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Specificity of sulfated polysaccharides to accelerate the inhibition of activated protein C by protein C inhibitor
THROMBOSIS RESEARCH 48; 179-185, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
SPECIFICITY OF SULFATED POLYSACCRARIDES TO ACCELERATE THE INHIBITION OF ACTIVATED PROTEIN C BY PROTEIN C INHIBITOR
Yoshiaki Kazama$ Masahiro Niway Ryoichi Yamagishit Kaoru Takahashit b Nobuo Sakuragawaf and Takehiko Koide aCentral Clinical Laboratory, Toyama Medical and Pharmaceutical University Toyama 930-01, Japan b Department of Biochemistry, Niigata University School of Medicine, Niigata 951, Japan (Received 8.1.1987; Accepted in revised form 13.8.1987 by Editor M. Matsuda)
ABSTRACT The ability of various sulfated polysaccharides to activate protein C inhibitor (PCI) and the effect of molecular weight (Mr) and sulfur content of dextran sulfates were investigated. Besides dextran sulfate, highly sulfated polysaccharides such as chondroitin polysulfates 1 and 5, and pentosan polysulfate were more active than heparin in enhancing the activated protein C inhibition by PCI. The molecular weight and the sulfur content of dextran sulfate were critical for the second-order rate constant of the reaction and for the optimal concentration of the polysaccharide, respectively. These results suggest that the carboxyl groups of polysaccharides are not necessarily required, but some sulfate groups within polymers may play a critical role in the interaction with PCI.
INTRODUCTION Protein C inhibitor (PCI) is the only known inhibitor of activated protein C (APC) in human plasma (1). PC1 was originally described in 1980 by Marlar and Griffin (Z), and first purified by Suzuki -et al. in 1983 (1). PC1 inhibits APC by forming a 1:l molar complex. Recently, the amino acid sequence of human PC1 deduced from the cDNA sequence was determined (3). Key words: Protein C inhibitor, Activated protein C, sulfated polysaccharide, dextran sulfate 179
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PCI, like antithrombin III (AT III) and heparin cofactor II (HC II), is the one of the heparin-dependent plasma inhibitors, since the rate of APC inhibition by PC1 is accelerated by heparin (4). Although the levels of heparin required to activate PC1 are considerably higher than those used clinically, the evidence that PC1 inhibits thrombin, factor Xa (4) and urokinase (5) as well as APC suggests that PC1 is the one of the important plasma protease inhibitors that regulate blood coagulation and fibrinolysis. The exact mechanism involved in the regulation of APC including the role of PC1 and the clinical significance of this inhibitor have not been clarified yet. Here, we report the activation of PC1 by various sulfated polysaccharides including synthetic ones, which we found that some synthetic polysaccharides with a high sulfur cvntent activated PCT.
MATERIALS
AND METHODS
