects out from the C sheet in the body of the inhibitor. Although there has been some question as to whether the 393/399/255 constraint observed in the native and other ATIII structures (7Jin L. Abrahams J. Skinner R. Petitou M. Pike R. Carrell R. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (629) Google Scholar, 11Skinner R. Chang W. Jin L. Pei X. Huntington J. Abrahams J.-P. Carrell R. Lomas D. J. Mol. Biol. 1998; 283: 9-14Crossref PubMed Scopus (93) Google Scholar) could be a consequence of dimerization between two ATIII molecules in the unit cells of the crystals and the incorporation of the reactive loop polypeptide of one molecule into β-sheet C of the other molecule, the existence of stabilizing 393/399/255 interactions under physiological conditions is supported by solution studies of increased P1 accessibility to the enzyme peptidylarginine deiminase in the presence of heparin pentasaccharide (7Jin L. Abrahams J. Skinner R. Petitou M. Pike R. Carrell R. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (629) Google Scholar, 12Pike R. Potempa J. Skinner R. Fitton H. McGraw W. Travis J. Owen M. Jin L. Carrell R. J. Biol. Chem. 1997; 272: 19652-19655Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Therefore, it has been proposed that a constraint on reactive site loop movement and P1 accessibility imposed by internal bonding with the side chain of arginine 393 contributes to the low inhibitory activity of native conformation ATIII and that release of this constraint is an important step in the activation of ATIII by heparin cofactor (5Carrell R. Stein P. Fermi G. Wardell M. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 7Jin L. Abrahams J. Skinner R. Petitou M. Pike R. Carrell R. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (629) Google Scholar, 12Pike R. Potempa J. Skinner R. Fitton H. McGraw W. Travis J. Owen M. Jin L. Carrell R. J. Biol. Chem. 1997; 272: 19652-19655Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The role of the P1 constraint in heparin activation of ATIII has been previously addressed in several studies. Two studies utilizing fluorescent reporter groups attached at the P1 position concluded that heparin binding to antithrombin does not greatly alter solvent accessibility (13Chuang Y. Swanson R. Raja S. Bock S. Olson S. J. Biol. Chem. 2001; 40: 6670-6679Google Scholar, 14Futamara A. Beechem J. Gettins P. Biochemistry. 2001; 40: 6680-6687Crossref PubMed Scopus (20) Google Scholar) or motional freedom of the P1 side chain (14Futamara A. Beechem J. Gettins P. Biochemistry. 2001; 40: 6680-6687Crossref PubMed Scopus (20) Google Scholar). Work with ATIII variants in which the P1 arginine 393 was replaced with a series of nonionic residues or histidine also imply that the P1 residue is similarly accessible to proteinases in the native and heparin-activated states (13Chuang Y. Swanson R. Raja S. Bock S. Olson S. J. Biol. Chem. 2001; 40: 6670-6679Google Scholar). However, because of the critical role of the P1 residue in serpin target enzyme interactions, the approach of substituting the P1 amino acid in these studies produced a concomitant change in the target proteinase specificity, and it was not possible to directly address the role of the P1 constraint with respect to the normal physiological targets of ATIII, thrombin and factor Xa. As described in this report, an alternative strategy for investigating the role of the P1 constraint in modulating ATIII anticoagulant activity involves alanine substitution of the negatively charged Glu255 residue that projects from the serpin body and interacts with the P1 and P6′ arginines. This approach has the advantages of maintaining the normal P1 arginine and wild-type reactive loop sequence and therefore permits investigation of the heparin cofactor mechanism to be conducted with respect to the normal physiological targets of ATIII. Using this approach we have quantitated the contributions of three different factors (the P1 and P14 reactive loop constraints and the approximation effect) to heparin cofactor activation of thrombin and factor Xa and have shown that at this of the for modulating ATIII inhibition of these enzymes of the P1 constraint plays a minor role, of the P14 constraint plays no role, and approximation plays a role in heparin of ATIII thrombin inhibition. In contrast, of the P1 constraint plays no role, of the P14 constraint plays a role, and approximation plays a minor role in heparin of factor Xa inhibition. The E255A was on a to heparin binding and facilitate A. X. P. B. I. Olson S. Bock S. J. PubMed Scopus Google Scholar). was as previously described Bock S. in Scholar) as the as the and as the mutant was by sequence of the it was to the of the sequence in by and in the cells with carrying the for or were and of cells and at the of the cells had to were to heparin and with a The was and the were for heparin cofactor were and to a second heparin which was as described but with the addition of polyethylene glycol to the activity from the second heparin were polyethylene glycol and by The was to a with and the ATIII was with a of in activity were and and by of ATIII was by was from the of ATIII B. Bjork I. J. Scopus Google Scholar). of the heparin from were in of and on a were by the L. Google Scholar), and the of in by fluorophore-assisted carbohydrate electrophoresis P. J. PubMed Scopus Google Scholar) high affinity heparin by S. Olson and I. from were and to a with with for antithrombin were with a at of the the of low and high affinity from the high affinity heparin were and and high affinity heparin was at affinity heparin were by ATIII. The to as heparin in these studies is high affinity heparin as were conducted at in In the absence of the ionic