Biochimica et Biophysica Acta, 1038 (1990) 231-239
231
Elsevier BBAPRO 33617
Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro Tetsuro Y a m a m o t o 1, Yoko Shibuya 2, Norikazu Nishino 3, Hiroaki Okabe 2 and Takeshi K a m b a r a i t Department of Allergy, Institute for Medical Immunology and 2 Department of Laboratory Medicine, Kumamoto University Medical School, Kumamoto, and ~ Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu (Japan)
(Received 19 September1989)
Key words: Hagemanfactor; Kallikrein-kininsystem; Bacterialproteinase; Pseudomonalinfection; Pseudomonalelastase; ( P. aeruginosa )
Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 3 7 ° C ) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONI-ICOCH(CH2C6Hs)CO-Ala-Gly-NH 2, at a concentration as low as 10 pM. In this activation Hageman factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation w e r e kca t w. 5 . 8 " 1 0 - 3 s - m , K m = 4.3 • 1 0 - 7 M and k ~ t / K m = 1.4 • 1 0 4 M - t . s - i . This K m value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 p g / m l ) , the activation rate by the elastase increased several fold, with the kinetic parameters of k , t = 1 3 . 9 . 1 0 -a s - t , K m = 1.6" 10 -7 M and kcat//Km---8.5 " 104 M - ! . s - I . These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.
Introduction Hageman factor (blood coagulation factor XII) has been shown to be an initiation factor of the intrinsic and extrinsic blood coagulation pathway, kallikreinkinin pathway, and fibrinolytic pathway, as well as the complement pathway (Refs. 1 and 2; only the newest reviews are cited as reference). However, considerable research progress made during the past 20 years has given rise to major doubts about the contribution of the Hageman factor-dependent system to thrombosis and hemostasis [1]. Most evidence suggest important roles of
Abbreviations: MCA, 7-amino-4-methylcoumarylamide; AMC, aminomethyl coumarin; SDS, sodium dodecyl sulfate; Abz, 2aminobenzoyl; Nba, 4-nitrobenzylamide; HFa, activated Hageman factor; HF, Hageman factor. Correspondence: T. Yamamoto, Department of Allergy, Institute for Medical Immunology, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto 860, Japan.
the pathways, especially the Hageman factor/kallikrein-kinin system, in the pathogenesis of inflammatory response [1,2]. Indeed, it has been demonstrated in guinea pigs that the Hageman factor/kallikrein-kinin system is one of the strongest mediator systems of vascular permeability enhancement, as it is seen in inflammatory response [3,4]. On the clinical side, participation of the system has been speculated in infectious diseases, especially with G r a m -negative bacteremia, since significant consumption of components of the system was occasionally observed in such bacteremia [5,6]. Hageman factor is a serine proteinase zymogen, therefore, conversion to an active-form proteinase is required when the system is initiated. Most of the research on the Hageman factor activation has been focussed on that by endogenous proteinases in plasma, such as plasma kallikrein [7-11], coagulation factor XIa [8], plasmin [8,12] and activated Hageman factor itself [9,13-16]. It was revealed in the research that: sufficient Hageman factor activation by the plasma proteinases
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232 occurred only in the presence of negatively charged substance [8-16], and high molecular mass kininogen was also required under some conditions [11,13]; the activation occurred due to a limited proteolysis of Hageman factor molecule; and the strongest Hageman factor activator in plasma was kallikrein. Hageman factor activation by the Gram-negative bacterial lipopolysaccharide, endotoxin [17], was assumed due to its function as the negatively charged substance. However, natural occurrence of the Hageman factor activation by the endogenous proteinases on negatively charged substances is still in a controversy. For instance, the rate of activation by kallikrein is very much influenced by the concentration of the negatively charged substance [11] and the ionic strength of the buffer used [10,18]; the rate is considerably slow or at physiological ionic strength (I = 0.14-0.15) [18]. In addition to the requirement of limited proteolysis for the Hageman factor activation, when the contribution of the system in the Gram-negative bacterial infections is considered, it must be reasonable to speculate an in situ occurrence of the Hageman factor activation with enzymes secreted by the pathogenic microorganisms. Indeed, we have recently found that the elastase and alkaline proteinase or the 56 kDa proteinase secreted by Pseudomonas aeruginosa or Serratia marcescens, respectively, activated guinea pig Hageman factor in vitro [19,20], as well as in vivo; in the guinea pig skin, this activation was succeeded by the kallikrein-kinin generation followed by the vascular permeability enhancement reaction [20,21]. In the present project, we selected P. aeruginosa elastase and alkaline proteinase as representatives of Gram-negative bacterial proteinases, since both enzymes activate guinea pig Hageman factor despite being zinc-dependent metal enzymes [22], and since the pseudomonal proteinases were recently considered to be important enzymes in the development of chronic lung disease in cystic fibrosis patients [23-25]. We investigated whether human Hageman factor was activated by the pseudomonal proteinases in vitro at physiological ionic strength ( I = 0.14), and whether the activation rate with the elastase was enhanced in the presence of a negatively charged substance, such as dextran sulfate. We also evaluated the elastase in regard to the kinetic parameters of the enzymatic activation in the presence and absence of the negatively charged substance. A part of this work was presented in XIIth Congress of the International Society on Thrombosis and Haemostasis [26].
(Fukuchiyama, Japan). Pro-Phe-Arg-methylcoumarylamide (MCA), Boc-GIn-GIy-Arg-MCA, z-Phe-ArgMCA and aminomethyl coumarin (AMC) were purchased from Peptide Institute (Minoh, Japan). Crystallized and lyophilized bovine serum albumin was purchased from Sigma Chemical Co. (St. Louis, MO). Dextran sulfate sodium salt (molecular mass about 500 kDa) was obtained from Nakarai Chemicals (Kyoto, Japan). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) and Nakarai Chemicals. Gradient polyacrylamide slab gel (4-20% acrylamide) was a product of Tefco Co. (Tokyo, Japan). Disposable polystyrene plastic cuvettes for fluorescence spectrophotometer were products of Elkay Products (Shrewsbury, MA). HONHCOCH(CHzC6Hs)CO-AIa-Gly-NH 2 and Abz-Ala-Gly-Leu-Ala-Nba were synthesized as reported previously [27].
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) Slab SDS-polyacrylamide gel electrophoresis was performed with gradient acrylamide (4-20%) gels according to the method of Laemmli [28]. The gels were stained with 0.8% Coomassie brilliant blue R-250. To make the molecular mass standard curve, a commercial kit (Electrophoresis calibration kit, Pharmacia) was used.
Preparation of plasma proteins The isolation of Hageman factor from human plasma was carried out essentially as described by Fujikawa and Davie [29], with a substitution of the chromatography with CM-cellulose by a high-performance liquid chromatography (HPLC, LKB system) with a CMToyopearl column (Tosoh). The final material had the specific activity with 51.4 clotting units per mg protein which was comparable to the previous report [29]. Hageman factor thus prepared was stored at - 80 ° C in 10 mM acetate buffer containing 300 mM NaCI and 0.02% NaN 3 (pH 5.5). Plasma kallikrein was prepared from fresh frozen human plasma using a soybean trypsin inhibitor affinity column, after activation by an acetone treatment, according to the method of Nagase and Barrett [30]. B-Form activated Hageman factor was prepared from the same material as used for the kallikrein preparation by the method of Hojima et al. [31], with a substitution of the isoelectric focusing by a HPLC step using a DEAE-5PW column (Tosoh). The final products were apparently pure, as judged by slab SDS-polyacrylamide gel electrophoresis with Coomassie blue staining.