Materials: The following materials were used: Heparin (unfractionated standard heparin; grade 1, 160 USP units/mg) and polybrene from Sigma Chemical Co. (St. Louis, MO); pentcsan polysulfate [Mr=2 kDa, sulfur (S) content=17.5%] from Bennechemie (Munich, Germany); dermatan sulfate, heparan sulfate, chondroitin polysulfate 1 (Mr=lO kDa, S content=15.4%), and chondroitin polysulfate 5 (Mr=lO kDa, S content=12.6%) from Seikagaku Kogyo (Tokyo); low molecular weight (Mr) heparin (Kabi-2165, Mr=4-5 kDa) from KabiVitrum AB (Stockholm, Sweden), Boc-Leu-Ser-Thr-Arg-MCA from Protein Research Foundation (Osaka); dextran sulfate (Mr=500 kDa, S content=17+1%) from Pharmacia Fine Chemicals (Uppsala, Sweden) and dextran sulfates (Mr=420 kDa, S content=17.8% & 3.4%; Mr=4 kDa, S content=17-20% & 3-6%) generously supplied by Dr. H. Tamura (Kowa Co. Ltd., Tckyo). In this paper, dextran sulfate refers to a preparation of Mr=500 kDa, S content=17+1%, unless otherwise specified. Proteins: Human APC was kindly provided by Dr. Walter Kisiel (University of New Mexico). Human PC1 was purified from fresh frozen plasma as described by Suzuki -et al. (1, 6), which included barium citrate adsorption, ammonium sulfate fractionation (50-70X saturation), dextran sulfateSepharose 4B chromatography, DEAE-Sephacel chromatography, and heparinSepharose CL-6B chromatography. Kinetics of the inhibition of APC by PCI: Reactions were performed at 37'C in polystylene tubes. The reaction mixture contained PC1 (1.12 X 10s7 M), sulfated polysaccharide (O-1000 pg/ml), and APC (1.08 X 10m8 M) in 150 ~1 of 0.05 M Tris-HCl (pH 8.0) containing 0.1% bovine serum albumin (BSA) and 0.1 M NaCl. APC was added last to initiate the reaction. After incubation for 1 min in the presence of or for 15 min in the absence of sulfated polysaccharides, 2.5 ml of 100 nM Boc-Leu-Ser-Thr-Arg-MCA (peptide-MCA) dissolved in 0.05 M Tris-HCl (pH 8.0) containing 0.1 M NaCl, 2 mM CaC12, and polybrene (0.4 mg) (substrate buffer) was added to the mixture. Substrate hydrolysis was terminated after 3 min by the addition of 0.5 ml of 50% acetic acid. Subsequently, the fluorescence of 7-amino-4-methyl-coumarin liberated was measured with excitation at 380 nm and emission at 440 nm. Sulfated polysaccharides and polybrene did not give any significant effect on the hydrolysis of the substrate by APC under these conditions. The inhibition of APC by PC1 proceeds as a second-order reaction
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shown by the following equation: k
PC1 + APC
I PCI*APC complex
where k is the second-order rate constant for the reaction. Under the conditions used in which the concentration of PC1 is in large excess of that of APC, the equation for a pseudo-first-order reactions can be applied and k is calculated from the equation:
k=
1 t [PCI]
WC10 In [APC],
where [APC]O=initial APC concentration (1.08 X 10 -8 M), [APClt= residual APC concentration at time t, [PCI]=concentration of PC1 (1.12 X lo-' M).
RESULTS AND DISCUSSION The ability of various sulfated polysaccharides to increase the rate of APC inhibition by PCI was investigated. Fig. 1 shows the residual APC
Sulfatedpolysaccharides( pg/ml) Fig. 1 Activation of PSI by sulfated polysaccharides: In 150 ~1 of reaction mixture, APC (1.08 X 10Wu M) was incubated at 3°C with PC1 (5.6 X 10-U M) in the presence of either unfractionated heparin (O), dermatan sulfate (A), heparan sulfate (V), dextran sulfate (O), CPS-1 (Cl), CPS-5 (m), pentosan polysulfa_te (*), or low Mr heparin (*). After 3 min, 1.5 ml of 100 nM peptideMCA in the substrate buffer without polybrene was added. After an additional 5 min of incubation, 1.5 ml of 50 % acetic acid was added to stop the reaction The residual APC activity was determined as described in "MATERIALS AND METHODS".
