Steven T. Olson

Mechanism of Heparin Activation of Antithrombin ROLE OF INDIVIDUAL RESIDUES OF THE PENTASACCHARIDE ACTIVATING SEQUENCE IN THE RECOGNITION OF NATIVE AND ACTIVATED STATES OF ANTITHROMBIN

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of ∼15–75% but did not affect the oligosaccharide’s ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I.

Exosite I of the blood clotting proteinase, thrombin, mediates interactions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins. Specific binding of a fluorescein-labeled derivative of the COOH-terminal dodecapeptide of hirudin ([5F] Hir54–65) to exosite I was used to probe changes in the function of the regulatory site accompanying inactivation of thrombin by its physiological serpin inhibitor, antithrombin. Fluorescence-monitored equilibrium binding studies showed that [5F]Hir54–65 and Hir54–65 bound to human α-thrombin with dissociation constants of 26 ± 2 nm and 38 ± 5 nm, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (≥200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54–65 occurred with the same time-course as the loss of thrombin catalytic activity. Binding of [5F] Hir54–65 and Hir54–65 to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7- and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir54–65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the rate-limiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.

Regulation of Thrombin by Antithrombin and Heparin Cofactor II

Thrombin plays a pivotal role in blood coagulation. It cleaves fibrinogen, a reaction that initiates the formation of the fibrin gel, which constitutes the framework of the blood clot. It also activates the cofactors factor V and factor VIII of the clotting system, thereby greatly accelerating the coagulation process. When bound to thrombomodulin on the endothelial cell surface, thrombin also activates protein C, which then inactivates the two cofactors and impedes blood clotting. In addition, thrombin activates factor XIII, leading to cross-linking of the fibrin gel. It is thus apparent that accurate regulation of thrombin activity is important in maintaining normal hemostasis.

Deconvolution of the Fluorescence Emission Spectrum of Human Antithrombin and Identification of the Tryptophan Residues That Are Responsive to Heparin Binding

Heparin causes an allosterically transmitted conformational change in the reactive center loop of antithrombin and a 40% enhancement of tryptophan fluorescence. We have expressed four human antithrombins containing single Trp → Phe mutations and determined that the fluorescence of antithrombin is a linear combination of the four tryptophans. The contributions to the spectrum of native antithrombin at 340 nm were 8% for Trp-49, 10% for Trp-189, 19% for Trp-225, and 63% for Trp-307. Trp-225 and Trp-307 accounted for the majority of the heparin-induced fluorescence enhancement, contributing 37 and 36%, respectively. Trp-49 and Trp-225 underwent spectral shifts of 15 nm to blue and 5 nm to red, respectively, in the antithrombin-heparin complex. The blue shift for Trp-49 is consistent with partial burial by contact with heparin, whereas the red shift for Trp-225 and large enhancement probably result from increased solvent access upon heparin-induced displacement of the contact residue Ser-380. The enhancement for Trp-307 may result from the heparin-induced movement of helix H seen in the crystal structure. The time-resolved fluorescence properties of individual tryptophans of wild-type antithrombin were also determined using the four variants and showed that Trp-225 and Trp-307 experienced the largest change in lifetime upon heparin binding, providing support for the steady-state fluorescence deconvolution.

Role of Regulatory Exosite I in Binding of Thrombin to Human Factor V, Factor Va, Factor Va Subunits, and Activation Fragments

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue byN α-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54–65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 ± 1.3 and 1.1 ± 0.4 μm and fluorescence enhancements of 182 ± 41 and 127 ± 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.

Quantitative evaluation of solution equilibrium binding interactions by affinity partitioning: application to specific and nonspecific protein-heparin interactions.

A variation of the quantitative affinity chromatography (QAC) method of Winzor, Chaiken, and co-workers for the analysis of protein-ligand interactions has been developed and used to characterize sequence-specific and nonspecific protein-heparin interactions relevant to blood coagulation. The method allows quantitation of the binding of two components, A and B, from the competitive effect of one component, B, on the partitioning of the other component, A, between an immobilized acceptor phase and solution phase at equilibrium. Under the conditions employed, the differences in total A concentrations yielding an equivalent degree of saturation of the immobilized acceptor in the absence and presence of B defines the concentration of A bound to B in solution, thereby enabling conventional Scatchard or nonlinear least-squares analysis of the A-B equilibrium interaction. Like the QAC method, quantitation of the competitor interaction does not depend on the nature of the affinity matrix interaction, which need only be described empirically. The additional advantage of the difference method is that only the total rather than the free competitor ligand concentration need be known. The method requires that the partitioning component A be univalent, but allows for multivalency in the competitor, B, and can in principle be used to study binding interactions involving nonidentical, interacting, or nonspecific overlapping sites. Both the binding constant and the stoichiometry for the specific antithrombin-heparin interaction as well as the apparent binding constant for the nonspecific thrombin-heparin interaction at low thrombin binding densities obtained using this technique were in excellent agreement with values determined using spectroscopic probes.

Role of Protein Conformational Changes, Surface Approximation and Protein Cofactors in Heparin-Accelerated Antithrombin-Proteinase Reactions

Antithrombin functions as the principal plasma protein inhibitor of most blood coagulation proteinases.1,2 The essential role of this inhibitor in regulating the activity of these proteinases in vivo is indicated from the well-established link between inherited or acquired deficiencies of antithrombin and the tendency to develop thrombotic disease. Antithrombin is a member of the serpin superfamily of protein proteinase inhibitors and its main target enzymes include the blood coagulation factors IXa, Xa and thrombin. This and other serpins are distinguished from other family members in that their reactions with target enzymes are greatly accelerated by the binding of heparin or heparan sulfate glycosaminoglycans. This property is chiefly responsible for the anticoagulant activity of heparin and has suggested a role for endogenous heparin and heparan sulfate in the regulation of blood coagulation proteinases by antithrombin. In this article, we will review our present understanding of the relationship between antithrombin structure and function based on currently available evidence. Our discussion will focus principally on two areas: 1) the mechanism by which antithrombin and other serpins inhibit their target proteinases; and 2) the molecular basis of heparin’s accelerating effect on antithrombin-proteinase reactions.

Apparent formation of sodium dodecyl sulfate-stable complexes between serpins and 3,4-dichloroisocoumarin-inactivated proteinases is due to regeneration of active proteinase from the inactivated enzyme.

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thøgersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859–14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and β-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, α1-proteinase inhibitor (α1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the α1PI reactive center P1-P1′ bond. Quantitation of kinetic constants for DCI and α1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by α1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.