Materials and Methods
Assay of Hageman factor activation Substances Crystalline elastase and alkaline proteinase from P. aeruginosa were obtained from Nagase Biochemicals
The assay was performed in 50 mM Tris-HC1 buffer (pH 8.0 at 25°C and pH 7.8 at 37°C) containing 130 mM NaC1, 0.1 mg/ml bovine serum albumin and 0.02%
233 N a N 3 in the presence of 200 # M of the substrate of activated Hageman factor in the plastic cuvette at 37 o C. The substrate Pro-Phe-Arg-MCA or Boc-Gln-Gly-ArgM C A was used with the pseudomonal enzymes or with plasma kallikrein, respectively. In the usual assay, aliquots of the stock solutions of Hageman factor and one of the enzymes were added, respectively, to the mixture of the substrate and buffer in the cuvette. Liberation of A M C from the peptide-MCA substrate was continuously recorded on the chart recorder, using the fluorescence spectrophotometer, with the wavelengths of excitation at 380 nm and emission at 440 nm. The amount of A M C liberated increased in a parabolic fashion during the assay period. The velocity of hydrolysis of the peptide-MCA substrate (A increase of A M C molar concentration per 60 s) at each corresponding time point was obtained from the recorded data. The molar concentration of activated Hageman factor at each time point ([HFa]t) was then calculated using the following equation: [HFaIt = [HF]0 x EUt/EUm~, where, [HF]0 is the initial molar concentration of Hageman factor, EUmax is the amidolytic enzyme units of activated Hageman factor when totally activated with each enzyme used and EUt is the enzyme units of activated Hageman factor at each time point. One enzyme unit is defined as the amount of activated Hageman factor which releases A M C 1 g M per min, in the assay conditions described above. To examine the values of the kinetic parameters of Hageman factor activation by the elastase or kallikrein, three or four different concentrations of Hageman factor ([HF]0), between 40 and 300 #M, were used. At each concentration, the [HFa]t values were plotted vs. time, and the rate of the activated Hageman factor generation was obtained from the slope of the plot. The values of the Michaelis constant (Km) and the maximum velocity (V) in the Michaelis-Menten equation were obtained from the double-reciprocal plot of the rate vs. Hageman factor concentration, as the best-fit values by the method of least squares with Taylar expansion, according to Sakoda and Hiromi [32]. The molar concentration of the elastase with the catalytic activity was determined from the catalytic rate of the Abz-Ala-Gly-Leu-Ala-Nba substrate, according to the kinetic parameters previously reported [27]. Molar concentration of active plasma kallikrein was calculated using the kinetic parameters on z-Phe-Arg-MCA as previously reported [33]. In the inhibition experiment with the elastase inhibitor, the elastase was initially incubated for 2 min with HONHCOCH(CH2C6Hs)CO-Ala-Gly-NH2 under the same assay conditions as described above. The peptideM C A substrate and Hageman factor were then added.
Results Each experiment described below was performed at least twice to confirm the reproducibility.
Enzymatic activation of Hageman factor in the absence of negatively charged substance The Hageman factor preparation used had a minor hydrolytic activity on the substrate, Pro-Phe-Arg-MCA or Boc-Gln-Gly-Arg-MCA, probably due to a contamination of the active form Hageman factor. The proportion of the spontaneous activity was about 5% of the total Pro-Phe-Arg-MCA hydrolytic potency of the preparation. The minor spontaneous activity did not change during incubation up to 30 min. Therefore, an activation of Hageman factor by activated Hageman factor (so called auto-activation) was negligible under the assay condition used. In the initial experiment, the purified Hageman factor (183 nM) was incubated with 5 nM of the pseudomonal proteinases or plasma kallikrein, under the assay conditions used. While pseudomonal elastase signifi-
75!
5O
25
0
5
10
15
20
T i m e (rain)
Fig. 1. Activation of Hageman factor by Pseudomonas aeruginosa elastase. The purified Hageman factor (final concentration 183 nM) was incubated with the elastase in a total volume of 1 ml in 50 mM Tris-HC1 buffer (pH 7.8) containing 130 mM NaC1,0.1 mg/ml bovine serum albumin and 0.02% NaN3 under the presence of 200/~M of the substrate of activated Hageman factor (Pro-Phe-Arg-MCA)in a plastic cuvette, at 37o C. The amidolytic activity of the activated Hageman factor was then continuouslymeasured and the molar concentration of activated Hageman factor at each time point, [HFa]t, was calculated as described under 'Materials and Methods'. The final concentration of the elastase used was 5 (A), 9.3 (11)and 18.6 nM (e). The same experiment was carried out using the elastase (final concentration 18.6 nM) previously treated with the inhibitor, HONHCOCH(CH2C6H5)CO-AIa-Gly-NH2 (10 tiM), for 2 min at 37 o C (zx).
234
Fig. 2. Time-course of limited proteolysis of Hageman factor by the elastase. The Hageman factor (366 nM) was incubated with the elastase (20 nM) in the same buffer as was used for the enzymatic assay (pH 7.8, I = 0.14), only lacking bovine serum albumin, at 37 ° C. At 0, 2, 5, 10, 20, 30 and 60 min, 30/~1 aliquots were taken and mixed with 5 btl of the boiled 10% sodium dodecyl sulfate (SDS) containing 8 M urea and tracking dye, with or without 2% fl-mercaptoethanol, were boiled for 2 min, and were analyzed by the gradient (4-20% acrylamide) slab polyacrylamide gel under non-reducing condition (A) and reducing condition (B) in the presence of SDS. The molecular mass of each band shown in the right-band column in (B) was obtained from the standard curve, by the use of the commercial kit with molecular mass (K) marker proteins.
c a n t l y a c t i v a t e d H a g e m a n factor, neither p s e u d o m o n a l alkaline p r o t e i n a s e n o r kaUikrein possessed the activation capacity. A typical p a t t e r n of the H a g e m a n factor activation b y the elastase is shown in Fig. 1. A f t e r a short lag period, the a m i d o l y t i c c a p a c i t y a t t r i b u t e d to a c t i v a t e d H a g e m a n factor a p p e a r e d a n d g r a d u a l l y increased, d e p e n d i n g u p o n the i n c u b a t i o n time. T h e velocity of the H a g e m a n factor activation d e p e n d e d u p o n the dose o f the elastase a d d e d at the final concentration, from 5 - 2 0 nM. T h e H a g e m a n factor activation c a p a c i t y of the elastase was b l o c k e d b y t r e a t m e n t with H O N H C O C H ( C H 2 C t H s ) C O - A l a - G I y - N H 2, a specific i n h i b i t o r of the elastase, at the c o n c e n t r a t i o n as low as 1 0 / ~ M (Fig. 1). T h e sensitivity to this i n h i b i t o r of the H a g e m a n factor activating c a p a c i t y of the elastase was c o m p a r a b l e to that o f the h y d r o l y t i c c a p a c i t y to A b z - A l a - G l y - L e u - A l a N b a , a synthetic s u b s t r a t e of the elastase ( d a t a n o t shown). F r o m these results, it was c o n c l u d e d that P. aeruginosa elastase a c t i v a t e d h u m a n H a g e m a n factor. W h e n a high dose of p s e u d o m o n a l alkaline p r o teinase (75 n M ) was used, a w e a k b u t significant H a g e m a n factor activation was observed. However, 80% of this a c t i v a t i o n c a p a c i t y was d i m i n i s h e d with the elastase i n h i b i t o r at the c o n c e n t r a t i o n 50 # M . T h e elastase inh i b i t o r at this c o n c e n t r a t i o n d i d not affect the original e n z y m a t i c activity of p s e u d o m o n a l alkaline proteinase. Therefore, it was suggested that the m a j o r p r o p o r t i o n o f the H a g e m a n f a c t o r - a c t i v a t i n g c a p a c i t y o b s e r v e d with P. aeruginosa alkaline p r o t e i n a s e was p r o b a b l y d u e to a c o n t a m i n a t i o n of the elastase in the p r e p a r a t i o n used.
Time-course of limited proteolysis of Hageman factor during the activation by the elastase T o investigate the r e l a t i o n s h i p b e t w e e n the H a g e m a n factor a c t i v a t i o n a n d the l i m i t e d p r o t e o l y s i s o f the H a g e m a n factor molecule, H a g e m a n factor (366 n M ) was i n c u b a t e d with the elastase (20 n M ) in a s s a y b u f f e r lacking b o v i n e s e r u m a l b u m i n , at 3 7 ° C . A t v a r i o u s times, aliquots were t a k e n a n d a n a l y z e d for the a m i d o -
20
,r,
~10
o
i
,o
do
3'0 Time
,'o
do
!
6o
(min)
Fig. 3. Time-course of the Hageman factor activation by the elastase. In the experiment shown in Fig. 2, 50/.tl aliquots, taken at 0, 2, 10, 20, 30 and 60 min, were incubated with 10 #l of the inhibitor of the elastase (final concentration 10 #M) and were assayed for the amidolytic activity on Pro-Phe-Arg-MCA in the same way as Fig. 1.
235 lytic activity to Pro-Phe-Arg-MCA in the presence of the inhibitor of the elastase (10 /~M), and for the molecular change by slab SDS-polyacrylamide gel electrophoresis analysis. As shown in Fig. 2, the 78 k D a molecule was initially cleaved into 40 k D a fragment(s), then about a half of the 40 k D a molecule was further cleaved into a 30 k D a fragment. The appearance of the 30 k D a fragment was slightly earlier in the reducing condition with fl-mercaptoethanol than in the non-reducing condition. The electrophoretic mobility of the 30 k D a fragment was not distinguishable from that of the t - f o r m activated Hageman factor (data not shown). In this time-course experiment, chronological appearance of the amidolytic activity attributed to the activated Hageman factor is shown in Fig. 3. The time-course of the appearance of the amidolytic activity seemed to correlate to that of the 30 k D a fragment in the reduced SDS-gel. These results indicated that the Hageman factor molecule was initially cleaved at the middle, then the fragment bearing the carboxy terminal region was further processed resulting in the activated Hageman factor generation.
