Joseph D. Shore

Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin.

Publisher Summary This chapter discusses the kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Such approaches should be applicable also to the study of other serpin-proteinase reactions either in the absence or presence of similar glycosaminoglycan effectors. The chapter also discusses the purification and properties of antithrombin. Antithrombin is easily purified from plasma by a procedure based on affinity chromatography on matrix-linked heparin. Antithrombin inactivates a target proteinase by forming an equimolar, tight complex in which the active site of the enzyme is inaccessible to substrates. The purity of the antithrombin preparation should be assessed by the determination of the apparent stoichiometry of inhibition of thrombin. Both discontinuous and continuous assay methods can be used to measure the rates of inhibition of clotting proteinases by antithrombin. In the discontinuous method, the proteinase is incubated with antithrombin and samples of the reaction mixture are taken at various times for measurement of residual enzyme activity.

Serpin-Protease Complexes Are Trapped as Stable Acyl-Enzyme Intermediates

The serine protease inhibitors of the serpin family are an unusual group of proteins thought to have metastable native structures. Functionally, they are unique among polypeptide protease inhibitors, although their precise mechanism of action remains controversial. Conflicting results from previous studies have suggested that the stable serpin-protease complex is trapped in either a tight Michaelis-like structure, a tetrahedral intermediate, or an acyl-enzyme. In this report we show that, upon association with a target protease, the serpin reactive-center loop (RCL) is cleaved resulting in formation of an acyl-enzyme intermediate. This cleavage is coupled to rapid movement of the RCL into the body of the protein bringing the inhibitor closer to its lowest free energy state. From these data we suggest a model for serpin action in which the drive toward the lowest free energy state results in trapping of the protease-inhibitor complex as an acyl-enzyme intermediate.

Characterization of the Binding of Different Conformational Forms of Plasminogen Activator Inhibitor-1 to Vitronectin IMPLICATIONS FOR THE REGULATION OF PERICELLULAR PROTEOLYSIS

Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into β-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with β-anhydrotrypsin, which does not undergo rearrangement of β-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to β-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1·plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.

Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator, is an important regulator of the blood fibrinolytic system. Elevated plasma levels of PAI-1 are associated with thrombosis, and high levels of PAI-1 within platelet-rich clots contribute to their resistance to lysis by t-PA. Consequently, strategies aimed at inhibition of PAI-1 may prove clinically useful. This study was designed to test the hypothesis that a 14-amino acid peptide, corresponding to the PAI-1 reactive center loop (residues 333-346), can rapidly inhibit PAI-1 function. PAI-1 (0.7 microM) was incubated with peptide (55 microM) at 37 degrees C. At timed intervals, residual PAI-1 activity was determined by addition of reaction mixture samples to t-PA and chromogenic substrate. The T1/2 of PAI-1 activity in the presence of peptide was 4 +/- 3 min compared to a control T1/2 of 98 +/- 18 min. The peptide also inhibited complex formation between PAI-1 and t-PA as demonstrated by SDS-PAGE analysis. However, the capacity of the peptide to inhibit PAI-1 bound to vitronectin, a plasma protein that stabilizes PAI-1 activity, was markedly attenuated. Finally, the peptide significantly enhanced in vitro lysis of platelet-rich clots and platelet-poor clots containing recombinant PAI-1. These results indicate that a 14-amino acid peptide can rapidly inactivate PAI-1 and accelerate fibrinolysis in vitro. These studies also demonstrate that PAI-1 function can be directly attenuated in a physiologic setting and suggest a novel approach for augmenting fibrinolysis in vivo.

Accelerated Conversion of Human Plasminogen Activator Inhibitor-1 to Its Latent Form by Antibody Binding

The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its β-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA)·PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probeN,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9·NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3–1.8 × 104 m −1 s−1), resulting in a 4000-fold faster inactivation at 4.2 μm antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody.

The Use of Fluorescent Probes to Characterize Conformational Changes in the Interaction between Vitronectin and Plasminogen Activator Inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central β-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1′ or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydryl-reactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1′ bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.

