Biol. Chem. Hoppe-Seyler Vol. 373, pp. 503-508, July 1992
Heparin Interferes with the Inhibition of Neutrophil Elastase by its Physiological Inhibitors Bernard FALLER, Klaus FROMMHERZ and Joseph G. BIETH Universite Louis Pasteur de Strasbourg, INSERM U 237, Laboratoire d'Enzymologie, Facult6 de Pharmacie, 74 route du Rhin, F-67400 Dlkirch, France
Summary Heparin depresses the second-order rate constant kass for the inhibition of neutrophil elastase by 04-proteinase inhibitor. For high and low molecular weight heparin the decrease in kass is 290-fold and 40-fold, respectively. This is due to a tight binding of the polymer to elastase : K^ = 3.3 nM or 89 nM for high or low molecular weight heparin respectively. In contrast heparin increases the rate of inhibition of elastase by mucus proteinase inhibitor. For low molecular weight heparin, there is a 27-fold increase in kass. This is due to a strong binding of the polymer to the inhibitor (K^ = 50 nM) which undergoes a conformational change. Introduction Neutrophil elastase (NE) cleaves a large number of proteins including elastin and coagulation factors [1]. Its excessive release by activated neutrophils may lead to lung emphysema or blood coagulation disorders. Plasma proteins are normally protected against NE-mediated proteolysis by αιΡΙ, a 53 kDa irreversible inhibitor; lung proteins are shielded from NE by oqPI (lower respiratory tract) and MPI (upper respiratory tract). The latter is an 11.7 kDa reversible elastase inhibitor. Heparin, a naturally occurring sulfated polysaccharide, is a mixture of polymers composed of disacharide units with three sulfated groups per unit. Some heparin chains also contain oversulfated pentasaccharide domains that react with antithrombin III with resultant acceleration of the rate of thrombin inhibition [2]. Heparin has been shown to bind and partially inhibit NE [3]. Heparin is given to patients undergoing hemodialysis and in the course of septic shock. In these two situations neutrophil activation with intravascular release of NE may occur. The question is then : does heparin interfere with the inhibition of NE by plasma Enzymes: Elastase (EC 3.4. 4.7), Trypsin (EC 3.4.4.4) Abbreviations: NE, neutrophil elastase ; αχΡΙ, arproteinase inhibitor ; MPI, mucus proteinase inhibitor also called secretory leucoprotease inhibitor, SLPI.
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504
B. Faller, K. Frommherz and J.G. Bieth
Vol. 373 (1992)
? On the other hand, the lung contains large amounts of glycosaminoglycans (the lung is a commercial source of heparin).This raises the question whether the rate constants for the inhibition of NE by α j PI and MPI which have been measured in vitro in the absence of heparin, are altered or not by heparin.
Results Effect of heparin on the NE + ajPI association We have measured k^, the association rate constant for the inhibition of elastase by ajPI [4] in the absence and presence of commercial high molecular weight heparin (Mr = 13.5-15 kDa) and low molecular weight heparin (Mr = 3.7 kDa). The rate constant decreased with the concentration heparin and then reached a plateau. The maximal decreases in kass are reported in table 1 together with the maximal effects produced by other glycosaminoglycans. Table 1 shows that high molecular weight heparin is seven-fold more efficient in depressing kass and acts at a much lower concentration than low molecular weight heparin. The other glycosaminoglycans are less effective. Table 1. Maximal effects of various glycosaminogycans on the rate constant kass for the inhibiton of NE by α ι PI Glycosaminoglycan Nature none H-heparina L-heparina dermatan sulfate^ chondroitrin-4-sulfateb chondroitrin-6-sulfateb hyaluronic acidc
kass without heparin
Concentration
_
0.21 μΜ 5.4 μΜ 0.2 mg / ml 0.2 mg / ml 0.4 mg / ml 1 mg / ml
kass with heparin
289 40 6.8 5.4 5.9 2.0
a
three sulfate groups per disaccharide unit, H and L stand for high and low molecular weight b one sulfate group per disaccharide unit, no molecular mass available c no sulfate, no molecular mass available
The heparin effect did not take place in the presence of l M NaCl or protamine sulfate. On the other hand, heparin did not depress kass for the reaction of αχΡΙ with porcine pancreatic trypsin or elastase but very strongly decreased the kass for the α^ΡΙ- cathepsin G binding (see table 2).