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activity observed after a 3-min incubation of APC (1.08 X 10s8 M) with PC1 (5.6 X 10m8 M) in the presence of various sulfated polysaccharides. Heparin and dextran sulfate (DS) but not heparan sulfate nor dermatan sulfate accelerated the APC inhibition by PCI. Dextran sulfate, a synthetic polymer of sulfated glucose containing no carboxyl groups, was most active among the sulfated polysaccharides tested in the present study, accelerating the APC inhibition by PC1 at least two times more than heparin. These results are consistent with those reported by Suzuki -et al. (6). Besides heparin and dextran sulfate, chondroitin polysulfate 1 (CPS-1), chondroitin polysulfate 5 (CPS-5), pentosan polysulfate, and low Mr heparin activated PC1 effectively. It is noteworthy that both CPS-1 and CPS-5 as well as pentosan polysulfate which lack iduronic acid but contain increased amounts of sulfate groups were more active than heparin in enhancing the APC inhibition by PCI, in contrast to the result by Suzuki (7) that chondroitin sulfates A and C had no appreciable effects on the reaction. Pentosan polysulfate is a semisynthetic polymer prepared by sulfation of pentosan extracted from beech wood shavings and considered to be a good activator of HC II (8). These results suggest that some sulfate groups within polymers may play a critical role but the carboxyl groups of polymers are not necessarily required in the interaction with PCI. Fig. 2 shows the effects of concentrations of these sulfated polysaccharides on the second-order rate constant of the APC inhibition by PCI. In
St_dfatecipdysaccharides
( D9 /ml )
Fig. 2 Kinetics of APC inhibition by PC1 in the prese ce of sulfated polysaccharides: -8 In 150 pl of reaction mixture, APC (1.08 X 10 M) was incubated at 37'C with PC1 (1.12 x 10-7 M) in the presence of either unfractionated heparin (O), dextran sulfate (o), CPS-1 (Cl), CPS-5 (m), pentosan polysulfate (*), or lcw Mr heparin (*). After 1 min, 2.5 ml of 100 nM peptide-MCA in the substrate buffer was added. After an additional 3-mir-rincubation, 0.5 ml of 50% acetic acid was added. The residual APC activity was determined and the second-order rate constant (k) was calculated as described in "MATERIALS AND METHODS".
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the absence of sulfated polysaccharides, the reaction was slow, giving the rate constant of 2.6 X lo5 M-'min-l. However, it was accelerated about 60-fold in the presence of 2-10 pg/ml of each sulfated polysaccharide except for heparin, giving the maximum rate constant of 1.5-1.7 X lo7 M-lmin-1. As much as 30-100 pgfml of unfractionated heparin was required to obtain the maximal 20-fold acceleration, while an optimal concentration of low Mr heparin to a similar extent of the maximal acceleration was about 10 times less than that of unfractionated heparin. These effects of sulfated polysaccharides on the APC inhibition by PC1 are generally similar to those in the thrombin inhibition by AT III and HC II (9). However, it should be pointed out that, different from the cases of AT III and HC II, heparin is a rather weak activator of PCI. Since dextran sulfate showed the highest efficiency as an activator of PCI, we further investigated the relationship between the activity of dextran sulfate and its molecular size and/or sulfur content. The effects of high and low Mr dextran sulfates (DS-420 kDa and DS-4 kDa, respectively), each with two different sulfur contents (3-6% and 17-20%), on the second-order rate constant of the APC inhibition by PC1 are shown in Fig. 3. The maximum second-order rate constants in the resence of DS-420 kDa and DS-4 kDa were 1.6 X lo7 M-lmin-1 and 0.9-1.1 X 107 M-lmin-1 respectively regardless of sulfur contents. On the other hand, dextran &fate with a iulfur content of 17-20% and 3-6% gave an optimal range of l-10 pg/ml and lo-100 pg/ml, respectively, regardless of its molecular size. These results indicate that the Mr and the sulfur content of dextran sulfate were critical for the second-order rate constants of the reaction and for the optimal concentration of the polysaccharide, respectively.
OC
lo-* 10-l 1 IO IO2 Sulfated polysaccharides ( 1-19 / ml )
IO3
Fig. 3 Kinetics of APC inhibition by PC1 in the presence of dextran sulfates: In 150 pl of reaction mixture, APC (1.08 X lo-8 M) was incubated with PC1 (1.12 X lo-' M) in the presence at 37°C: OMr=420 kDa, sulfur (S) content =3.4%; ?? Mr=420 kDa, S content=17.8%; OMr=4 kDa, S content=3-6%; WMr= 4 kDa, S content=l7-20%. The residual APC activity was determined and the second-order rate constant (k) was calculated as described in "MATERIALS AND METHODS".