10
,: 0
5
B
Enzymatic activation of Hageman factor in the presence of dextran sulfate The effect of dextran sulfate, a negatively charged surface, on the activation of Hageman factor by pseudomonal elastase was examined. Under assay conditions described above ( p H 7.8, I = 0.14, 37 ° C, 200 /~M of peptide-MCA substrate) the auto-activation in the H a g e m a n factor preparation was not detectable, even in the presence of dextran sulfate (0.3-10 /~g/ml). The effect of varying doses of dextran sulfate on the Hageman factor activation was shown in Fig. 5. In the experiments, three different concentrations of Hageman factor between 48 and 277 n M were used. There is an apparent range of optimum concentration of dextran sulfate for the activation (0.3-3.0 /~g/ml), and the en-
15
400
320
Kinetics of Hageman factor activation by pseudomonal elastase In this experiment, four different concentrations of Hageman factor in the range of 46 to 277 nM, and 5 n M of the elastase, were used. Plots of the activated H a g e m a n factor concentration generated with time, by the elastase, are shown in Fig. 4A. After the initial lag period, the rate of the activated Hageman factor generation became constant for at least 20 min. During the constant period, the dots plotted gave straight lines, with the correlation coefficients ( r ) greater than 0.99. A double-reciprocal plot of these rates vs. Hageman factor concentrations and the least squares line with Taylar expansion is shown in Fig. 4B. Values of 5.8.10 -3 s -1 and 427 nM for kcat and Km, respectively, were obtained.
10 Time (rain)
T 240 C
",p-
160
80
I
-0.02
0.00 II(HF)
I
I
0.02
0.04
I
(nM - 1 }
Fig. 4. (A) Progress curves for activation of Hageman factor by the
elastase in the absence of dextran sulfate. Hageman factor was incubated at 37 °C with the elastase (final concentration 5 nM) and the amidolytie activity of the activated Hageman factor was measured as in Fig. 1. The initial concentrations of Hageman factor were 46 (v), 92 (&), 185 (ll) and 277 nM (e). (B) Lineweaver-Burk plot of the activation of Hageman factor by the elastase. The rates of the activated Hageman factor formation presented in (A) were plotted. The broken line was obtained by the method of least squares, and the solid line was the best-fit values obtained by the method of least squares with Taylar expansion. hancement effect was cancelled with a higher dose of the substance, such as 10 /~g/ml. In the presence of dextran sulfate at a concentration of 3 v g / m l , the Hageman factor activation with the elastase was enhanced about 5-fold.
Kinetics of Hageman factor activation by elastase and kallikrein in the presence of dextran sulfate Kinetic analysis was performed in the same way as in
236
y
0.0 1
__,----o----
0
1
0.3 DxS
_+
3
10
( ughl)
Fig. 5. Effect of dextran sulfate on the activation of Hageman factor by the elastase. Hageman factor was incubated with the elastase (5 nM) as in Fig. 1, but in the presence of various concentrations of dextran sulfate, up to 10 pg/ml. The initial concentrations of Hageman factor were 46 (A), 185 (0) and 277 nM (0). 0-0, denotes the result of the same experiment, using plasma kalhkrein (5 nM) with 185 nM Hageman factor. The vertical axis is the apparent maximum velocity of the activated Hageman factor release expressed in nM of activated Hageman factor per s.
the absence of dextran sulfate at pH 7.8, I = 0.14 and 37 o C. Plots of the concentration of activated Hageman factor generated by the elastase with time in the presence of dextran sulfate at the concentration of 3 pg/ml are shown in Fig. 6A. A Lineweaver-Burk plot and the least-squares line with Taylar expansion is shown in Fig. 6B. Values of k,,, and K, obtained are shown in Table I. At a concentration of dextran sulfate of 0.3 pg/ml, an increment of k,,, value was apparent, and at higher concentrations (3 pg/ml), the decrement of K,,, value became significant. In the presence of dextran sulfate (3 pg/ml) the Hageman factor activation by plasma kallikrein was clear and rapid, as shown in Fig. 7. In this case the apparent lag phase for the activation was not observed. TABLE
I
Kinetic parameters
of enzymatic activation of Hageman factor
Assay was carried out in 50 mM Tris-HCl buffer (pH 7.8) containing 130 mM NaCl (I=O.14), 0.1 mg/ml bovine serum albumin and 0.02% NaN, in the presence of Pro-Phe-Arg-MCA (in the case of pseudomonal elastase) or Boc-Gin-Gly-Arg-MCA (in the case of plasma kallikrein).
0
5
10 (min)
Time
0
15
I
I =
32 t/
I
-0.02
/
I 0.00 1 /(HF)
I
0.02
I
0.04
(r&l -‘I
Fig. 6. (A) Progress curves for activation of Hageman factor by the elastase in the presence of dextran sulfate. Hageman factor was incubated with the elastase (5 nM) as for Fig. 1, but with the addition of dextran sulfate (3 pg/ml). The initial concentrations of Hageman factor were 46 (A), 185 (m) and 277 nM (0). (B) Lineweaver-Burk plot of the Hageman factor activation by the elastase in the presence of dextran sulfate. The rates of the activated Hageman factor formation presented in (A) were plotted. The broken line was obtained by the method of least-squares, and the solid line was the best-fit values obtained by the method of least squares with Taylar expansion.
The activation Activator
DxS a (pg/ml)
K, (nM)
k,,, (10d3 s-t)
L/K, (lo4 M-‘.s-t)
Elastase Elastase Elastase Kallikrein Kallikrein
0 0.3 3.0 3.0 3.0
427 367 163 36 57
5.81 13.89 13.89 1.57 169.30
1.36 3.78 8.52 4.37 295.90
b
a DxS, dextran sulfate. b The same assay conditions except that 60 mh4 NaCl instead of 130 mM NaCl, were used.
k,,,
capacity of the kallikrein especially the influenced by ionic strength of the used under the presence of dextran sulfate. At ionic strength such as I = 0.075, the k,,,/K, was 70-fold higher than that at the physiological strength, I = 0.14 (Table I).
value was rather
buffer lower value ionic
Discussion (I = 0.075),
Pseudomonal elastase, which is continuously secreted by the bacterium, was demonstrated to be a major
237 A
,. 4
1
0
2.5
B
5.0 Time (min)
j
300 240
180
,
I -0.02
7.5
0.00
i
i
0.02
0.04
II(HF) (nM-1)
Fig. 7. (A) Progress curves for activation of Hageman factor by plasma kallikrein in the presence of dextran sulfate. Hageman factor was incubated as for Fig. 1, but with kallikrein (5 nM) instead of the elastase, and with dextran sulfate (3 /~g/ml). The initial concentrations of Hageman factor were 46 (A), 92 (11) and 185 nM (I). (B) Lineweaver-Burk plot of the Hageman factor activation by kallikrein in the presence of dextran sulfate. The rates of the activated Hageman faster formation presented in (A) were plotted. The broken line was obtained by the method of least squares, and the solid line was the best-fit values obtained by the method of least squares with Taylar expansion.
tissue destructive pathogen in pseudomonal infection [34]. Indeed an intra corneal injection of 1.5 #g of the proteinase caused severe liquefactive necrosis in guinea pigs [20]. However, an intra cutaneous injection of the same dose of the enzyme induced an enhancing vascular permeability reaction [35] instead of the tissue necrosis, concomitant with an extravascular mobilization of
plasma proteinase inhibitors [36]. Since the permeability enhancement was mediated by the H a g e m a n f a c t o r / kallikrein-kinin pathway [20], an extravascular Hageman factor activation by the elastase or by some other bacterial proteinases is supposed to be an early step in host defence against bacterial infection at the surface tissues such as skin [37]. In this sense, a diffuse distribution of H a g e m a n factor at extravascular tissue space in normal guinea pig skin has been demonstrated by means of immunohistochemistry [38]. The amounts of guinea pig Hageman factor in plasma and extravascular skin tissue previously observed were 1 2 0 / ~ g / m l and 9 v g / g wet tissue, respectively [39]. On the assumption that the the interstitial tissue volume is 50 ml per 100 g wet skin tissue [40], the average concentration of Hageman factor in the interstitial tissue fluid of the guinea pig skin is calculated to be 15% of the plasma concentration. Applying this percent factor to the h u m a n system, the concentration of H a g e m a n factor in the h u m a n tissue fluid is assumed to be 4 - 6 / ~ g / m l (about 70 nM), since the plasma concentration reported is 24-40 /~g/ml (about 500 nM) [2]. The present study demonstrated in the activation of human Hageman factor with the elastase in vitro. In this reaction at least two cleavages of H a g e m a n factor molecules were observed. The second cleavage seemed to cause the activation. Therefore, there is a possibility that the apparent kinetic parameters in Table I for the second cleavage reaction m a y be composite ones, containing a competition effect by the first cleavage of remaining native H a g e m a n factor molecules. However, such influence in the competition of the kinetics of the second reaction seems to be minor, even if present, as the pattern of the second cleavage on SDS-polyacrylamide gel electrophoresis began to appear after the first cleavage was almost finished (Fig. 2), and because the rate of the activated Hageman factor generation in the enzymatic assay actually became constant after the initial lag period (Fig. 4). Therefore, the kinetic parameters described in Table I could be regarded as mainly those of the activation (second cleavage) step. The Michaelis constant of the enzymatic activation in the absence of a negatively charged surface was 427 nM. It is similar to the plasma H a g e m a n factor concentration and only 6-fold higher than the supposed concentration in interstitial tissue fluid. These values might support a possible occurrence of the enzymatic initiation of the H a g e m a n factor-dependent pathways by pseudomonal elastase even at interstitial tissue space and under the absence of a negatively charged substance. The initiation of this system must result in the permeability enhancement as described above. In such cases, the initial phase of the permeability enhancement reaction caused, might in a sense work as a positive feedback loop by increasing the local extravascular H a g e m a n factor concentration as the substrate.
