PubMed Health⌕ Search

Biomedical subjects

S T Olson

Publications and source records attributed to S T Olson.

At least 19 recordsLinked to original sources
PubMed1996

Mechanism of heparin activation of antithrombin. Evidence for reactive center loop preinsertion with expulsion upon heparin binding.

A heparin-induced conformational change is required to convert antithrombin from a slow to a fast inhibitor of factor Xa. It has been proposed [van Boeckel et al. (1994) Nat. Struct. Biol. 1, 423-425] that the reactive center residue P14 is inserted into beta-sheet A in native antithrombin and is displaced from the beta-sheet by heparin binding, thereby altering the conformation of the reactive center and making it a better target for factor Xa binding. To test this hypothesis, we have characterized a P14 serine --> tryptophan antithrombin variant. From changes in tryptophan fluorescence upon heparin binding, increased affinity for heparin, and partial activation of the variant against factor Xa, we conclude that the proposed mechanism of heparin activation is correct with respect to loop expulsion and that it may consequently be possible to create more highly activated antithrombin variants through suitable hinge region substitutions.

Antithrombin III↗
PubMed1996

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. N alpha-[(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 (delta Fmax/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 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 approximately 6-fold higher affinity of SK for the native Pg zymogen compared to the labeled Pg analog. Saturating levels of epsilon-aminocaproic acid reduced the affinity of SK for labeled [Glu]Pg1 by approximately 2-fold and lowered the fluorescence enhancement to 1.8 +/- 0.1, whereas the affinity of SK for labeled [Lys]Pg1 was reduced by approximately 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.

Enzyme Precursors↗
PubMed1995

Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.

The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to alpha 1-proteinase inhibitor (alpha 1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser-->Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding energy to serpin-trypsin interactions and is essential for inducing the serpin conformational change involved in the trapping of enzyme in stable complexes.

Animals↗
PubMed1995

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Animals↗
PubMed1995

Kinetic characterization of the proteinase binding defect in a reactive site variant of the serpin, antithrombin. Role of the P1' residue in transition-state stabilization of antithrombin-proteinase complex formation.

To elucidate the role of the P1' residue of the serpin, antithrombin (AT), in proteinase inhibition, the source of the functional defect in a natural Ser-394-->Leu variant, AT-Denver, was investigated. AT-Denver inhibited thrombin, Factor IXa, plasmin, and Factor Xa with second order rate constants that were 430-, 120-, 40-, and 7-fold slower, respectively, than those of native AT, consistent with an altered specificity of the variant inhibitor for its target proteinases. AT-Denver inhibited thrombin and Factor Xa with nearly equimolar stoichiometries and formed SDS-stable complexes with these proteinases, indicating that the diminished inhibitor activity was not due to an enhanced turnover of the inhibitor as a substrate. Binding and kinetic studies showed that heparin binding to AT-Denver as well as heparin accelerations of AT-Denver-proteinase reactions were normal, consistent with the P1' mutation not affecting the heparin activation mechanism. Resolution of the two-step reaction of AT-Denver with thrombin revealed that the majority of the defective function was localized in the second reaction step and resulted from a 190-fold decreased rate constant for conversion of a noncovalent proteinase-inhibitor encounter complex to a stable, covalent complex. Little or no effects of the mutation on the binding constant for encounter complex formation or on the rate constant for stable complex dissociation were evident. These results support a role for the P1' residue of antithrombin in transition-state stabilization of a substrate-like attack of the proteinase on the inhibitor-reactive bond following the formation of a proteinase-inhibitor encounter complex but prior to the conformational change leading to the trapping of proteinase in a stable, covalent complex. Such a role indicates that the P1' residue does not contribute to thermodynamic stabilization of AT-proteinase complexes and instead favors a kinetic stabilization of these complexes by a suicide substrate reaction mechanism.

Antithrombins↗
PubMed1995

Mechanism of acceleration of antithrombin-proteinase reactions by low affinity heparin. Role of the antithrombin binding pentasaccharide in heparin rate enhancement.