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Dextran sulfate has previously been reported by Suzuki (6) as a potent activator of the APC inhibition by PCI. However, his results were somehow different from ours. He showed that dextran sulfates of Mr&lO,OOO (DS 10, 50 and 200 kDa) with about 18% sulfur content accelerated the APC inhibition by PCI, among which DS 10 kDa was most active, followed by DS 200 kDa, and those of Mr&7,500 (DS 3.5 and 7.5 kDa) with 5.3% sulfur content were essentially inactive. However, our results showed that dextran sulfates with high Mr gave high k values and those with high sulfur content gave a maximal k value at lower concentration. DS-420 kDa and DS-4 kDa increased k about 60-fold and 40-fold, respectively, and in either case, dextran sulfate with 17-20% sulfur was 10 times more effective than that with 3-6% sulfur, and DS-4 kDa with 3-6% sulfur content was still as active as unfractionated heparin (Figs. 2 and 3). As the concentrations of sulfated polysaccharides increased about the optimal levels, their effectiveness in accelerating APCfPCI reaction appears to decrease (Figs. 2 and 3). This phenomenon is compatible with those observed in the case of thrombin inhibition by AT III and HC II in the presence of heparin, and that by HC II in the presence of dermatan sulfate (10). These observations are indicative of a template model in which both enzyme and inhibitor have to bind to the same molecule of sulfated polysaccharide for activation to occur (11). To elucidate the exact mechanism involved in the reactions of enzymes and inhibitors associated with sulfated polysaccharides, further studies will be needed.
ACKNOWLEDGEMENTS We thank Dr. Walter Kisiel, University of New Mexico for kindly providing human APC, and Dr. Hideaki Tamura, Kowa Co. Ltd. (Tokyo) for kindly providing dextran sulfates.
REFERENCES 1. SUZUKI, K., NISHIOKA, J. and HASHIMOTO, S. Protein C inhibitor: purification from human plasma and characterization. J. Biol. Chem., 9, 163-168, 1983. 2. MARLAR, R. A. and GRIFFIN, J. H. combined Factor V/VIII deficiency 1189, 1980.
Deficiency of protein C inhibitor in disease. J. Clin. Invest., 6&, 1186-
3. SUZUKI, K., DEYASHIKI, Y., NISHIOKA, J., KIJRACHI, K., AKIRA, M., YAMAMOTO, S. and HASHIMOTO, S. Characterization of a cDNA for human protein C inhibitor: a new member of the plasma serine protease inhibitor superfamily. J. Biol. Chem., 262, 611-616,1987. 4. SUZUKI, K., NISHIOKA, J. and HASHIMOTO, S. Mechanism of inhibition of activated protein C by protein C inhibitor. J. Biochem. (Tokyo), 95, 187-195, 1984.
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5.
HEEB, M. J., ESPANA, F., GEIGER, M., COLLEN, D., STUMP, D. C. and GRIFFIN, J. H. Immunological similarities between plasma and urinary protein C inhibitors (PCIs) and urinary urokinase inhibitor (UKI). (abstr.). Thromb. Haemostas., 58, 277, 1987.
6.
SUZUKI, K. Biochemistry and physiology of protein C inhibitor. In: Protein C, biological and medical aspects. Witt, I. (Ed.), BerlinNew York, Walter de Gruyter, 1985, pp 43-58.
7.
SUZUKI, K. Activated protein C inhibitor. 154-161, 1984.
8.
SCULLY, M. F. and KAKKAR, V. V. Identification of heparin cofactor II as the principal plasma cofactor for the antithrombotic activity of pentosan polysulphate (SP 54). Thromb. Res., 36, 187-194, 1984.
9.
YAMAGISHI, R., NIWA, M., KONDO, S., SAKURAGAWA, N. and KOIDE, T. Purification and biological property of heparin cofactor II: activation of heparin cofactor II and antithrombin III by dextran sulfate and various glycosaminoglycans. lhromb. Res., 36, 633-642, 1984.