238 It has been demonstrated that the strongest Hageman factor activator in plasma was kallikrein [7,8]. In the present study only negligible activation was observed with kallikrein in the absence of a negatively charged surface. It is basically consistent with the previous reports, including the data for the bovine system [8]. By the use of different assay systems and conditions, kinetic parameters for the activation of the human system with kallikrein in the absence of a negatively charged substance were reported from two laboratories as follows: k c a t = 1 • 10 -2 s -1, K m = 1.1 • 10 -5 M, and k c a t / K m = 9.6- 102 M -1 • s -a [9]; k 1 = k c a t / K m = 1.57103 M -1- s -1 [10]. The value of kcat/K m for pseudomonal elastase presently observed is more than 10-fold higher than for those with kallikrein. It has been established that the Hageman factor activation with kallikrein was very much enhanced by the presence of a negatively charged surface [1,2]. Dextran sulfate (molecular mass 500 kDa) has been used as the negatively charged soluble substance for kinetic analysis of the Hageman factor activation [8-10]. The most sufficient effect of the negatively charged substance could be observed when Hageman factor and kallikrein both bound on the substance in a condensed manner [1]. In this case, the binding force among them is assumed to be a major ionic interaction. Therefore, it requires the following conditions; sufficient net negative charge and molecular size of the substance; a suitable molar ratio between the substance and the proteins; and low ionic strength of the environment. The values of the kinetic parameters observed in the present study for the activation with kallikrein are lower than those reported previously, such as, kcat = 5.7 s -1, g m = 5.1 • 10 -7 M and k c a t / K m = l . 1 2 " l O 7 M -1 "s -1 [9] and k I = kcat/g m = 5.34- 1 0 6 M - 1 . s-1 (with sulfatide instead of dextran sulfate [10]). It is probably attributable to the difference in ionic strength of the buffer used. We used a buffer with physiological ionic strength ( I = 0.14), while a buffer with a lower ionic strength, such as I = 0.075 or 0.10, respectively, was previously used. By using the low ionic strength buffer ( I = 0.075), we also observed much higher values for the kinetic parameters (Table I) as had been previously reported. Therefore, the enhancing effect of the negatively charged substrates is partial in tile physiological ionic strength and in the absence of high molecular mass kininogen, since only Hageman factor, and not kallikrein, might bind to dextran sulfate, as observed with sulfatide [10]. In the present study, dextran sulfate also enhanced the Hageman factor activation by pseudomonal elastase, to a maximum of 5-fold. In the case with the elastase, the molar ratio between dextran sulfate and Hageman factor (therefore, the density of the latter molecule on the former molecule) appears to be significantly important. The concentration of dextran sulfate,
10/~g/ml, under which the enhancement effect was still observed for the activation by kallikrein to the same extent as in the case of 3/~g/ml, inhibited the activation by the elastase (Fig. 5). Pseudomonal elastase with acidic molecular nature (pI is 5.9 [41]) might be prevented to interact with Hageman factor molecule on dextran sulfate, if a wide area with sulfated sites on this substance remained uncovered, keeping the negative charge. It is still a matter of controversy which the negatively charged natural substance in vivo is, although sulfatide [8], heparin, condroitin sulfate E [42] and others [2] have been suggested as candidates. We have examined the effect of pseudomonal endotoxin of commercial source on the Hageman factor activation by the elastase. However, we have not yet obtained the positive enhancing effect. The active Hageman factor moiety generated in the limited proteolysis by the elastase seemed to have the apparent molecular mass of 30 kDa (Fig. 2), and the mobility of this protein band in the SDS-polyacrylamide gel electrophoresis was indistinguishable from that of the fl-form activated Hageman factor. With the fl-form activated Hageman factor, the strong prekallikrein activating capacity, but lack of effect on the coagulation factor XI and subsequent intrinsic blood coagulation pathway, was demonstrated [2]. It was consistent with the result of our preliminary experiment, in which the elastase did not initiate coagulation in human plasma (unpublished data). Therefore, the Hageman factor activated by the elastase might work mainly as the initiator of the kallikrein-kinin pathway. Since an important role of the P. aeruginosa elastase in the pathogenesis of cystic fibrosis has been pronounced [23-25], it must be of importance to examine the state of the Hageman factor/kallikrein-kinin system in the patients of this genetic disease. P. aerugmosa alkaline proteinase negligibly activated Hageman factor in the present study. This data differents from that reported on the guinea pig Hageman factor activation. This proteinase activated guinea pig Hageman factor quite well, both in vitro and in vivo [20]. At the present time, we have no explanation for this species difference. It is a speculation that there might be a difference of an amino acid(s) in the sequence of the human and the guinea pig Hageman factor molecule, around the essential cleavage site for the activation.
Acknowledgements The authors are grateful to Dr. Tanase, Kumamoto University Medical School for the fruitful discussion. This work was supported in part by grants from Japanese Ministry of Education, Science and Culture.
239
References 1 Kaplan, A.P. and Silverberg, M (1987) Blood 70, 1-15. 2 Kozin, F. and Cochrane, C.G. (1988) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J.I., Goldstein, I.M. and Snyderman, R., eds.), pp. 101-127, Raven Press, New York. 3 Yamamoto, T. and Cochrane, C.G. (1981) Am. J. Pathol. 105, 164-175. 4 Yamamoto, T., Kozono, K., Kambara, T. and Cochrane, C.G. (1988) Biochim. Biophys. Acta 966, 196-206. 5 Mason, J.W., Kleeberg, U., Dolan, P. and Colman, R.W. (1970) Ann. Intern. Med. 73, 545-551. 60'DonneU, T.F., Jr., Clowes, G.H.A., Jr., Talamo, R.C. and Colman, R.W. (1976) Surg. Gynecol. Obstet. 143, 539-545. 7 Cochrane, C.G., Revak, S.D. and Wuepper, K.D. (1973) J. Exp. Med. 138, 1564-1583. 8 Fujikawa, K., Heimark, R.L., Kurachi, K. and Davie, E.W. (1980) Biochemistry 19, 1322-1330. 9 Tankersley, D.L. and Finlayson, J.S. (1984) Biochemistry 23, 273279. 10 Rozing, J., Tans, G. and Griffin, J.H. (1985) Eur. J. Biochem. 151, 531-538. 11 Shimada, T., Sugo, T., Kato, H., Yoshida, K. and Iwanaga, S. (1985) J. Biochem. 97, 429-439. 12 Kaplan, A.P. and Austen, K.F. (1971) J. Exp. Med. 133, 696-712. 13 Wiggins, R. and Cochrane, C.G. (1979) J. Exp. Med. 150, 11221133. 14 Miller, G., Silverberg, M. and Kaplan, A.P. (1980) Biochem. Biophys. Rcs. Commun. 92, 803-810. 15 Silverberg, M., Dunn, J.T., Garen, L. and Kaplan, A.P. (1980) J. Biol. Chem. 255, 7281-7286. 16 Tans, G., Rosing, J. and Griffin, J.H. (1983) Y Biol. Chem. 258, 8215-8222. 17 Morrison, D.C. and Cochrane, C.G. (1974) J. Exp. Med. 140, 797-811. 18 Tankersley, D.L., Alving, B.M. and Finlayson, J.S. (1983) Blood 62, 448-456. 19 Matsumoto, K., Yamamoto, T., Kamata, R. and Maeda, H. (1984) J. Biochem. 96, 739-749. 20 Molla, A., Yamamoto, T., Akaike, T., Miyoshi, S. and Maeda, H. (1989) J. Biol. Chem. 264, 10589-10594.