The role of the sequence-specific pentasaccharide region of high affinity heparin (HAH) in heparin acceleration of antithrombin-proteinase reactions was elucidated by determining the accelerating mechanism of low affinity heparin (LAH) lacking this sequence. LAH was shown to be free of HAH (< 0.001%) from the lack of exchange of added fluorescein-labeled HAH into LAH after separating the polysaccharides by antithrombin-agarose chromatography. Fluorescence titrations showed that LAH bound to antithrombin with a 1000-fold weaker affinity (KD 19 +/- 6 microM) and 5-6-fold smaller fluorescence enhancement (8 +/- 3%) than HAH. LAH accelerated the antithrombin-thrombin reaction with a bell-shaped dependence on heparin concentration resembling that of HAH, but with the bell-shaped curve shifted to approximately 100-fold higher polysaccharide concentrations and with a approximately 100-fold reduced maximal accelerating effect. Rapid kinetic studies indicated these differences arose from a reverse order of assembly of an intermediate heparin-thrombin-antithrombin ternary complex and diminished ability of LAH to bridge antithrombin and thrombin in this complex, as compared to HAH. By contrast, LAH and HAH both accelerated the antithrombin-factor Xa reaction with a simple saturable dependence on heparin or inhibitor concentrations which paralleled the formation of an antithrombin-heparin binary complex. The maximal accelerations of the two heparins in this case correlated with the inhibitor fluorescence enhancements induced by the polysaccharides, consistent with the accelerations arising from conformational activation of antithrombin. 1H NMR difference spectroscopy of antithrombin complexes with LAH and HAH and competitive binding studies were consistent with LAH accelerating activity being mediated by binding to the same site on the inhibitor as HAH. These results demonstrate that LAH accelerates antithrombin-proteinase reactions by bridging and conformational activation mechanisms similar to those of HAH, with the reduced magnitude of LAH accelerations resulting both from a decreased antithrombin affinity and the inability to induce a full activating conformational change in the inhibitor.

Antithrombins↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition.

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.

Base Sequence↗
PubMed1994

Surface-induced alterations in the kinetic pathway for cleavage of human high molecular weight kininogen by plasma kallikrein.

We have studied the cleavage of human high molecular weight kininogen (HK) by plasma kallikrein in the absence and presence of the surfaces, dextran sulfate (DxSO4) and sulfatides. Using a combination of SDS-polyacrylamide gel electrophoresis, Western blotting with polyclonal antibodies that specifically recognize the COOH terminus of the bradykinin moiety, and high pressure liquid chromatography analyses of the cleavage reaction, we have identified two intermediates in the formation of bradykinin from intact kininogen and demonstrated that alternative cleavage pathways are followed in the absence and presence of surfaces. The COOH-terminal bradykinin cleavage occurred first both in the absence and presence of DxSO4, producing a 103-kDa HK intermediate consisting of disulfide-linked heavy and light chains that retained the kinin moiety. In the presence of DxSO4, this was followed exclusively by the NH2-terminal bradykinin cleavage and release of kinin with no apparent change in molecular mass. Subsequently, a slower cleavage of an 8-kDa peptide from the amino terminus of the HK light chain occurred to form a 95-kDa end product. In contrast to this sequential cleavage pattern, NH2-terminal bradykinin and light chain cleavages occurred randomly in the absence of DxSO4, resulting in the production of an additional 95-kDa intermediate that retained bradykinin but had lost the 8-kDa peptide from the HK light chain. Comparison of the relative rates of the three kallikrein cleavages in the absence and presence of DxSO4 indicated that the surface enhanced the rates of both bradykinin cleavages 2-4-fold, but inhibited the light chain cleavage rate approximately 10-fold, thereby accounting for the change from a partially random to a sequential cleavage pattern in the presence of the surface. Steady-state kinetic analysis revealed that DxSO4 enhanced the kcat/KM for bradykinin release by the rate-limiting NH2-terminal bradykinin cleavage by approximately 2-fold due exclusively to an increase in kcat. Sulfatides appeared to produce the same effects on the pattern of HK cleavages as DxSO4. Blocking of the nonactive site, i.e. exosite, interaction between kallikrein and HK with excess prekallikrein or a synthetic peptide containing the region of HK that interacts with the kallikrein exosite significantly reduced the rate of bradykinin release as well as HK cleavages detected by SDS-polyacrylamide gel electrophoresis either in the absence or presence of DxSO4, indicating that the exosite interaction facilitates bradykinin cleavage.