185
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10. TOLLEFSEN, D. M., PESTKA, C. A. and MONAFO, W. J. Activation of heparin cofactor II by dermatan sulfate. J. Biol. Chem., 258, 6713-6716, 1983. 11. GRIFFITH, M. J. Kinetics of the heparin-enhanced antithrombin III/thrombin reaction: evidence for a template model for the mechanism of action of heparin. J. Biol. Chem., 257, 7360-7365, 1982.
SPECIFICITY OF SULFATED POLYSACCRARIDES TO ACCELERATE THE INHIBITION OF ACTIVATED PROTEIN C BY PROTEIN C INHIBITOR
Yoshiaki Kazama$ Masahiro Niway Ryoichi Yamagishit Kaoru Takahashit b Nobuo Sakuragawaf and Takehiko Koide aCentral Clinical Laboratory, Toyama Medical and Pharmaceutical University Toyama 930-01, Japan b Department of Biochemistry, Niigata University School of Medicine, Niigata 951, Japan (Received 8.1.1987; Accepted in revised form 13.8.1987 by Editor M. Matsuda)
ABSTRACT The ability of various sulfated polysaccharides to activate protein C inhibitor (PCI) and the effect of molecular weight (Mr) and sulfur content of dextran sulfates were investigated. Besides dextran sulfate, highly sulfated polysaccharides such as chondroitin polysulfates 1 and 5, and pentosan polysulfate were more active than heparin in enhancing the activated protein C inhibition by PCI. The molecular weight and the sulfur content of dextran sulfate were critical for the second-order rate constant of the reaction and for the optimal concentration of the polysaccharide, respectively. These results suggest that the carboxyl groups of polysaccharides are not necessarily required, but some sulfate groups within polymers may play a critical role in the interaction with PCI.
INTRODUCTION Protein C inhibitor (PCI) is the only known inhibitor of activated protein C (APC) in human plasma (1). PC1 was originally described in 1980 by Marlar and Griffin (Z), and first purified by Suzuki -et al. in 1983 (1). PC1 inhibits APC by forming a 1:l molar complex. Recently, the amino acid sequence of human PC1 deduced from the cDNA sequence was determined (3). Key words: Protein C inhibitor, Activated protein C, sulfated polysaccharide, dextran sulfate 179
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PCI, like antithrombin III (AT III) and heparin cofactor II (HC II), is the one of the heparin-dependent plasma inhibitors, since the rate of APC inhibition by PC1 is accelerated by heparin (4). Although the levels of heparin required to activate PC1 are considerably higher than those used clinically, the evidence that PC1 inhibits thrombin, factor Xa (4) and urokinase (5) as well as APC suggests that PC1 is the one of the important plasma protease inhibitors that regulate blood coagulation and fibrinolysis. The exact mechanism involved in the regulation of APC including the role of PC1 and the clinical significance of this inhibitor have not been clarified yet. Here, we report the activation of PC1 by various sulfated polysaccharides including synthetic ones, which we found that some synthetic polysaccharides with a high sulfur cvntent activated PCT.