21 Kamata, R., Yarnamoto, T., Matsumoto, K. and Maeda, H. (1985) Infect. Immun. 48, 747-753. 22 Morihara, K. (1974) Adv. Enzymol. 41, 179-243. 23 Klinger, J.D., Tandler, B., Liedtke, C.M. and Boat, T.F. (1984) J. Clin. Invest. 74, 1669-1678. 24 Hingley, S.T., Hastie, A.T., Kueppers, F. and Higgins, M.L. (1986) Infect. Immun. 54, 379-385. 25 Niederman, M.S., Merril, W.W. and Polomski, L.M. (1986) Am. Rev. Respir. Dis. 133, 255-260. 26 Yamamoto, T., Shibuya, Y, Nishino, N., Okabe, H. and Kambara, T. (1989) Thromb. Haemost. 62, 295. 27 Nishino, N. and Powers, J.C. (1980) J. Biol. Chem. 255, 3482-3486. 28 Laemmli, U.K. (1970) Nature 227, 680-685. 29 Fujikawa, K. and Davie, E.W. (1981) Methods Enzymol. 80, 198-211. 30 Nagase, H. and Barrett, A.J. (1981) Biochem. J. 193, 187-192. 31 Hojima, Y., Tankersley, D.L., Miller-Andersson, Pierce, J.V. and Pisano, J.J. (1980) Thromb. Res. 18, 417-430. 32 Sakoda, M. and Hiromi, K. (1976) J. Biochem. 80, 547-555. 33 Kawabata, S., Miura, T., Morita, T., Kato, H., Fujikawa, K., Iwanaga, S., Takada, K., Kimura, T. and Sakakibara, S. (1988) Eur. J. Biochem. 172, 17-25. 34 Morihara, K. and Tsuzuki, H. (1977) Infect. Immun. 15, 679-685. 35 Molla, A., Matsumura, Y., Yamamoto, T., Okamura, R. and Maeda, H. (1987) Infect. Immun. 55, 2509-2517. 36 lshimatsu, T., Yamamoto, T., Kozono, K. and Kambara, T. (1984) Am. J. Pathol. 115, 57-69. 37 Yamamoto, T. (1985) in Chemical Mediators of Inflammation and Immunity (Cohen, S,, Hayashi, H., Saito, K. and Takada, A., eds.), pp. 129-143, Academic Press, Tokyo. 38 Tsuruta, J., Yamamoto, T. and Kambara, T. (1986) Kinins IV, Part B (Greenbaum, L.M. and Margolius, H.S., eds.), pp. 63-70, Plenum Press, New York. 39 Yamamoto, T. and Cochrane, C.G. (1982) Am. J. Pathol. 107, 127-134. 40 Aukland, K. and Nicolaysen, G. (1981) Physiol. Rev. 61,556-643. 41 Morihara, K., Tsuzuki, H., Oka, T., Inoue, H. and Ebata, M. (1965) J. Biol. Chem. 240, 3295-3304. 42 Hojima, Y., Cochrane, C.G., Wiggins, R.C., Austen, K.F. and Stevens, R.L. (1984) Blood 63, 1453-1459.
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Elsevier BBAPRO 33617
Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro Tetsuro Y a m a m o t o 1, Yoko Shibuya 2, Norikazu Nishino 3, Hiroaki Okabe 2 and Takeshi K a m b a r a i t Department of Allergy, Institute for Medical Immunology and 2 Department of Laboratory Medicine, Kumamoto University Medical School, Kumamoto, and ~ Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu (Japan)
(Received 19 September1989)
Key words: Hagemanfactor; Kallikrein-kininsystem; Bacterialproteinase; Pseudomonalinfection; Pseudomonalelastase; ( P. aeruginosa )
Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 3 7 ° C ) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONI-ICOCH(CH2C6Hs)CO-Ala-Gly-NH 2, at a concentration as low as 10 pM. In this activation Hageman factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation w e r e kca t w. 5 . 8 " 1 0 - 3 s - m , K m = 4.3 • 1 0 - 7 M and k ~ t / K m = 1.4 • 1 0 4 M - t . s - i . This K m value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 p g / m l ) , the activation rate by the elastase increased several fold, with the kinetic parameters of k , t = 1 3 . 9 . 1 0 -a s - t , K m = 1.6" 10 -7 M and kcat//Km---8.5 " 104 M - ! . s - I . These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.
Introduction Hageman factor (blood coagulation factor XII) has been shown to be an initiation factor of the intrinsic and extrinsic blood coagulation pathway, kallikreinkinin pathway, and fibrinolytic pathway, as well as the complement pathway (Refs. 1 and 2; only the newest reviews are cited as reference). However, considerable research progress made during the past 20 years has given rise to major doubts about the contribution of the Hageman factor-dependent system to thrombosis and hemostasis [1]. Most evidence suggest important roles of
Abbreviations: MCA, 7-amino-4-methylcoumarylamide; AMC, aminomethyl coumarin; SDS, sodium dodecyl sulfate; Abz, 2aminobenzoyl; Nba, 4-nitrobenzylamide; HFa, activated Hageman factor; HF, Hageman factor. Correspondence: T. Yamamoto, Department of Allergy, Institute for Medical Immunology, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto 860, Japan.
the pathways, especially the Hageman factor/kallikrein-kinin system, in the pathogenesis of inflammatory response [1,2]. Indeed, it has been demonstrated in guinea pigs that the Hageman factor/kallikrein-kinin system is one of the strongest mediator systems of vascular permeability enhancement, as it is seen in inflammatory response [3,4]. On the clinical side, participation of the system has been speculated in infectious diseases, especially with G r a m -negative bacteremia, since significant consumption of components of the system was occasionally observed in such bacteremia [5,6]. Hageman factor is a serine proteinase zymogen, therefore, conversion to an active-form proteinase is required when the system is initiated. Most of the research on the Hageman factor activation has been focussed on that by endogenous proteinases in plasma, such as plasma kallikrein [7-11], coagulation factor XIa [8], plasmin [8,12] and activated Hageman factor itself [9,13-16]. It was revealed in the research that: sufficient Hageman factor activation by the plasma proteinases
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
232 occurred only in the presence of negatively charged substance [8-16], and high molecular mass kininogen was also required under some conditions [11,13]; the activation occurred due to a limited proteolysis of Hageman factor molecule; and the strongest Hageman factor activator in plasma was kallikrein. Hageman factor activation by the Gram-negative bacterial lipopolysaccharide, endotoxin [17], was assumed due to its function as the negatively charged substance. However, natural occurrence of the Hageman factor activation by the endogenous proteinases on negatively charged substances is still in a controversy. For instance, the rate of activation by kallikrein is very much influenced by the concentration of the negatively charged substance [11] and the ionic strength of the buffer used [10,18]; the rate is considerably slow or at physiological ionic strength (I = 0.14-0.15) [18]. In addition to the requirement of limited proteolysis for the Hageman factor activation, when the contribution of the system in the Gram-negative bacterial infections is considered, it must be reasonable to speculate an in situ occurrence of the Hageman factor activation with enzymes secreted by the pathogenic microorganisms. Indeed, we have recently found that the elastase and alkaline proteinase or the 56 kDa proteinase secreted by Pseudomonas aeruginosa or Serratia marcescens, respectively, activated guinea pig Hageman factor in vitro [19,20], as well as in vivo; in the guinea pig skin, this activation was succeeded by the kallikrein-kinin generation followed by the vascular permeability enhancement reaction [20,21]. In the present project, we selected P. aeruginosa elastase and alkaline proteinase as representatives of Gram-negative bacterial proteinases, since both enzymes activate guinea pig Hageman factor despite being zinc-dependent metal enzymes [22], and since the pseudomonal proteinases were recently considered to be important enzymes in the development of chronic lung disease in cystic fibrosis patients [23-25]. We investigated whether human Hageman factor was activated by the pseudomonal proteinases in vitro at physiological ionic strength ( I = 0.14), and whether the activation rate with the elastase was enhanced in the presence of a negatively charged substance, such as dextran sulfate. We also evaluated the elastase in regard to the kinetic parameters of the enzymatic activation in the presence and absence of the negatively charged substance. A part of this work was presented in XIIth Congress of the International Society on Thrombosis and Haemostasis [26].
(Fukuchiyama, Japan). Pro-Phe-Arg-methylcoumarylamide (MCA), Boc-GIn-GIy-Arg-MCA, z-Phe-ArgMCA and aminomethyl coumarin (AMC) were purchased from Peptide Institute (Minoh, Japan). Crystallized and lyophilized bovine serum albumin was purchased from Sigma Chemical Co. (St. Louis, MO). Dextran sulfate sodium salt (molecular mass about 500 kDa) was obtained from Nakarai Chemicals (Kyoto, Japan). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) and Nakarai Chemicals. Gradient polyacrylamide slab gel (4-20% acrylamide) was a product of Tefco Co. (Tokyo, Japan). Disposable polystyrene plastic cuvettes for fluorescence spectrophotometer were products of Elkay Products (Shrewsbury, MA). HONHCOCH(CHzC6Hs)CO-AIa-Gly-NH 2 and Abz-Ala-Gly-Leu-Ala-Nba were synthesized as reported previously [27].