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Vol. 373 (1992)
Heparin Interferes with Inhibition of Neutrophil Elastase
505
Table 2. Effect of high molecular weight heparin on proteinase-inhibitor association rate constants kass. The molar ratio of heparin to proteinase was 23 except for the NE + MPI system where it was 8 Proteinase
Inhibitor
k^s without heparin kass with heparin
NE pancreatic elastase pancreatic trypsin cathepsine G NE NE
cqPI ttlPI oqPI ajPI eglinc MPI
290 1 1 3000 164 0.1
Affinity chromatography on Heparin-Sepharose showed that o^PI did not interact with heparin whereas both NE and cathepsin G were tightly bound to the affinity column. Use was made of the partial inhibition of NE activity by heparin to measure the stoichiometry and the dissociation constant of the heparin-NE complexes [5]. Table 3 shows that one mol of high molecular weight heparin binds 3 mol of NE with a Kd of 3.3 nM whereas the stoichiometry is 1 : 1 and the Kj is 89 nM for low molecular weight heparin. Table 3. Binding stoichiometries and equilibrium dissociation constants Kd of heparin-MPI complexes and heparin-NE complexes at pH 7.4 and 25° C. H and L stand for high and low molecular weight. H-heparin Stoichiometry a
Kd(nM)
4 3
6±2 3.3 ±1
MPI NE a
L-heparin Stoichiometry a 1 1
Kd(nM) 50±9 89 ±12
mol protein bound per mol heparin
Affinity chromatography on Heparin-Sepharose also showed that pancreatic trypsin and elastase formed loose complexes with heparin. Thus the kass for the cqPI-proteinase association is only reduced if the proteinase tightly binds heparin (H): fast NE + o^PI ^ NE-o^PI H:NE+
slow ttjPI
^ H : N E - c^PI
Several heparins used in clinical care have been tested for their ability to affect the elastase / cqPI binding. Standard heparin, a mixture of high molecular weight chains, Brought to you by | Purdue University Libraries Authenticated Download Date | 5/29/15 6:25 AM
506
B. Faller, K. Frommherz and J. G. Bieth
Vol. 373 (1992)
strongly impairs the inhibition of elastase by α^ΡΙ when tested as a dose used in clinical care. Low molecular weight heparin are less effective. The data described above have been published recently [6]. Unpublished results indicate that heparin promotes the formation of a high affinity reaction intermediate between NE and 04 PL This was demonstrated using the progress curve method [7] and a fast kinetic accessory. NE was added to mixtures of a^PI, heparin and substrate and the release of product was recorded with a stopped-flow apparatus. A large range of a^PI concentration was used. Up to 6 μΜ ctjPI no intermediate could be detected in the absence of heparin, i.e.: NE+ α Ρχ Ι
k ass -^
NE-oCPI
In contrast, with a saturating concentration of low molecular weight heparin, a reaction intermediate Η : NE - a^PI* could be detected : H r N E + o ^1P I
ki
k * 2 v -Χ Η : NF - la PI -^ Η : Ν1Ε - α η Ρ Ι
^ k~[ KJ* ( = k_i I kj) was found to be 3 χ ΙΟ'8 Μ and k2, 6 χ ΙΟ"3 s-1. Thus, heparin has a beneficial effect on the NE + 04 PI association in that it promotes the rapid formation of an intermediate with a fairly high affinity. It has however, a detrimental effect because the intermediate decays very slowly into the stable complex (tj/2 ~ 2 min). The overall effect (k2 / KJ* = 2 χ 105 M'1 s'1) is of course a slo wing-down of the rate of inhibition (compare k2 / KJ* = 2 χ 105 M"1 s'1 in the presence of heparin to kass = 8 χ ΙΟ6 Μ'1 s'1 in the absence of the polymer). Effect of heparin on the NE + MPI association When the heparin effect was assessed on two low molecular weight reversible NE inhibitors, divergent effects were observed (table 2). The kass for the NE + eglin c system was depressed whereas that for the NE + MPI association was enhanced. The former did not bind heparin; the latter did. Heparin strongly increased the intrinsic fluorescence of MPI. Use was made of this property to measure the stoichiometry and the K^ of the heparin MPI complexes. Table 3 shows that MPI tightly binds the two heparins. The polymer profoundly alters a number of spectroscopic properties of Trp30, the single Trp residue of MPI : the quantum yield and the fluorescence lifetime strongly increase and there is significant blue-shift of the maximum emission wavelength. These data strongly suggest that heparin alters the conformation of MPI. A non- saturating concentration of high molecular weight heparin increases the kass for the NE + MPI association by a factor of 10 (table 2). On the other hand, a low molecular weight heparin concentration able to saturate both NE and MPI increases both kass an by a factor of about 30 as shown in the following schemes where Η stands for heparin :