MATERIALS
AND METHODS
Materials: The following materials were used: Heparin (unfractionated standard heparin; grade 1, 160 USP units/mg) and polybrene from Sigma Chemical Co. (St. Louis, MO); pentcsan polysulfate [Mr=2 kDa, sulfur (S) content=17.5%] from Bennechemie (Munich, Germany); dermatan sulfate, heparan sulfate, chondroitin polysulfate 1 (Mr=lO kDa, S content=15.4%), and chondroitin polysulfate 5 (Mr=lO kDa, S content=12.6%) from Seikagaku Kogyo (Tokyo); low molecular weight (Mr) heparin (Kabi-2165, Mr=4-5 kDa) from KabiVitrum AB (Stockholm, Sweden), Boc-Leu-Ser-Thr-Arg-MCA from Protein Research Foundation (Osaka); dextran sulfate (Mr=500 kDa, S content=17+1%) from Pharmacia Fine Chemicals (Uppsala, Sweden) and dextran sulfates (Mr=420 kDa, S content=17.8% & 3.4%; Mr=4 kDa, S content=17-20% & 3-6%) generously supplied by Dr. H. Tamura (Kowa Co. Ltd., Tckyo). In this paper, dextran sulfate refers to a preparation of Mr=500 kDa, S content=17+1%, unless otherwise specified. Proteins: Human APC was kindly provided by Dr. Walter Kisiel (University of New Mexico). Human PC1 was purified from fresh frozen plasma as described by Suzuki -et al. (1, 6), which included barium citrate adsorption, ammonium sulfate fractionation (50-70X saturation), dextran sulfateSepharose 4B chromatography, DEAE-Sephacel chromatography, and heparinSepharose CL-6B chromatography. Kinetics of the inhibition of APC by PCI: Reactions were performed at 37'C in polystylene tubes. The reaction mixture contained PC1 (1.12 X 10s7 M), sulfated polysaccharide (O-1000 pg/ml), and APC (1.08 X 10m8 M) in 150 ~1 of 0.05 M Tris-HCl (pH 8.0) containing 0.1% bovine serum albumin (BSA) and 0.1 M NaCl. APC was added last to initiate the reaction. After incubation for 1 min in the presence of or for 15 min in the absence of sulfated polysaccharides, 2.5 ml of 100 nM Boc-Leu-Ser-Thr-Arg-MCA (peptide-MCA) dissolved in 0.05 M Tris-HCl (pH 8.0) containing 0.1 M NaCl, 2 mM CaC12, and polybrene (0.4 mg) (substrate buffer) was added to the mixture. Substrate hydrolysis was terminated after 3 min by the addition of 0.5 ml of 50% acetic acid. Subsequently, the fluorescence of 7-amino-4-methyl-coumarin liberated was measured with excitation at 380 nm and emission at 440 nm. Sulfated polysaccharides and polybrene did not give any significant effect on the hydrolysis of the substrate by APC under these conditions. The inhibition of APC by PC1 proceeds as a second-order reaction
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shown by the following equation: k
PC1 + APC
I PCI*APC complex
where k is the second-order rate constant for the reaction. Under the conditions used in which the concentration of PC1 is in large excess of that of APC, the equation for a pseudo-first-order reactions can be applied and k is calculated from the equation:
k=
1 t [PCI]
WC10 In [APC],
where [APC]O=initial APC concentration (1.08 X 10 -8 M), [APClt= residual APC concentration at time t, [PCI]=concentration of PC1 (1.12 X lo-' M).
RESULTS AND DISCUSSION The ability of various sulfated polysaccharides to increase the rate of APC inhibition by PCI was investigated. Fig. 1 shows the residual APC
Sulfatedpolysaccharides( pg/ml) Fig. 1 Activation of PSI by sulfated polysaccharides: In 150 ~1 of reaction mixture, APC (1.08 X 10Wu M) was incubated at 3°C with PC1 (5.6 X 10-U M) in the presence of either unfractionated heparin (O), dermatan sulfate (A), heparan sulfate (V), dextran sulfate (O), CPS-1 (Cl), CPS-5 (m), pentosan polysulfa_te (*), or low Mr heparin (*). After 3 min, 1.5 ml of 100 nM peptideMCA in the substrate buffer without polybrene was added. After an additional 5 min of incubation, 1.5 ml of 50 % acetic acid was added to stop the reaction The residual APC activity was determined as described in "MATERIALS AND METHODS".