Blood Proteins↗
PubMed1994

Engineering plasminogen activator inhibitor 1 mutants with increased functional stability.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serine protease inhibitor (Serpin) superfamily, is the primary inhibitor of the plasminogen activators tPA and uPA. PAI-1 is produced in an active form but converts to an inactive or latent form with a half-life of approximately 1 h at pH 7.5, 37 degrees C. This study describes the construction, expression, and characterization of PAI-1 mutants with increased functional stability. Three mutations that disrupt an ion pair, present in latent PAI-1, between Arg-30 and Glu-350 (P4'), were introduced into recombinant PAI-1. All three mutant proteins maintained normal functional activity against both uPA and tPA. However, the half-life of each purified PAI-1 mutant was extended compared to the 1.1 h observed for wild-type PAI-1 (wtPAI-1) (1.2 h for Glu-350-->Arg, 2.0 h for Glu-350-->Pro, and 2.1 h for the Arg-30-->Glu mutation). An additional PAI-1 variant containing a second mutation designed to potentially reconstitute the ion pair (Arg-30-->Glu, Glu-350-->Arg) failed to restore the wild-type half-life. Circular dichroism spectra analysis indicated that the active and latent forms of wtPAI-1 and all four mutants contained similar secondary structural elements. Thermal stability determinations showed that latent wtPAI-1 was much more structurally stable than the active conformation. However, the latent form for all four mutants was significantly less stable than the corresponding wtPAI-1 conformer. This is the first report of PAI-1 mutants which have been specifically engineered to produce enhanced functional stability.

Base Sequence↗
PubMed1993

High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: role for antithrombin in the regulation of kallikrein.

The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with heparin on the regulation of kallikrein by the heparin-activated inhibitor, antithrombin, were investigated. H-kininogen, at levels sufficient to fully complex kallikrein, greatly potentiated the acceleration of antithrombin inhibition of kallikrein produced by heparin with high affinity for antithrombin. At I = 0.15, pH 7.4, 25 degrees C, kininogen thus maximally increased the heparin enhancement of the second-order rate constant for the antithrombin-kallikrein reaction from 13-fold (1.6 x 10(2) M-1 s-1 to 2.1 x 10(3) M-1 s-1) to 1200-fold (1.9 x 10(5) M-1 s-1). In contrast, H-kininogen had no effect on the antithrombin-kallikrein reaction in the absence of heparin, nor did the protein enhance the rate constants of 1.7 x 10(4) and 3.4 x 10(4) M-1 s-1 for kallikrein reactions with its primary plasma inhibitors C1-inhibitor and alpha 2-macroglobulin, respectively, in the absence or presence of heparin. Consistent with these results, SDS gel electrophoresis of the 125I-labeled kallikrein-inhibitor complexes formed in a mixture of these kallikrein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha 2-macroglobulin for kallikrein, accounting for 54% of the total kallikrein complexes, only when both heparin and H-kininogen were present. Similarly, the presence of therapeutic levels of heparin (approximately 1 unit/mL) in normal, factor XII-deficient, and prekallikrein-deficient plasmas enhanced the rate of inactivation of added kallikrein by 2.3-fold and significantly altered the partitioning of radiolabeled kallikrein from predominantly C1-inhibitor and alpha 2-macroglobulin complexes (86-92%) to mostly antithrombin complexes (50-53%). Experiments in antithrombin-deficient and H-kininogen-deficient plasmas confirmed that the enhanced kallikrein inactivation rate and predominant formation of antithrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kallikrein inhibition in plasma remained significant (approximately 40-70%) at optimal concentrations of unfractionated or size- and antithrombin affinity-fractionated heparin, in the presence of plasma levels of calcium and zinc ions, at 37 degrees C, and with minimal plasma dilution. These results suggest that antithrombin and H-kininogen may play important roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.