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Vol. 373 (1992)
Heparin Interferes with Inhibition of Neutrophil Elastase
507
6 ™-i -i 2.8 χ 10 M s NE + MPI _^ NE-MPI
-4 .1 1.2x10 s
7.7x10
M
" s"1
H : N E + H:MPI
^ H:NE-MPI:H 4 x l O ~ s" 1
Discussion Our data suggest that intravenous administration of standard heparin causes plasma ocjPI to behave like a slow-binding NE inhibitor whose kass (3.4 χ ΙΟ4 M"1 s"1) is close to that of oxidized ajPI [4] which is unable to prevent elastolysis in vivo [1]. NE-mediated intravascular proteolysis may therefore occur in patients with neutrophil activation (septicemia, hemodialysis) who are under heparin therapy. It is noteworthy that decreased activities of a number of coagulation factors have been observed in patients with severe septicemia [8, 9]. Since ocjPI and eglin c do not bind heparin whereas MPI does, it is likely that the increased rate of NE + MPI association is due to the ability of the polymer to bind the latter inhibitor. The effect of heparin on the NE / MPI system is reminiscent of that observed by others on the thrombin / antithrombin III pair : heparin changes the conformation of antithrombin [10] and accelerates the inhibition of thrombin by this inhibitor [11]. There are, however, quantitative differences in the effect of heparin on the two inhibitors : (i) the intrinsic fluorescence of antithrombin increases by 40 % only [10] (ii) the kass for the thrombin-antithrombin association is increased 2300-fold [11]. MPI and α ι PI are the physiologic NE inhibitors that protect the lung against the tissue-destructive function of accidentally liberated NE [1]. This protecting function may be expressed in a quantitative way through dt, the delay time of inhibition, i.e. the time required to almost fully inhibit NE in vivo : dt = 5 / kass [Io] where [Io] is the in vivo inhibitor concentration [12]. The lower dt, the less substrate hydrolysis during the inhibition process, hence, the more efficient the antielastase protection. MPI and o^PI have about equal kass values in the absence of heparin [6, 13]. On the other hand, the molar ratio of the former to the latter is about 0.1 in the lower respiratory tract [14]. Hence, dt(MPI) ~ 10 dt(ociPI) so that MPI appears to play a minor antielastase function in the lower respiratory tract as compared with ocjPI. Low molecular weight heparin increases kass(MPI) by a factor of 27 and decreases kass(ctiPI) by a factor of 40. Thus, if heparin were present in the lung at a concentration sufficient to saturate both NE and MPI, one would have dt(MPI) «0.1 ), i.e. MPI would very favorably compete with a^PI for the binding of NE, and Brought to you by | Purdue University Libraries Authenticated Download Date | 5/29/15 6:25 AM
508
B. Faller, K. Frommherz and J.G. Bieth
Vol. 373 (1992)
would therefore be the most efficient NE inhibitor of the lower respiratory tract despite its low concentration. Heparin is abundant in the lung since this organ is a commercial source of the polymer [15]. Lung mast cells which secrete heparin [15], are found in large quantities in the alveolar walls, the sites where NE attacks lung matrix proteins to induce emphysematous lesions [16]. MPI, which is commonly thought to play an antielastase function in the upper respiratory tract only [16], might therefore be an important antielastase of the lower respiratory tract too. Acknowledgment We thank Synergen , Boulder Co for the gift of recombinant MPI. References 1. J. G. Bieth. (1986) Elastase: catalytic and biological properties, in: Regulation of Matrix accumulation (Mecham, R.P. ed.) pp 217-320, Academic Press New York . 