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activity observed after a 3-min incubation of APC (1.08 X 10s8 M) with PC1 (5.6 X 10m8 M) in the presence of various sulfated polysaccharides. Heparin and dextran sulfate (DS) but not heparan sulfate nor dermatan sulfate accelerated the APC inhibition by PCI. Dextran sulfate, a synthetic polymer of sulfated glucose containing no carboxyl groups, was most active among the sulfated polysaccharides tested in the present study, accelerating the APC inhibition by PC1 at least two times more than heparin. These results are consistent with those reported by Suzuki -et al. (6). Besides heparin and dextran sulfate, chondroitin polysulfate 1 (CPS-1), chondroitin polysulfate 5 (CPS-5), pentosan polysulfate, and low Mr heparin activated PC1 effectively. It is noteworthy that both CPS-1 and CPS-5 as well as pentosan polysulfate which lack iduronic acid but contain increased amounts of sulfate groups were more active than heparin in enhancing the APC inhibition by PCI, in contrast to the result by Suzuki (7) that chondroitin sulfates A and C had no appreciable effects on the reaction. Pentosan polysulfate is a semisynthetic polymer prepared by sulfation of pentosan extracted from beech wood shavings and considered to be a good activator of HC II (8). These results suggest that some sulfate groups within polymers may play a critical role but the carboxyl groups of polymers are not necessarily required in the interaction with PCI. Fig. 2 shows the effects of concentrations of these sulfated polysaccharides on the second-order rate constant of the APC inhibition by PCI. In
St_dfatecipdysaccharides
( D9 /ml )
Fig. 2 Kinetics of APC inhibition by PC1 in the prese ce of sulfated polysaccharides: -8 In 150 pl of reaction mixture, APC (1.08 X 10 M) was incubated at 37'C with PC1 (1.12 x 10-7 M) in the presence of either unfractionated heparin (O), dextran sulfate (o), CPS-1 (Cl), CPS-5 (m), pentosan polysulfate (*), or lcw Mr heparin (*). After 1 min, 2.5 ml of 100 nM peptide-MCA in the substrate buffer was added. After an additional 3-mir-rincubation, 0.5 ml of 50% acetic acid was added. The residual APC activity was determined and the second-order rate constant (k) was calculated as described in "MATERIALS AND METHODS".
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the absence of sulfated polysaccharides, the reaction was slow, giving the rate constant of 2.6 X lo5 M-'min-l. However, it was accelerated about 60-fold in the presence of 2-10 pg/ml of each sulfated polysaccharide except for heparin, giving the maximum rate constant of 1.5-1.7 X lo7 M-lmin-1. As much as 30-100 pgfml of unfractionated heparin was required to obtain the maximal 20-fold acceleration, while an optimal concentration of low Mr heparin to a similar extent of the maximal acceleration was about 10 times less than that of unfractionated heparin. These effects of sulfated polysaccharides on the APC inhibition by PC1 are generally similar to those in the thrombin inhibition by AT III and HC II (9). However, it should be pointed out that, different from the cases of AT III and HC II, heparin is a rather weak activator of PCI. Since dextran sulfate showed the highest efficiency as an activator of PCI, we further investigated the relationship between the activity of dextran sulfate and its molecular size and/or sulfur content. The effects of high and low Mr dextran sulfates (DS-420 kDa and DS-4 kDa, respectively), each with two different sulfur contents (3-6% and 17-20%), on the second-order rate constant of the APC inhibition by PC1 are shown in Fig. 3. The maximum second-order rate constants in the resence of DS-420 kDa and DS-4 kDa were 1.6 X lo7 M-lmin-1 and 0.9-1.1 X 107 M-lmin-1 respectively regardless of sulfur contents. On the other hand, dextran &fate with a iulfur content of 17-20% and 3-6% gave an optimal range of l-10 pg/ml and lo-100 pg/ml, respectively, regardless of its molecular size. These results indicate that the Mr and the sulfur content of dextran sulfate were critical for the second-order rate constants of the reaction and for the optimal concentration of the polysaccharide, respectively.
OC
lo-* 10-l 1 IO IO2 Sulfated polysaccharides ( 1-19 / ml )
IO3
Fig. 3 Kinetics of APC inhibition by PC1 in the presence of dextran sulfates: In 150 pl of reaction mixture, APC (1.08 X lo-8 M) was incubated with PC1 (1.12 X lo-' M) in the presence at 37°C: OMr=420 kDa, sulfur (S) content =3.4%; ?? Mr=420 kDa, S content=17.8%; OMr=4 kDa, S content=3-6%; WMr= 4 kDa, S content=l7-20%. The residual APC activity was determined and the second-order rate constant (k) was calculated as described in "MATERIALS AND METHODS".