strength of was at was to achieve the higher ionic strength in the heparin binding studies. for and inhibition of thrombin and factor Xa in the absence and presence of pentasaccharide or heparin were by active thrombin and factor Xa with the ATIIIs at inhibitor to enzyme Pentasaccharide and active enzymes were by and high affinity heparin was as described heparin and pentasaccharide were at and respectively, to a the ATIII of inhibition antithrombin and target enzyme in were at for the of inhibition of the of the reaction were into of and the were of the reaction were to a enzyme activity was by of thrombin or factor Xa and the initial rate at enzyme activity was the of inhibition was by the to the to proteinase inhibition. In addition to of thrombin and factor Xa inhibitory the of ATIII and at different were by of the of the reaction on under for the of activity were under pseudo-first-order conditions and ATIII and target enzymes in from to ATIIIs and thrombin or were in in a of The was for thrombin and for of thrombin and activity were by of or pseudo-first-order rate were from the of a of enzyme of enzyme and inhibitor rate were by the observed pseudo-first-order rate by the ATIII several the was to the inhibition of thrombin and by and in the presence of of pentasaccharide and heparin. ATIII target enzyme from to and was with to the pseudo-first-order rate were for heparin or pentasaccharide from the of proteinase and heparin or pentasaccharide to the second-order rate constant for the inhibition by the or from S. Bjork I. Shore J. PubMed Scopus Google Scholar) for heparin binding at the ionic strength of the reaction were for the the by the of by the for the and equilibrium dissociation for pentasaccharide and heparin binding to ATIII were by the which the binding interaction S. Bjork I. Shore J. PubMed Scopus Google Scholar). were at with a an of and of for heparin polyethylene glycol to the ionic were for and the were to by with is the change at a heparin or pentasaccharide is the heparin or pentasaccharide is the is the ATIII and is the equilibrium dissociation The ATIII was by the ATIII for heparin binding antithrombin III was in the as previously described A. X. P. B. I. Olson S. Bock S. J. PubMed Scopus Google Scholar). The substitution of was produced on an to and heparin binding from the of two the wild-type is at residues Bock S. Biochemistry. PubMed Scopus Google Scholar). was from of cells by two of affinity on second heparin was necessary to ATIII which a of the antithrombin in and its on and were for and inhibition of thrombin and factor Xa were in the absence and presence of cofactor The for and inhibition of thrombin and factor Xa in the absence of heparin were in with and with heparin binding of by In the presence of E255A was for inhibition of thrombin and factor as was also observed for the molecule, and is for antithrombin S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of the mutant and that is and that the does not alter the mechanism or of target proteinase for and inhibition of thrombin and factor Xa in the absence and presence of of and factor Xa inhibition were at as described under Two to three were for and as for three and for two of in a and factor Xa inhibition were at as described under Two to three were for and as for three and for two from heparin at a higher ionic strength than its The of the increased E255A affinity for heparin was by of the dissociation equilibrium for binding to pentasaccharide and full-length high affinity heparin were conducted at an ionic strength of because the affinity of at physiological ionic strength was to The for heparin binding to the was with A. X. P. B. I. Olson S. Bock S. J. PubMed Scopus Google and B. I. Bock S. Olson S. Bjork I. Biochemistry. 1997; PubMed Scopus Google Scholar), that of was The for pentasaccharide binding to the was in with A. X. P. B. I. Olson S. Bock S. J. PubMed Scopus Google and B. I. Bock S. Olson S. Bjork I. Biochemistry. 1997; PubMed Scopus Google Scholar), that of was The similarly increased affinity for heparin and pentasaccharide binding that disruption of the interaction between the P1 arginine in the reactive loop and glutamic acid at the of ATIII leads to conformational in the pentasaccharide binding site than the of the that in a 5-fold increase in antithrombin affinity for heparin and equilibrium for pentasaccharide and full-length high affinity heparin binding to and at ionic strength A. X. P. B. I. Olson S. Bock S. J. PubMed Scopus Google B. I. Bock S. Olson S. Bjork I. Biochemistry. 1997; PubMed Scopus Google equilibrium were by at as described under for and for in of in a equilibrium were by at as described under for and for in of second-order rate for the inhibition of and its in the absence and presence of pentasaccharide and full-length high affinity heparin at and ionic strength second-order rate were from the of target proteinase activity and for the to the association In the absence of cofactor, the E255A substitution increased the second-order rate constant for inhibition of thrombin by a factor of compared with its parent, but there was no change in the rate of factor Xa inhibition. the native conformation of the E255A is for thrombin inhibition, but not factor Xa rate for and of and with thrombin and factor a Xa a rate for the inhibition of thrombin and factor Xa in the absence of cofactor and the presence of pentasaccharide and full-length high affinity heparin were at and as described under association rate a were for the by the apparent rate constant by in I. for three as Two in a rate for the inhibition of thrombin and factor Xa in the absence of cofactor and the presence of pentasaccharide and full-length high affinity heparin were at and as described under association rate a were for the by the apparent rate constant by in I. for three as of heparin cofactor activation for and inhibition of thrombin for the molecule and thrombin increased in the presence of as has been previously B. I. Bock S. Olson S. Bjork I. Biochemistry. 