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) Slab SDS-polyacrylamide gel electrophoresis was performed with gradient acrylamide (4-20%) gels according to the method of Laemmli [28]. The gels were stained with 0.8% Coomassie brilliant blue R-250. To make the molecular mass standard curve, a commercial kit (Electrophoresis calibration kit, Pharmacia) was used.
Preparation of plasma proteins The isolation of Hageman factor from human plasma was carried out essentially as described by Fujikawa and Davie [29], with a substitution of the chromatography with CM-cellulose by a high-performance liquid chromatography (HPLC, LKB system) with a CMToyopearl column (Tosoh). The final material had the specific activity with 51.4 clotting units per mg protein which was comparable to the previous report [29]. Hageman factor thus prepared was stored at - 80 ° C in 10 mM acetate buffer containing 300 mM NaCI and 0.02% NaN 3 (pH 5.5). Plasma kallikrein was prepared from fresh frozen human plasma using a soybean trypsin inhibitor affinity column, after activation by an acetone treatment, according to the method of Nagase and Barrett [30]. B-Form activated Hageman factor was prepared from the same material as used for the kallikrein preparation by the method of Hojima et al. [31], with a substitution of the isoelectric focusing by a HPLC step using a DEAE-5PW column (Tosoh). The final products were apparently pure, as judged by slab SDS-polyacrylamide gel electrophoresis with Coomassie blue staining.
Materials and Methods
Assay of Hageman factor activation Substances Crystalline elastase and alkaline proteinase from P. aeruginosa were obtained from Nagase Biochemicals
The assay was performed in 50 mM Tris-HC1 buffer (pH 8.0 at 25°C and pH 7.8 at 37°C) containing 130 mM NaC1, 0.1 mg/ml bovine serum albumin and 0.02%
233 N a N 3 in the presence of 200 # M of the substrate of activated Hageman factor in the plastic cuvette at 37 o C. The substrate Pro-Phe-Arg-MCA or Boc-Gln-Gly-ArgM C A was used with the pseudomonal enzymes or with plasma kallikrein, respectively. In the usual assay, aliquots of the stock solutions of Hageman factor and one of the enzymes were added, respectively, to the mixture of the substrate and buffer in the cuvette. Liberation of A M C from the peptide-MCA substrate was continuously recorded on the chart recorder, using the fluorescence spectrophotometer, with the wavelengths of excitation at 380 nm and emission at 440 nm. The amount of A M C liberated increased in a parabolic fashion during the assay period. The velocity of hydrolysis of the peptide-MCA substrate (A increase of A M C molar concentration per 60 s) at each corresponding time point was obtained from the recorded data. The molar concentration of activated Hageman factor at each time point ([HFa]t) was then calculated using the following equation: [HFaIt = [HF]0 x EUt/EUm~, where, [HF]0 is the initial molar concentration of Hageman factor, EUmax is the amidolytic enzyme units of activated Hageman factor when totally activated with each enzyme used and EUt is the enzyme units of activated Hageman factor at each time point. One enzyme unit is defined as the amount of activated Hageman factor which releases A M C 1 g M per min, in the assay conditions described above. To examine the values of the kinetic parameters of Hageman factor activation by the elastase or kallikrein, three or four different concentrations of Hageman factor ([HF]0), between 40 and 300 #M, were used. At each concentration, the [HFa]t values were plotted vs. time, and the rate of the activated Hageman factor generation was obtained from the slope of the plot. The values of the Michaelis constant (Km) and the maximum velocity (V) in the Michaelis-Menten equation were obtained from the double-reciprocal plot of the rate vs. Hageman factor concentration, as the best-fit values by the method of least squares with Taylar expansion, according to Sakoda and Hiromi [32]. The molar concentration of the elastase with the catalytic activity was determined from the catalytic rate of the Abz-Ala-Gly-Leu-Ala-Nba substrate, according to the kinetic parameters previously reported [27]. Molar concentration of active plasma kallikrein was calculated using the kinetic parameters on z-Phe-Arg-MCA as previously reported [33]. In the inhibition experiment with the elastase inhibitor, the elastase was initially incubated for 2 min with HONHCOCH(CH2C6Hs)CO-Ala-Gly-NH2 under the same assay conditions as described above. The peptideM C A substrate and Hageman factor were then added.
Results Each experiment described below was performed at least twice to confirm the reproducibility.
Enzymatic activation of Hageman factor in the absence of negatively charged substance The Hageman factor preparation used had a minor hydrolytic activity on the substrate, Pro-Phe-Arg-MCA or Boc-Gln-Gly-Arg-MCA, probably due to a contamination of the active form Hageman factor. The proportion of the spontaneous activity was about 5% of the total Pro-Phe-Arg-MCA hydrolytic potency of the preparation. The minor spontaneous activity did not change during incubation up to 30 min. Therefore, an activation of Hageman factor by activated Hageman factor (so called auto-activation) was negligible under the assay condition used. In the initial experiment, the purified Hageman factor (183 nM) was incubated with 5 nM of the pseudomonal proteinases or plasma kallikrein, under the assay conditions used. While pseudomonal elastase signifi-
75!
5O
25
0
5
10
15
20
T i m e (rain)
Fig. 1. Activation of Hageman factor by Pseudomonas aeruginosa elastase. The purified Hageman factor (final concentration 183 nM) was incubated with the elastase in a total volume of 1 ml in 50 mM Tris-HC1 buffer (pH 7.8) containing 130 mM NaC1,0.1 mg/ml bovine serum albumin and 0.02% NaN3 under the presence of 200/~M of the substrate of activated Hageman factor (Pro-Phe-Arg-MCA)in a plastic cuvette, at 37o C. The amidolytic activity of the activated Hageman factor was then continuouslymeasured and the molar concentration of activated Hageman factor at each time point, [HFa]t, was calculated as described under 'Materials and Methods'. The final concentration of the elastase used was 5 (A), 9.3 (11)and 18.6 nM (e). The same experiment was carried out using the elastase (final concentration 18.6 nM) previously treated with the inhibitor, HONHCOCH(CH2C6H5)CO-AIa-Gly-NH2 (10 tiM), for 2 min at 37 o C (zx).
234
Fig. 2. Time-course of limited proteolysis of Hageman factor by the elastase. The Hageman factor (366 nM) was incubated with the elastase (20 nM) in the same buffer as was used for the enzymatic assay (pH 7.8, I = 0.14), only lacking bovine serum albumin, at 37 ° C. At 0, 2, 5, 10, 20, 30 and 60 min, 30/~1 aliquots were taken and mixed with 5 btl of the boiled 10% sodium dodecyl sulfate (SDS) containing 8 M urea and tracking dye, with or without 2% fl-mercaptoethanol, were boiled for 2 min, and were analyzed by the gradient (4-20% acrylamide) slab polyacrylamide gel under non-reducing condition (A) and reducing condition (B) in the presence of SDS. The molecular mass of each band shown in the right-band column in (B) was obtained from the standard curve, by the use of the commercial kit with molecular mass (K) marker proteins.
c a n t l y a c t i v a t e d H a g e m a n factor, neither p s e u d o m o n a l alkaline p r o t e i n a s e n o r kaUikrein possessed the activation capacity. A typical p a t t e r n of the H a g e m a n factor activation b y the elastase is shown in Fig. 1. A f t e r a short lag period, the a m i d o l y t i c c a p a c i t y a t t r i b u t e d to a c t i v a t e d H a g e m a n factor a p p e a r e d a n d g r a d u a l l y increased, d e p e n d i n g u p o n the i n c u b a t i o n time. T h e velocity of the H a g e m a n factor activation d e p e n d e d u p o n the dose o f the elastase a d d e d at the final concentration, from 5 - 2 0 nM. T h e H a g e m a n factor activation c a p a c i t y of the elastase was b l o c k e d b y t r e a t m e n t with H O N H C O C H ( C H 2 C t H s ) C O - A l a - G I y - N H 2, a specific i n h i b i t o r of the elastase, at the c o n c e n t r a t i o n as low as 1 0 / ~ M (Fig. 1). T h e sensitivity to this i n h i b i t o r of the H a g e m a n factor activating c a p a c i t y of the elastase was c o m p a r a b l e to that o f the h y d r o l y t i c c a p a c i t y to A b z - A l a - G l y - L e u - A l a N b a , a synthetic s u b s t r a t e of the elastase ( d a t a n o t shown). F r o m these results, it was c o n c l u d e d that P. aeruginosa elastase a c t i v a t e d h u m a n H a g e m a n factor. W h e n a high dose of p s e u d o m o n a l alkaline p r o teinase (75 n M ) was used, a w e a k b u t significant H a g e m a n factor activation was observed. However, 80% of this a c t i v a t i o n c a p a c i t y was d i m i n i s h e d with the elastase i n h i b i t o r at the c o n c e n t r a t i o n 50 # M . T h e elastase inh i b i t o r at this c o n c e n t r a t i o n d i d not affect the original e n z y m a t i c activity of p s e u d o m o n a l alkaline proteinase. Therefore, it was suggested that the m a j o r p r o p o r t i o n o f the H a g e m a n f a c t o r - a c t i v a t i n g c a p a c i t y o b s e r v e d with P. aeruginosa alkaline p r o t e i n a s e was p r o b a b l y d u e to a c o n t a m i n a t i o n of the elastase in the p r e p a r a t i o n used.