2. Casu, B., Oreste P., Torri, G., Zoppetti, G., Choay, J.JLormeau, J.C., Petitou, M. & Sinoy, P. (1981) Biochem. J. 197, 599-605. 3. Redini, F., Tixier, J.M., Petitou, M., Choay, J., Robert, L. & Hornebeck, W. (1988) Biochem. J. 252. 515-519. 4. Beatty, K., Bieth, J.G. & Travis, J.(1980) J. Biol. Chem. 255, 3931-3934. 5. Baici, A., Salgam, P., Fehr, K. & B ni, Α. α980) Biochem. Pharmacol. 29. 17231727. 6. Frommherz, K.J., Faller, B. & Bieth, J.G. Π99Ρ J. Biol. Chem. 266.15356-15362. 7. Morrison, J.F. & Walsh, C.T. (1988) Adv. Enzvmol. Related. Areas Mol. Biol 61. 201-301. 8. Egbring, R., Schmidt, W., Fuchs, G. & Havemann, K. (1977) Blood 49, 219-231. 9. Duswald, K..H., Jochum, M., Schramm, W. & Fritz, H. (1985) Surgery 98, 892 899. 10. Olson, S.T.& Shore, J.D. Π98Ρ J. Biol. Chem. 255. 11065-11072. 11. Jordan, R.E., Beeler, D.& Rosenberg, R. (1979) J. Biol. Chem. 254. 2902-2913. 12. Bieth, J.G. (1980) Bull. Europ. Phvsiopath. Respir. 16 (suppl.) 183-195. 13. Boudier, C.& Bieth, J.G. (1989^ Biochim. Biophvs. Acta. 995. 36-41.14.15.16. 14. Gast, Α., Dieteman-Molard, Α., Pelletier, Α., Pauli, G.& Bieth, J.G. (1990) Am. Rev. Respir. Dis. 141. 880-883. 15. Clark, J.G., K hn, C, McDonald, J.A.& Mecham, R.P. (1983) in: International review of connective tissue research (Hall, D.A., and Jackson, D.S. eds) vol. 10, pp. 249-331, Academic Press, New York. 16. Janoff, A. (1988) in:Inflammation: Basic Principles and Clinical Correlates (Gallin, J.I., Goldstein, I.M., and Snyderman, R. eds.) pp 803-814, Raven Press, New York.
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Heparin Interferes with the Inhibition of Neutrophil Elastase by its Physiological Inhibitors Bernard FALLER, Klaus FROMMHERZ and Joseph G. BIETH Universite Louis Pasteur de Strasbourg, INSERM U 237, Laboratoire d'Enzymologie, Facult6 de Pharmacie, 74 route du Rhin, F-67400 Dlkirch, France
Summary Heparin depresses the second-order rate constant kass for the inhibition of neutrophil elastase by 04-proteinase inhibitor. For high and low molecular weight heparin the decrease in kass is 290-fold and 40-fold, respectively. This is due to a tight binding of the polymer to elastase : K^ = 3.3 nM or 89 nM for high or low molecular weight heparin respectively. In contrast heparin increases the rate of inhibition of elastase by mucus proteinase inhibitor. For low molecular weight heparin, there is a 27-fold increase in kass. This is due to a strong binding of the polymer to the inhibitor (K^ = 50 nM) which undergoes a conformational change. Introduction Neutrophil elastase (NE) cleaves a large number of proteins including elastin and coagulation factors [1]. Its excessive release by activated neutrophils may lead to lung emphysema or blood coagulation disorders. Plasma proteins are normally protected against NE-mediated proteolysis by αιΡΙ, a 53 kDa irreversible inhibitor; lung proteins are shielded from NE by oqPI (lower respiratory tract) and MPI (upper respiratory tract). The latter is an 11.7 kDa reversible elastase inhibitor. Heparin, a naturally occurring sulfated polysaccharide, is a mixture of polymers composed of disacharide units with three sulfated groups per unit. Some heparin chains also contain oversulfated pentasaccharide domains that react with antithrombin III with resultant acceleration of the rate of thrombin inhibition [2]. Heparin has been shown to bind and partially inhibit NE [3]. Heparin is given to patients undergoing hemodialysis and in the course of septic shock. In these two situations neutrophil activation with intravascular release of NE may occur. The question is then : does heparin interfere with the inhibition of NE by plasma Enzymes: Elastase (EC 3.4. 4.7), Trypsin (EC 3.4.4.4) Abbreviations: NE, neutrophil elastase ; αχΡΙ, arproteinase inhibitor ; MPI, mucus proteinase inhibitor also called secretory leucoprotease inhibitor, SLPI.