Antithrombins↗
PubMed1993

Parallel mechanisms of high molecular weight kininogen action as a cofactor in kallikrein inactivation and prekallikrein activation reactions.

The mechanism by which high molecular weight kininogen (H-kininogen) potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin [Olson, S. T., Sheffer, R., & Francis, A. M. (1993) Biochemistry (preceding paper in this issue)] was investigated at I = 0.15, pH 7.4, 25 degrees C. Single-chain, two-chain, and light-chain, but not heavy-chain, forms of H-kininogen were similarly effective in potentiating the heparin-accelerated antithrombin-kallikrein reaction, indicating that the light-chain region of the protein was responsible for promoting kallikrein inactivation and that cleavage of H-kininogen did not significantly affect this promoting activity. H-kininogen potentiation increased in a saturable manner with increasing kininogen concentration, reflecting a KD (23 +/- 8 nM) similar to that previously measured for H-kininogen binding to kallikrein by equilibrium methods. Both H-kininogen-stimulated and unstimulated heparin rate enhancements initially increased with increasing heparin concentration in a manner corresponding to the saturation of antithrombin with heparin (KD = 10-30 nM). However, at higher heparin concentrations, the stimulated but not the unstimulated heparin rate enhancement decreased in parallel with the saturation of a protein-heparin interaction with a KD (0.4 +/- 0.2 microM) comparable to that directly measured for the H-kininogen-heparin interaction (2.0 +/- 0.2 microM). These results implied that H-kininogen stimulation required the formation of a quaternary complex in which antithrombin and H-kininogen-kallikrein complex were bound to the same heparin chain. In keeping with this interpretation, a synthetic heparin pentasaccharide representing the antithrombin binding sequence accelerated the antithrombin-kallikrein reaction to an extent similar to that of full-length heparin chains containing this sequence, but the pentasaccharide acceleration was not stimulated by H-kininogen. The importance of H-kininogen-kallikrein complex binding to heparin for kininogen stimulation was further indicated from the marked salt dependence of the second-order rate constant for the association of H-kininogen-kallikrein complex but not free kallikrein with antithrombin-heparin complex, under conditions where saturation of the two binary complexes was maintained. Kinetic analyses of antithrombin-kallikrein reactions as a function of the inhibitor concentration indicated that the KD for an initial antithrombin-kallikrein encounter complex was decreased 20-fold by heparin binding to antithrombin and an additional 200-fold by H-kininogen also binding to kallikrein. By contrast, rate constants for the conversion of the encounter complex to a stable complex were comparable for all reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Heparin↗
PubMed1993

Transmission of conformational change from the heparin binding site to the reactive center of antithrombin.

Heparin greatly increases the rates at which antithrombin inhibits target proteinases. An important part of this rate acceleration is a heparin-induced conformational change in antithrombin. To answer the question of whether or not this change is transmitted to the reactive center, we have prepared a recombinant P1 mutant of antithrombin, R393C, labeled the cysteine with nitrobenzofuran (NBD) fluorophore, and examined the perturbation of NBD fluorescence intensity as a function of bound sulfated oligosaccharide. Two high-affinity heparins, low-affinity heparin, and dextran sulfate were used. We found (i) that binding to antithrombin of all these oligosaccharides resulted in transmission of conformational change to P1 in the reactive center, (ii) that these oligosaccharides all gave enhancements of the rate of inhibition of factor Xa beyond any contribution from surface approximation, and (iii) that the degree of perturbation of P1 correlated with the enhancement of the rate of factor Xa inhibition that was not due to surface approximation.

Antithrombins↗
PubMed1993

Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.

Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.

Amino Acid Sequence↗
PubMed1993

Surface-independent acceleration of factor XII activation by zinc ions. I. Kinetic characterization of the metal ion rate enhancement.