Time-course of limited proteolysis of Hageman factor during the activation by the elastase T o investigate the r e l a t i o n s h i p b e t w e e n the H a g e m a n factor a c t i v a t i o n a n d the l i m i t e d p r o t e o l y s i s o f the H a g e m a n factor molecule, H a g e m a n factor (366 n M ) was i n c u b a t e d with the elastase (20 n M ) in a s s a y b u f f e r lacking b o v i n e s e r u m a l b u m i n , at 3 7 ° C . A t v a r i o u s times, aliquots were t a k e n a n d a n a l y z e d for the a m i d o -
20
,r,
~10
o
i
,o
do
3'0 Time
,'o
do
!
6o
(min)
Fig. 3. Time-course of the Hageman factor activation by the elastase. In the experiment shown in Fig. 2, 50/.tl aliquots, taken at 0, 2, 10, 20, 30 and 60 min, were incubated with 10 #l of the inhibitor of the elastase (final concentration 10 #M) and were assayed for the amidolytic activity on Pro-Phe-Arg-MCA in the same way as Fig. 1.
235 lytic activity to Pro-Phe-Arg-MCA in the presence of the inhibitor of the elastase (10 /~M), and for the molecular change by slab SDS-polyacrylamide gel electrophoresis analysis. As shown in Fig. 2, the 78 k D a molecule was initially cleaved into 40 k D a fragment(s), then about a half of the 40 k D a molecule was further cleaved into a 30 k D a fragment. The appearance of the 30 k D a fragment was slightly earlier in the reducing condition with fl-mercaptoethanol than in the non-reducing condition. The electrophoretic mobility of the 30 k D a fragment was not distinguishable from that of the t - f o r m activated Hageman factor (data not shown). In this time-course experiment, chronological appearance of the amidolytic activity attributed to the activated Hageman factor is shown in Fig. 3. The time-course of the appearance of the amidolytic activity seemed to correlate to that of the 30 k D a fragment in the reduced SDS-gel. These results indicated that the Hageman factor molecule was initially cleaved at the middle, then the fragment bearing the carboxy terminal region was further processed resulting in the activated Hageman factor generation.
10
,: 0
5
B
Enzymatic activation of Hageman factor in the presence of dextran sulfate The effect of dextran sulfate, a negatively charged surface, on the activation of Hageman factor by pseudomonal elastase was examined. Under assay conditions described above ( p H 7.8, I = 0.14, 37 ° C, 200 /~M of peptide-MCA substrate) the auto-activation in the H a g e m a n factor preparation was not detectable, even in the presence of dextran sulfate (0.3-10 /~g/ml). The effect of varying doses of dextran sulfate on the Hageman factor activation was shown in Fig. 5. In the experiments, three different concentrations of Hageman factor between 48 and 277 n M were used. There is an apparent range of optimum concentration of dextran sulfate for the activation (0.3-3.0 /~g/ml), and the en-
15
400
320
Kinetics of Hageman factor activation by pseudomonal elastase In this experiment, four different concentrations of Hageman factor in the range of 46 to 277 nM, and 5 n M of the elastase, were used. Plots of the activated H a g e m a n factor concentration generated with time, by the elastase, are shown in Fig. 4A. After the initial lag period, the rate of the activated Hageman factor generation became constant for at least 20 min. During the constant period, the dots plotted gave straight lines, with the correlation coefficients ( r ) greater than 0.99. A double-reciprocal plot of these rates vs. Hageman factor concentrations and the least squares line with Taylar expansion is shown in Fig. 4B. Values of 5.8.10 -3 s -1 and 427 nM for kcat and Km, respectively, were obtained.
10 Time (rain)
T 240 C
",p-
160
80
I
-0.02
0.00 II(HF)
I
I
0.02
0.04
I
(nM - 1 }
Fig. 4. (A) Progress curves for activation of Hageman factor by the
elastase in the absence of dextran sulfate. Hageman factor was incubated at 37 °C with the elastase (final concentration 5 nM) and the amidolytie activity of the activated Hageman factor was measured as in Fig. 1. The initial concentrations of Hageman factor were 46 (v), 92 (&), 185 (ll) and 277 nM (e). (B) Lineweaver-Burk plot of the activation of Hageman factor by the elastase. The rates of the activated Hageman factor formation presented in (A) were plotted. The broken line was obtained by the method of least squares, and the solid line was the best-fit values obtained by the method of least squares with Taylar expansion. hancement effect was cancelled with a higher dose of the substance, such as 10 /~g/ml. In the presence of dextran sulfate at a concentration of 3 v g / m l , the Hageman factor activation with the elastase was enhanced about 5-fold.
Kinetics of Hageman factor activation by elastase and kallikrein in the presence of dextran sulfate Kinetic analysis was performed in the same way as in
236
y
0.0 1
__,----o----
0
1
0.3 DxS
_+
3
10
( ughl)
Fig. 5. Effect of dextran sulfate on the activation of Hageman factor by the elastase. Hageman factor was incubated with the elastase (5 nM) as in Fig. 1, but in the presence of various concentrations of dextran sulfate, up to 10 pg/ml. The initial concentrations of Hageman factor were 46 (A), 185 (0) and 277 nM (0). 0-0, denotes the result of the same experiment, using plasma kalhkrein (5 nM) with 185 nM Hageman factor. The vertical axis is the apparent maximum velocity of the activated Hageman factor release expressed in nM of activated Hageman factor per s.
the absence of dextran sulfate at pH 7.8, I = 0.14 and 37 o C. Plots of the concentration of activated Hageman factor generated by the elastase with time in the presence of dextran sulfate at the concentration of 3 pg/ml are shown in Fig. 6A. A Lineweaver-Burk plot and the least-squares line with Taylar expansion is shown in Fig. 6B. Values of k,,, and K, obtained are shown in Table I. At a concentration of dextran sulfate of 0.3 pg/ml, an increment of k,,, value was apparent, and at higher concentrations (3 pg/ml), the decrement of K,,, value became significant. In the presence of dextran sulfate (3 pg/ml) the Hageman factor activation by plasma kallikrein was clear and rapid, as shown in Fig. 7. In this case the apparent lag phase for the activation was not observed. TABLE
I
Kinetic parameters
of enzymatic activation of Hageman factor
Assay was carried out in 50 mM Tris-HCl buffer (pH 7.8) containing 130 mM NaCl (I=O.14), 0.1 mg/ml bovine serum albumin and 0.02% NaN, in the presence of Pro-Phe-Arg-MCA (in the case of pseudomonal elastase) or Boc-Gin-Gly-Arg-MCA (in the case of plasma kallikrein).
0
5
10 (min)
Time
0
15
I
I =
32 t/
I
-0.02
/
I 0.00 1 /(HF)
I
0.02
I
0.04
(r&l -‘I
Fig. 6. (A) Progress curves for activation of Hageman factor by the elastase in the presence of dextran sulfate. Hageman factor was incubated with the elastase (5 nM) as for Fig. 1, but with the addition of dextran sulfate (3 pg/ml). The initial concentrations of Hageman factor were 46 (A), 185 (m) and 277 nM (0). (B) Lineweaver-Burk plot of the Hageman factor activation by the elastase in the presence of dextran sulfate. The rates of the activated Hageman factor formation presented in (A) were plotted. The broken line was obtained by the method of least-squares, and the solid line was the best-fit values obtained by the method of least squares with Taylar expansion.