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Dextran sulfate has previously been reported by Suzuki (6) as a potent activator of the APC inhibition by PCI. However, his results were somehow different from ours. He showed that dextran sulfates of Mr&lO,OOO (DS 10, 50 and 200 kDa) with about 18% sulfur content accelerated the APC inhibition by PCI, among which DS 10 kDa was most active, followed by DS 200 kDa, and those of Mr&7,500 (DS 3.5 and 7.5 kDa) with 5.3% sulfur content were essentially inactive. However, our results showed that dextran sulfates with high Mr gave high k values and those with high sulfur content gave a maximal k value at lower concentration. DS-420 kDa and DS-4 kDa increased k about 60-fold and 40-fold, respectively, and in either case, dextran sulfate with 17-20% sulfur was 10 times more effective than that with 3-6% sulfur, and DS-4 kDa with 3-6% sulfur content was still as active as unfractionated heparin (Figs. 2 and 3). As the concentrations of sulfated polysaccharides increased about the optimal levels, their effectiveness in accelerating APCfPCI reaction appears to decrease (Figs. 2 and 3). This phenomenon is compatible with those observed in the case of thrombin inhibition by AT III and HC II in the presence of heparin, and that by HC II in the presence of dermatan sulfate (10). These observations are indicative of a template model in which both enzyme and inhibitor have to bind to the same molecule of sulfated polysaccharide for activation to occur (11). To elucidate the exact mechanism involved in the reactions of enzymes and inhibitors associated with sulfated polysaccharides, further studies will be needed.
ACKNOWLEDGEMENTS We thank Dr. Walter Kisiel, University of New Mexico for kindly providing human APC, and Dr. Hideaki Tamura, Kowa Co. Ltd. (Tokyo) for kindly providing dextran sulfates.
REFERENCES 1. SUZUKI, K., NISHIOKA, J. and HASHIMOTO, S. Protein C inhibitor: purification from human plasma and characterization. J. Biol. Chem., 9, 163-168, 1983. 2. MARLAR, R. A. and GRIFFIN, J. H. combined Factor V/VIII deficiency 1189, 1980.
Deficiency of protein C inhibitor in disease. J. Clin. Invest., 6&, 1186-
3. SUZUKI, K., DEYASHIKI, Y., NISHIOKA, J., KIJRACHI, K., AKIRA, M., YAMAMOTO, S. and HASHIMOTO, S. Characterization of a cDNA for human protein C inhibitor: a new member of the plasma serine protease inhibitor superfamily. J. Biol. Chem., 262, 611-616,1987. 4. SUZUKI, K., NISHIOKA, J. and HASHIMOTO, S. Mechanism of inhibition of activated protein C by protein C inhibitor. J. Biochem. (Tokyo), 95, 187-195, 1984.
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5.
HEEB, M. J., ESPANA, F., GEIGER, M., COLLEN, D., STUMP, D. C. and GRIFFIN, J. H. Immunological similarities between plasma and urinary protein C inhibitors (PCIs) and urinary urokinase inhibitor (UKI). (abstr.). Thromb. Haemostas., 58, 277, 1987.
6.
SUZUKI, K. Biochemistry and physiology of protein C inhibitor. In: Protein C, biological and medical aspects. Witt, I. (Ed.), BerlinNew York, Walter de Gruyter, 1985, pp 43-58.
7.
SUZUKI, K. Activated protein C inhibitor. 154-161, 1984.
8.
SCULLY, M. F. and KAKKAR, V. V. Identification of heparin cofactor II as the principal plasma cofactor for the antithrombotic activity of pentosan polysulphate (SP 54). Thromb. Res., 36, 187-194, 1984.
9.
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