1997; PubMed Scopus Google Scholar). This is in to which was compared with the molecule, and exhibited no further activation upon addition of the pentasaccharide. However, in the presence of full-length high affinity the rates of thrombin inhibition by and increased by to The E255A not increase the rate of factor Xa inhibition. and its were to by pentasaccharide and full-length heparin for inhibition, the heparin-dependent of was than for the that the E255A substitution the rate of interaction with factor Xa. The P1 arginine and arginine in the reactive loop of ATIII with glutamic acid residue which and out from the body of the molecule The of this was to the role of the 393/399/255 reactive constraint on antithrombin heparin cofactor Because the P1 arginine and the reactive loop polypeptide to the interaction of ATIII with its target we to associated with the of reactive loop residues and to an E255A substitution mutant that is in the of the interaction on the serpin body side of the This to the of 393/399/255 reactive body in the of normal physiological thrombin and factor Xa. the of the P1 and P14 reactive loop constraints and pentasaccharide and full-length heparin binding on rates of ATIII thrombin and factor Xa inhibition. In this the rates of thrombin or factor Xa inhibition by ATIII variants with wild-type or E255A reactive site in the absence and presence of pentasaccharide or full-length high affinity heparin. The of the of the to the of the association rate for enzyme a and that removal of the 393/399/255 constraint the rate of association with thrombin 5-fold but has no on the rate of factor Xa inhibition. of and that upon release of the P14 constraint by binding of the rate of factor Xa inhibition there is no increase in the rate of reaction with thrombin was achieved by removal of the 393/399/255 these that thrombin with native ATIII In contrast, the (13Chuang Y. Swanson R. Raja S. Bock S. Olson S. J. Biol. Chem. 2001; 40: 6670-6679Google Scholar) or equilibrium between native ATIII is of no consequence with respect to factor Xa release of the P14 constraint by of the pentasaccharide binding site is to increase the rate of the factor Xa Therefore, on the basis of these we that under inhibition conditions in the absence of the key initial interactions of the antithrombin reactive loop with thrombin in the P1 the key initial interactions with factor Xa in a of the reactive loop that is by P14 as previously proposed by (13Chuang Y. Swanson R. Raja S. Bock S. Olson S. J. Biol. Chem. 2001; 40: 6670-6679Google Scholar, 14Futamara A. Beechem J. Gettins P. Biochemistry. 2001; 40: 6680-6687Crossref PubMed Scopus (20) Google Scholar), in an ATIII factor Xa binding that is by the conformational of and that of the pentasaccharide with residues the rate of thrombin inhibition by the E255A but has a on further accelerating the rate of factor Xa inhibition. The of the accelerating for full-length heparin pentasaccharide to observed for the molecule and antithrombin III and B. I. Bock S. Olson S. Bjork I. Biochemistry. 1997; PubMed Scopus Google Scholar). the in III and the contributions of the reactive loop constraint, the reactive loop P14 constraint, and to heparin activation of antithrombin III thrombin and factor Xa inhibition. respect to thrombin inhibition, release of the P1 constraint makes a contribution to accelerating the reaction with release of the P14 constraint plays no role at all. In the of it is approximation of the inhibitor and thrombin by full-length heparin that is for the activation of thrombin inhibition. In contrast, the P1 constraint plays no role and approximation plays a minor role, in heparin cofactor inhibition of factor Xa. it is the protein conformational change associated with of the pentasaccharide binding site that heparin activation of ATIII factor Xa inhibition. of full-length high affinity heparin and pentasaccharide binding to and in and protein conformational of the P1 The increased affinity of for pentasaccharide and full-length high affinity heparin that the 393/399/255 reactive loop constraint is for stabilizing a conformation of the inhibitor with affinity for heparin. In the further for between the reactive loop and the pentasaccharide binding site on D. of the 393/399/255 constraint in the mutant was associated with 5-fold in the for full-length heparin and pentasaccharide. in full-length heparin binding affinity have been observed for and ATIII P1 (13Chuang Y. Swanson R. Raja S. Bock S. Olson S. J. Biol. Chem. 2001; 40: 6670-6679Google Scholar, M. P. D. D. 1991; PubMed Scopus Google Scholar). the increased full-length heparin of the and P1 substitution show that release of the 393/399/255 constraint is associated with a protein conformational change the binding site for full-length heparin. However, the 5-fold increased pentasaccharide affinity for the to its conformational between the reactive loop and the pentasaccharide binding This that have the conformational from the of on of the pentasaccharide binding site the and of the reactive loop P. Fan B. B. I. Olson S. Biochemistry. PubMed Scopus Google Scholar). In on an antithrombin E255A mutant that the P1 reactive loop constraint that the for heparin cofactor of thrombin and factor Xa inhibition distinct and and that activation of this important anticoagulant protein involves than the increased of its P1 Bjork and Olson for and of and heparin and for the use of