The activation Activator
DxS a (pg/ml)
K, (nM)
k,,, (10d3 s-t)
L/K, (lo4 M-‘.s-t)
Elastase Elastase Elastase Kallikrein Kallikrein
0 0.3 3.0 3.0 3.0
427 367 163 36 57
5.81 13.89 13.89 1.57 169.30
1.36 3.78 8.52 4.37 295.90
b
a DxS, dextran sulfate. b The same assay conditions except that 60 mh4 NaCl instead of 130 mM NaCl, were used.
k,,,
capacity of the kallikrein especially the influenced by ionic strength of the used under the presence of dextran sulfate. At ionic strength such as I = 0.075, the k,,,/K, was 70-fold higher than that at the physiological strength, I = 0.14 (Table I).
value was rather
buffer lower value ionic
Discussion (I = 0.075),
Pseudomonal elastase, which is continuously secreted by the bacterium, was demonstrated to be a major
237 A
,. 4
1
0
2.5
B
5.0 Time (min)
j
300 240
180
,
I -0.02
7.5
0.00
i
i
0.02
0.04
II(HF) (nM-1)
Fig. 7. (A) Progress curves for activation of Hageman factor by plasma kallikrein in the presence of dextran sulfate. Hageman factor was incubated as for Fig. 1, but with kallikrein (5 nM) instead of the elastase, and with dextran sulfate (3 /~g/ml). The initial concentrations of Hageman factor were 46 (A), 92 (11) and 185 nM (I). (B) Lineweaver-Burk plot of the Hageman factor activation by kallikrein in the presence of dextran sulfate. The rates of the activated Hageman faster formation presented in (A) were plotted. The broken line was obtained by the method of least squares, and the solid line was the best-fit values obtained by the method of least squares with Taylar expansion.
tissue destructive pathogen in pseudomonal infection [34]. Indeed an intra corneal injection of 1.5 #g of the proteinase caused severe liquefactive necrosis in guinea pigs [20]. However, an intra cutaneous injection of the same dose of the enzyme induced an enhancing vascular permeability reaction [35] instead of the tissue necrosis, concomitant with an extravascular mobilization of
plasma proteinase inhibitors [36]. Since the permeability enhancement was mediated by the H a g e m a n f a c t o r / kallikrein-kinin pathway [20], an extravascular Hageman factor activation by the elastase or by some other bacterial proteinases is supposed to be an early step in host defence against bacterial infection at the surface tissues such as skin [37]. In this sense, a diffuse distribution of H a g e m a n factor at extravascular tissue space in normal guinea pig skin has been demonstrated by means of immunohistochemistry [38]. The amounts of guinea pig Hageman factor in plasma and extravascular skin tissue previously observed were 1 2 0 / ~ g / m l and 9 v g / g wet tissue, respectively [39]. On the assumption that the the interstitial tissue volume is 50 ml per 100 g wet skin tissue [40], the average concentration of Hageman factor in the interstitial tissue fluid of the guinea pig skin is calculated to be 15% of the plasma concentration. Applying this percent factor to the h u m a n system, the concentration of H a g e m a n factor in the h u m a n tissue fluid is assumed to be 4 - 6 / ~ g / m l (about 70 nM), since the plasma concentration reported is 24-40 /~g/ml (about 500 nM) [2]. The present study demonstrated in the activation of human Hageman factor with the elastase in vitro. In this reaction at least two cleavages of H a g e m a n factor molecules were observed. The second cleavage seemed to cause the activation. Therefore, there is a possibility that the apparent kinetic parameters in Table I for the second cleavage reaction m a y be composite ones, containing a competition effect by the first cleavage of remaining native H a g e m a n factor molecules. However, such influence in the competition of the kinetics of the second reaction seems to be minor, even if present, as the pattern of the second cleavage on SDS-polyacrylamide gel electrophoresis began to appear after the first cleavage was almost finished (Fig. 2), and because the rate of the activated Hageman factor generation in the enzymatic assay actually became constant after the initial lag period (Fig. 4). Therefore, the kinetic parameters described in Table I could be regarded as mainly those of the activation (second cleavage) step. The Michaelis constant of the enzymatic activation in the absence of a negatively charged surface was 427 nM. It is similar to the plasma H a g e m a n factor concentration and only 6-fold higher than the supposed concentration in interstitial tissue fluid. These values might support a possible occurrence of the enzymatic initiation of the H a g e m a n factor-dependent pathways by pseudomonal elastase even at interstitial tissue space and under the absence of a negatively charged substance. The initiation of this system must result in the permeability enhancement as described above. In such cases, the initial phase of the permeability enhancement reaction caused, might in a sense work as a positive feedback loop by increasing the local extravascular H a g e m a n factor concentration as the substrate.
238 It has been demonstrated that the strongest Hageman factor activator in plasma was kallikrein [7,8]. In the present study only negligible activation was observed with kallikrein in the absence of a negatively charged surface. It is basically consistent with the previous reports, including the data for the bovine system [8]. By the use of different assay systems and conditions, kinetic parameters for the activation of the human system with kallikrein in the absence of a negatively charged substance were reported from two laboratories as follows: k c a t = 1 • 10 -2 s -1, K m = 1.1 • 10 -5 M, and k c a t / K m = 9.6- 102 M -1 • s -a [9]; k 1 = k c a t / K m = 1.57103 M -1- s -1 [10]. The value of kcat/K m for pseudomonal elastase presently observed is more than 10-fold higher than for those with kallikrein. It has been established that the Hageman factor activation with kallikrein was very much enhanced by the presence of a negatively charged surface [1,2]. Dextran sulfate (molecular mass 500 kDa) has been used as the negatively charged soluble substance for kinetic analysis of the Hageman factor activation [8-10]. The most sufficient effect of the negatively charged substance could be observed when Hageman factor and kallikrein both bound on the substance in a condensed manner [1]. In this case, the binding force among them is assumed to be a major ionic interaction. Therefore, it requires the following conditions; sufficient net negative charge and molecular size of the substance; a suitable molar ratio between the substance and the proteins; and low ionic strength of the environment. The values of the kinetic parameters observed in the present study for the activation with kallikrein are lower than those reported previously, such as, kcat = 5.7 s -1, g m = 5.1 • 10 -7 M and k c a t / K m = l . 1 2 " l O 7 M -1 "s -1 [9] and k I = kcat/g m = 5.34- 1 0 6 M - 1 . s-1 (with sulfatide instead of dextran sulfate [10]). It is probably attributable to the difference in ionic strength of the buffer used. We used a buffer with physiological ionic strength ( I = 0.14), while a buffer with a lower ionic strength, such as I = 0.075 or 0.10, respectively, was previously used. By using the low ionic strength buffer ( I = 0.075), we also observed much higher values for the kinetic parameters (Table I) as had been previously reported. Therefore, the enhancing effect of the negatively charged substrates is partial in tile physiological ionic strength and in the absence of high molecular mass kininogen, since only Hageman factor, and not kallikrein, might bind to dextran sulfate, as observed with sulfatide [10]. In the present study, dextran sulfate also enhanced the Hageman factor activation by pseudomonal elastase, to a maximum of 5-fold. In the case with the elastase, the molar ratio between dextran sulfate and Hageman factor (therefore, the density of the latter molecule on the former molecule) appears to be significantly important. The concentration of dextran sulfate,
10/~g/ml, under which the enhancement effect was still observed for the activation by kallikrein to the same extent as in the case of 3/~g/ml, inhibited the activation by the elastase (Fig. 5). Pseudomonal elastase with acidic molecular nature (pI is 5.9 [41]) might be prevented to interact with Hageman factor molecule on dextran sulfate, if a wide area with sulfated sites on this substance remained uncovered, keeping the negative charge. It is still a matter of controversy which the negatively charged natural substance in vivo is, although sulfatide [8], heparin, condroitin sulfate E [42] and others [2] have been suggested as candidates. We have examined the effect of pseudomonal endotoxin of commercial source on the Hageman factor activation by the elastase. However, we have not yet obtained the positive enhancing effect. The active Hageman factor moiety generated in the limited proteolysis by the elastase seemed to have the apparent molecular mass of 30 kDa (Fig. 2), and the mobility of this protein band in the SDS-polyacrylamide gel electrophoresis was indistinguishable from that of the fl-form activated Hageman factor. With the fl-form activated Hageman factor, the strong prekallikrein activating capacity, but lack of effect on the coagulation factor XI and subsequent intrinsic blood coagulation pathway, was demonstrated [2]. It was consistent with the result of our preliminary experiment, in which the elastase did not initiate coagulation in human plasma (unpublished data). Therefore, the Hageman factor activated by the elastase might work mainly as the initiator of the kallikrein-kinin pathway. Since an important role of the P. aeruginosa elastase in the pathogenesis of cystic fibrosis has been pronounced [23-25], it must be of importance to examine the state of the Hageman factor/kallikrein-kinin system in the patients of this genetic disease. P. aerugmosa alkaline proteinase negligibly activated Hageman factor in the present study. This data differents from that reported on the guinea pig Hageman factor activation. This proteinase activated guinea pig Hageman factor quite well, both in vitro and in vivo [20]. At the present time, we have no explanation for this species difference. It is a speculation that there might be a difference of an amino acid(s) in the sequence of the human and the guinea pig Hageman factor molecule, around the essential cleavage site for the activation.
Acknowledgements The authors are grateful to Dr. Tanase, Kumamoto University Medical School for the fruitful discussion. This work was supported in part by grants from Japanese Ministry of Education, Science and Culture.
239
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