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504
B. Faller, K. Frommherz and J.G. Bieth
Vol. 373 (1992)
? On the other hand, the lung contains large amounts of glycosaminoglycans (the lung is a commercial source of heparin).This raises the question whether the rate constants for the inhibition of NE by α j PI and MPI which have been measured in vitro in the absence of heparin, are altered or not by heparin.
Results Effect of heparin on the NE + ajPI association We have measured k^, the association rate constant for the inhibition of elastase by ajPI [4] in the absence and presence of commercial high molecular weight heparin (Mr = 13.5-15 kDa) and low molecular weight heparin (Mr = 3.7 kDa). The rate constant decreased with the concentration heparin and then reached a plateau. The maximal decreases in kass are reported in table 1 together with the maximal effects produced by other glycosaminoglycans. Table 1 shows that high molecular weight heparin is seven-fold more efficient in depressing kass and acts at a much lower concentration than low molecular weight heparin. The other glycosaminoglycans are less effective. Table 1. Maximal effects of various glycosaminogycans on the rate constant kass for the inhibiton of NE by α ι PI Glycosaminoglycan Nature none H-heparina L-heparina dermatan sulfate^ chondroitrin-4-sulfateb chondroitrin-6-sulfateb hyaluronic acidc
kass without heparin
Concentration
_
0.21 μΜ 5.4 μΜ 0.2 mg / ml 0.2 mg / ml 0.4 mg / ml 1 mg / ml
kass with heparin
289 40 6.8 5.4 5.9 2.0
a
three sulfate groups per disaccharide unit, H and L stand for high and low molecular weight b one sulfate group per disaccharide unit, no molecular mass available c no sulfate, no molecular mass available
The heparin effect did not take place in the presence of l M NaCl or protamine sulfate. On the other hand, heparin did not depress kass for the reaction of αχΡΙ with porcine pancreatic trypsin or elastase but very strongly decreased the kass for the α^ΡΙ- cathepsin G binding (see table 2).
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Vol. 373 (1992)
Heparin Interferes with Inhibition of Neutrophil Elastase
505
Table 2. Effect of high molecular weight heparin on proteinase-inhibitor association rate constants kass. The molar ratio of heparin to proteinase was 23 except for the NE + MPI system where it was 8 Proteinase
Inhibitor
k^s without heparin kass with heparin
NE pancreatic elastase pancreatic trypsin cathepsine G NE NE
cqPI ttlPI oqPI ajPI eglinc MPI
290 1 1 3000 164 0.1
Affinity chromatography on Heparin-Sepharose showed that o^PI did not interact with heparin whereas both NE and cathepsin G were tightly bound to the affinity column. Use was made of the partial inhibition of NE activity by heparin to measure the stoichiometry and the dissociation constant of the heparin-NE complexes [5]. Table 3 shows that one mol of high molecular weight heparin binds 3 mol of NE with a Kd of 3.3 nM whereas the stoichiometry is 1 : 1 and the Kj is 89 nM for low molecular weight heparin. Table 3. Binding stoichiometries and equilibrium dissociation constants Kd of heparin-MPI complexes and heparin-NE complexes at pH 7.4 and 25° C. H and L stand for high and low molecular weight. H-heparin Stoichiometry a
Kd(nM)
4 3
6±2 3.3 ±1
MPI NE a
L-heparin Stoichiometry a 1 1
Kd(nM) 50±9 89 ±12
mol protein bound per mol heparin
Affinity chromatography on Heparin-Sepharose also showed that pancreatic trypsin and elastase formed loose complexes with heparin. Thus the kass for the cqPI-proteinase association is only reduced if the proteinase tightly binds heparin (H): fast NE + o^PI ^ NE-o^PI H:NE+
slow ttjPI
^ H : N E - c^PI
Several heparins used in clinical care have been tested for their ability to affect the elastase / cqPI binding. Standard heparin, a mixture of high molecular weight chains, Brought to you by | Purdue University Libraries Authenticated Download Date | 5/29/15 6:25 AM
506
B. Faller, K. Frommherz and J. G. Bieth