The effect of zinc ions (Zn(II)) on the activation of factor XII in the absence of a procoagulant surface was investigated by initial velocity kinetic studies at I = 0.15, pH 7.4, and 25 degrees C. Zinc ions at concentrations greater than 160 microM potentiated 99-fold the kcat/KM for the activation of factor XII by kallikrein and, at an optimum concentration of 110 microM, accelerated 140-fold the apparent kcat/KM for factor XII autoactivation. High molecular weight kininogen had no effect on either metal-potentiated reaction. Analysis of the factor XII concentration dependence of initial activation rates revealed that Zn(II), at levels that saturate the effect, accelerates kallikrein activation of factor XII by lowering KM (from 52 to 7.3 microM) and raising kcat (from 2.6 to 31 min-1). For the autocatalytic activation reaction of factor XII in the presence of optimal Zn(II), apparent KM and kcat values of 2.4 microM and 0.041 min-1, respectively, were determined, but these parameters were not resolvable in the absence of the metal ion. Zinc ions minimally affected kallikrein enzymatic activity and inhibited factor XIIa enzymatic activity with KI values of 20-40 microM, suggesting that the rate-enhancing effects of the metal ion are due to interactions with the substrate (factor XII) rather than with the enzyme. The Zn(II) inhibition of factor XIIa enzymatic activity accounted for a decreased Zn(II) enhancement of factor XII autoactivation at high metal ion concentrations (> 110 microM). The Zn(II) concentration dependence of the acceleration of factor XII activation reactions were sigmoid and characterized by Hill coefficients of 3.3-4.3, suggesting that cooperative binding of at least four zinc ions to factor XII was responsible for the Zn(II) potentiating effect. The Zn(II) enhancement of the rates of factor XII activation decreased both above and below pH 7.4 with midpoint pH values of 6.5-7.0 and 8.0, consistent with histidine and possibly water ligands mediating Zn(II) binding to the protein. Despite an apparent weaker binding of Zn(II) to factor XII at pH 6.5, indistinguishable maximum accelerating effects of the metal ion were observed at saturation at this pH, indicating that the increased positive charge of factor XII resulting from protonation at the lower pH did not mimic the effect of Zn(II) binding. These results imply that zinc ions induce a conformational change in factor XII that makes it a better substrate for its enzyme activators.

Factor XII↗
PubMed1993

Surface-independent acceleration of factor XII activation by zinc ions. II. Direct binding and fluorescence studies.

To determine the role of Zn(II)-factor XII interactions in the rate-enhancing effect of Zn(II) on factor XII activation demonstrated in the preceding paper, equilibrium binding of zinc ions to factor XII, and the spectroscopic changes accompanying this binding were investigated. Equilibrium dialysis provided direct evidence for the binding of Zn(II) to factor XII. The binding data were consistent with 7.8 +/- 0.3 zinc ions binding with an indistinguishable Kd of 91 +/- 6 microM. Binding of Zn(II) was accompanied by a 10% quenching of the intrinsic protein fluorescence and a 2-nm red shift of the wavelength of maximum emission. These spectroscopic changes were specific for factor XII and were not observed with factor XIIa. The Zn(II) concentration dependence of factor XII fluorescence quenching was sigmoid and paralleled the Zn(II)-accelerating effect of factor XII activation by kallikrein and factor XIIa, indicating that the spectral change was reporting Zn(II)-factor XII interactions responsible for the enhanced activation rate. The apparent cooperativity of Zn(II) effects on factor XII fluorescence quenching and activation kinetics, and the apparent noncooperativity in Zn(II) binding to factor XII measured by equilibrium dialysis could be explained by a two-state model in which Zn(II) binding is linked to a conformational change in the protein. The Zn(II)-induced quenching of factor XII fluorescence exhibited a pH dependence consistent with the involvement of histidine residues in the binding of Zn(II). Dynamic quenching of factor XII protein fluorescence by iodide or acrylamide, in the absence and presence of Zn(II), revealed heterogeneity in the environment of the 13 tryptophan residues of factor XII that was markedly reduced by metal ion binding. Together, these results indicate that cooperative interactions of Zn(II) with factor XII induce structural changes in the zymogen that facilitate its proteolytic cleavage and activation.

Acrylamide↗