Substrate inhibition effects in the liver alcohol dehydrogenase reaction

Abstract Kinetic experiments with LADH + NAD+ + high concentrations of alcohols confirm earlier results [Acta Chem. Scand.15: 1834 (1961)] showing that the partial inhibition of the reaction velocity depends on the formation of a ternary LADH-NADH-alcohol complex. This can dissociate to LADH-alcohol + NADH, but at a slower rate than LADH-NADH → LADH + NADH. Propanol, butanol, and pentanol give more and more increasing effects, which are attributed to the lipophilic binding site of the enzyme. The dissociation constants of these complexes are given.

The effects of adenine nucleotides on NADH binding to mitochondrial malate dehydrogenase

Abstract The effects of adenine nucleotides on initial velocity and NADH binding have been studied with the malate dehydrogenase reaction. ATP, ADP, and AMP were inhibitors competitive with NADH and uncompetitive with oxaloacetate but caused only 50–60% inhibition at saturating concentrations. Direct fluorescence titrations indicated that saturating concentrations of the adenine nucleotides displaced 50–60% of the bound NADH from enzyme-NADH complex. Adenine and adenosine had no inhibitory effect but ADP-ribose caused complete inhibition and NADH dissociation. The possible mechanistic basis for these results and their physiological implications are discussed.

Analogs of Human Plasminogen That Are Labeled with Fluorescence Probes at the Catalytic Site of the Zymogen PREPARATION, CHARACTERIZATION, AND INTERACTION WITH STREPTOKINASE

Fluorescent analogs of the proteinase zymogen, plasminogen (Pg), which are specifically inactivated and labeled at the catalytic site have been prepared and characterized as probes of the mechanisms of Pg activation. The active site induced non-proteolytically in Pg by streptokinase (SK) was inactivated stoichiometrically with the thioester peptide chloromethyl ketone, Nα-[(acetylthio)acetyl]-(D-Phe)-Phe-Arg-CH2Cl; the thiol group generated subsequently on the incorporated inhibitor with NH2OH was quantitatively labeled with the fluorescence probe, 2-((4′-iodoacetamido)anilino)naphthalene-6-sulfonic acid; and the labeled Pg was separated from SK. Cleavage of labeled [Glu]Pg1 by urokinase-type plasminogen activator (uPA) was accompanied by a fluorescence enhancement (ΔFmax/Fo) of 2.0, and formation of 1% plasmin (Pm) activity. Comparison of labeled and native [Glu]Pg1 as uPA substrates showed that activation of labeled [Glu]Pg1 generated [Glu]Pm1 as the major product, while native [Glu]Pg1 was activated at a faster rate and produced [Lys]Pm1 because of concurrent proteolysis by plasmin. When a mixture of labeled and native Pg was activated, to include plasmin-feedback reactions, the zymogens were activated at equivalent rates. The lack of potential proteolytic activity of the Pg derivatives allowed their interactions with SK to be studied under equilibrium binding conditions. SK bound to labeled [Glu]Pg1 and [Lys]Pg1 with dissociation constants of 590 ± 110 and 11 ± 7 nM, and fluorescence enhancements of 3.1 ± 0.1 and 1.6 ± 0.1, respectively. Characterization of the interaction of SK with native [Glu]Pg1 by the use of labeled [Glu]Pg1 as a probe indicated a ∼6-fold higher affinity of SK for the native Pg zymogen compared to the labeled Pg analog. Saturating levels of ϵ-aminocaproic acid reduced the affinity of SK for labeled [Glu]Pg1 by ∼2-fold and lowered the fluorescence enhancement to 1.8 ± 0.1, whereas the affinity of SK for labeled [Lys]Pg1 was reduced by ∼98-fold with little effect on the enhancement. These results demonstrate that occupation of lysine binding sites modulates the affinity of SK for Pg and the changes in the environment of the catalytic site associated with SK-induced conformational activation. Together, these studies show that the labeled Pg derivatives behave as analogs of native Pg which report functionally significant changes in the environment of the catalytic site of the zymogen.

A kinetic study of ternary complexes in the mechanism of action of liver alcohol dehydrogenase

Abstract It is shown that the ternary complex dissociation constants K ER , aldehyde and K EO , alcohol can be determined under certain assumptions from the maximum reaction velocity with and without added product. The results obtained with ethanol-acetaldehyde were in the same range of magnitude as those obtained by Theorell and Yonetani in 1963 by equilibrium measurements. The results with higher aliphatic alcohols clearly indicated the importance of a lipophilic binding site for the substrate.