Vol. 373 (1992)
strongly impairs the inhibition of elastase by α^ΡΙ when tested as a dose used in clinical care. Low molecular weight heparin are less effective. The data described above have been published recently [6]. Unpublished results indicate that heparin promotes the formation of a high affinity reaction intermediate between NE and 04 PL This was demonstrated using the progress curve method [7] and a fast kinetic accessory. NE was added to mixtures of a^PI, heparin and substrate and the release of product was recorded with a stopped-flow apparatus. A large range of a^PI concentration was used. Up to 6 μΜ ctjPI no intermediate could be detected in the absence of heparin, i.e.: NE+ α Ρχ Ι
k ass -^
NE-oCPI
In contrast, with a saturating concentration of low molecular weight heparin, a reaction intermediate Η : NE - a^PI* could be detected : H r N E + o ^1P I
ki
k * 2 v -Χ Η : NF - la PI -^ Η : Ν1Ε - α η Ρ Ι
^ k~[ KJ* ( = k_i I kj) was found to be 3 χ ΙΟ'8 Μ and k2, 6 χ ΙΟ"3 s-1. Thus, heparin has a beneficial effect on the NE + 04 PI association in that it promotes the rapid formation of an intermediate with a fairly high affinity. It has however, a detrimental effect because the intermediate decays very slowly into the stable complex (tj/2 ~ 2 min). The overall effect (k2 / KJ* = 2 χ 105 M'1 s'1) is of course a slo wing-down of the rate of inhibition (compare k2 / KJ* = 2 χ 105 M"1 s'1 in the presence of heparin to kass = 8 χ ΙΟ6 Μ'1 s'1 in the absence of the polymer). Effect of heparin on the NE + MPI association When the heparin effect was assessed on two low molecular weight reversible NE inhibitors, divergent effects were observed (table 2). The kass for the NE + eglin c system was depressed whereas that for the NE + MPI association was enhanced. The former did not bind heparin; the latter did. Heparin strongly increased the intrinsic fluorescence of MPI. Use was made of this property to measure the stoichiometry and the K^ of the heparin MPI complexes. Table 3 shows that MPI tightly binds the two heparins. The polymer profoundly alters a number of spectroscopic properties of Trp30, the single Trp residue of MPI : the quantum yield and the fluorescence lifetime strongly increase and there is significant blue-shift of the maximum emission wavelength. These data strongly suggest that heparin alters the conformation of MPI. A non- saturating concentration of high molecular weight heparin increases the kass for the NE + MPI association by a factor of 10 (table 2). On the other hand, a low molecular weight heparin concentration able to saturate both NE and MPI increases both kass an by a factor of about 30 as shown in the following schemes where Η stands for heparin :
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Vol. 373 (1992)
Heparin Interferes with Inhibition of Neutrophil Elastase
507
6 ™-i -i 2.8 χ 10 M s NE + MPI _^ NE-MPI
-4 .1 1.2x10 s
7.7x10
M
" s"1
H : N E + H:MPI
^ H:NE-MPI:H 4 x l O ~ s" 1
Discussion Our data suggest that intravenous administration of standard heparin causes plasma ocjPI to behave like a slow-binding NE inhibitor whose kass (3.4 χ ΙΟ4 M"1 s"1) is close to that of oxidized ajPI [4] which is unable to prevent elastolysis in vivo [1]. NE-mediated intravascular proteolysis may therefore occur in patients with neutrophil activation (septicemia, hemodialysis) who are under heparin therapy. It is noteworthy that decreased activities of a number of coagulation factors have been observed in patients with severe septicemia [8, 9]. Since ocjPI and eglin c do not bind heparin whereas MPI does, it is likely that the increased rate of NE + MPI association is due to the ability of the polymer to bind the latter inhibitor. The effect of heparin on the NE / MPI system is reminiscent of that observed by others on the thrombin / antithrombin III pair : heparin changes the conformation of antithrombin [10] and accelerates the inhibition of thrombin by this inhibitor [11]. There are, however, quantitative differences in the effect of heparin on the two inhibitors : (i) the intrinsic fluorescence of antithrombin increases by 40 % only [10] (ii) the kass for the thrombin-antithrombin association is increased 2300-fold [11]. MPI and α ι PI are the physiologic NE inhibitors that protect the lung against the tissue-destructive function of accidentally liberated NE [1]. This protecting function may be expressed in a quantitative way through dt, the delay time of inhibition, i.e. the time required to almost fully inhibit NE in vivo : dt = 5 / kass [Io] where [Io] is the in vivo inhibitor concentration [12]. The lower dt, the less substrate hydrolysis during the inhibition process, hence, the more efficient the antielastase protection. MPI and o^PI have about equal kass values in the absence of heparin [6, 13]. On the other hand, the molar ratio of the former to the latter is about 0.1 in the lower respiratory tract [14]. Hence, dt(MPI) ~ 10 dt(ociPI) so that MPI appears to play a minor antielastase function in the lower respiratory tract as compared with ocjPI. Low molecular weight heparin increases kass(MPI) by a factor of 27 and decreases kass(ctiPI) by a factor of 40. Thus, if heparin were present in the lung at a concentration sufficient to saturate both NE and MPI, one would have dt(MPI) «0.1 ), i.e. MPI would very favorably compete with a^PI for the binding of NE, and Brought to you by | Purdue University Libraries Authenticated Download Date | 5/29/15 6:25 AM
508
B. Faller, K. Frommherz and J.G. Bieth
Vol. 373 (1992)
would therefore be the most efficient NE inhibitor of the lower respiratory tract despite its low concentration. Heparin is abundant in the lung since this organ is a commercial source of the polymer [15]. Lung mast cells which secrete heparin [15], are found in large quantities in the alveolar walls, the sites where NE attacks lung matrix proteins to induce emphysematous lesions [16]. MPI, which is commonly thought to play an antielastase function in the upper respiratory tract only [16], might therefore be an important antielastase of the lower respiratory tract too. Acknowledgment We thank Synergen , Boulder Co for the gift of recombinant MPI. References 1. J. G. Bieth. (1986) Elastase: catalytic and biological properties, in: Regulation of Matrix accumulation (Mecham, R.P. ed.) pp 217-320, Academic Press New York . 2. Casu, B., Oreste P., Torri, G., Zoppetti, G., Choay, J.JLormeau, J.C., Petitou, M. & Sinoy, P. (1981) Biochem. J. 197, 599-605. 3. Redini, F., Tixier, J.M., Petitou, M., Choay, J., Robert, L. & Hornebeck, W. (1988) Biochem. J. 252. 515-519. 4. Beatty, K., Bieth, J.G. & Travis, J.(1980) J. Biol. Chem. 255, 3931-3934. 5. Baici, A., Salgam, P., Fehr, K. & B ni, Α. α980) Biochem. Pharmacol. 29. 17231727. 6. Frommherz, K.J., Faller, B. & Bieth, J.G. Π99Ρ J. Biol. Chem. 266.15356-15362. 7. Morrison, J.F. & Walsh, C.T. (1988) Adv. Enzvmol. Related. Areas Mol. Biol 61. 201-301. 8. Egbring, R., Schmidt, W., Fuchs, G. & Havemann, K. (1977) Blood 49, 219-231. 9. Duswald, K..H., Jochum, M., Schramm, W. & Fritz, H. (1985) Surgery 98, 892 899. 10. Olson, S.T.& Shore, J.D. Π98Ρ J. Biol. Chem. 255. 11065-11072. 11. Jordan, R.E., Beeler, D.& Rosenberg, R. (1979) J. Biol. Chem. 254. 2902-2913. 12. Bieth, J.G. (1980) Bull. Europ. Phvsiopath. Respir. 16 (suppl.) 183-195. 13. Boudier, C.& Bieth, J.G. (1989^ Biochim. Biophvs. Acta. 995. 36-41.14.15.16. 14. Gast, Α., Dieteman-Molard, Α., Pelletier, Α., Pauli, G.& Bieth, J.G. (1990) Am. Rev. Respir. Dis. 141. 880-883. 15. Clark, J.G., K hn, C, McDonald, J.A.& Mecham, R.P. (1983) in: International review of connective tissue research (Hall, D.A., and Jackson, D.S. eds) vol. 10, pp. 249-331, Academic Press, New York. 16. Janoff, A. (1988) in:Inflammation: Basic Principles and Clinical Correlates (Gallin, J.I., Goldstein, I.M., and Snyderman, R. eds.) pp 803-814, Raven Press, New York.
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