Study of anticoagulant mechanism of low molecular weight heparin

THROMBOSIS RESEARCH 65; 801-808,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

STUDY OF ANTICOAGULANTMECHANISMOF LOW MOLECULARWEIGHTHEPARIN

Shuichiro Hamano*, Masahiko Nishiyama*, Hidetada Komatsu*, Hiroshi Mipata’, Shigeru Ikeda* and Nobuo Saknragawa** + Pharmacological Laboratories, Kissei Pharmaceutical Co. Ltd., Hotaka, Japan Clinical Laboratory Medicine, Toyama Medical and +*Department of Pharmaceutical University. Toyama, Japan (Received

159.1991; accepted in original form 9.12.1992 by Editor O.N. Ulutin) (Received by Executive Editorial Office 30.1.1992)

ABSTRACT The binding ability of low molecular weight heparin (FR-860). and conventional unfractionated heparin (UP-heparin) to factor Xa (F.Xa). thrombin and ATIK was investigated using FR-860- and UF-heparinSepharoses. FR-860 could not bind directly to F.Xa. FR-860 bound to thrombin and ATIII with stronger affinity to ATM than to thrombin. On the other hand, UF-heparin bound to F.Xa, thrombin and ATM with the strongest affinity to ATIlI followed by thrombin and F.Xa. AT III mediated the binding between F.Xa and FR-866 and accelerated the reaction between F.Xa and UF-heparin. On the other hand, ATM did not affect the binding between thrombin and FR-860 or UF-heparin. Diisopropyl fluorophosphate-treated thrombin inhibited the binding between ATM and FR-860. but not that between ATM and UF-heparin. These results suggest that the anti-F.Xa activity of FR-860 is mediated by AT KI. Furthermore, the difference of antithrombin activity between FR-860 and UF-heparin depends on the capability to form ternary complex of FR-860 or UF-heparin. ATM and thrombin.

INTRODUCTION It is known that conventional unfractionated heparin (UF-heparin. M.W.=lOOOO-15000). a sulfated glycosaminoglycan. converts antithrombin III (ATM) frow its progresslve form to au immediate form (1). This change of ATM exhibits antithrombotic properties. It has been reported that the low molecular weight heparin (LMW-heparin) Key

words

:

LMW-heparin, FR-860-Sepharose, thrombin. ATM 801

UP-heparin-Sepharose,

F.Xa,

802

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shows efficient antithrombotic properties by converting ATIU to an immediate form (2. 3). We also demonstrated the effects of LMW-heparin (FR-860, M.W. = 4400-5600). on a hemodialysis model (4) and on disseminated intravascular coagulation models (5). Furthermore, in our previous study, FR-860 showed an anti-factor Xa (F.Xal activity comparable to that of UF-heparin. But, FR-860 did not have an antithrombin activity as UF-heparin did (6. 7). This different activity between LMW-heparin and UF-heparin has been studied more detail. Holmer et al. (8) showed that various molecular weight heparin oligosaccharides exhibited different activity on blood coagulation factors. In this study, to explain the anticoagulant mechanism of FR-860. we investigated the binding ability of FR-860 and UF-heparin to F.Xa, thrombin and ATIK using FR-860- and UF-heparin-Sepharoses.

MATERIALS AND METHODS

Materials FR-860-Sepharose was coupled by reacting FR-860 (KabiVitrum) and epoxyactivated sepharose 6B (Pharmacia). UF-heparin-Sepharose was purchased from Pharmacia. F.Xa and thrombin were purified from rabbits plasma using DEAESepharose CL-6B (Pharmacia) and activated by Russell’s viper venom (Sigma) and F.Xa. respectively (9-11). ATIII was purified from rabbits plasma using UFheparin-Sepharose (12). Diisopropyl fluorophosphate-treated thrombin (DFPthrombin) was produced by incubating diisopropyl fluorophosphate (Sigma) with rabbit’s thrombin (13). Affinity chromatography F.Xa. thrombin or ATKI was applied to FR-860- and UF-heparin-Sepharoses for column chromatography. After rinsing with 50 mM Tris-EC1 buffer (pH 7.4). elution was performed using a 0 to 1 M NaCl linear gradient of the same buffer. One hundred y 1 of fractionated sample was mixed with 200 ~1 of synthetic substrates S-2222 (1 mM,Daiichi-Chemical) and S-2238 (1 mM.Daiichi-Chemical) to determine F.Xa and thrombin activities, respectively. AT IK activity was expressed as the antithrombin activity in the presence of an overdose of UF-heparin. Assay of the reaction between coagulation factors and FR-860- or UF-heparinSepharose Either F.Xa or thrombin was incubated for 5 min at room temperature as follows : A. only F.Xa or thrombin ; B. FR-860- or UF-heparin-Sepharose ; C. at ATKI ; D, FR-860- or UF-heparin-Sepharose with ATKI. After centrifugation 1500 x g for 2 min. the remaining activities of F.Xa and thrombin in the supernate were measured by mixing with 200 or1 of synthetic substrates S-2222 (1 mM) and S-2238 (1 mM). respectively. The remaining activity expresses the binding ability of FR-860 or UF-heparin to F.Xa and thrombin. Assay of the reaction between ATIU and FR-860- or UF-heparin-Sepharose in the presence of DFP-thrombin After FR-860- and UF-heparin-Sepharoses were treated with DFP-thrombin.

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each precipitate was incubated with ATllI for 5 min at room temperature. The remaining activity of ATIII in the supemate was expressed as the antithrombin activity in the presence of an overdose of BF-heparin. Statistics Statistical differences between the results of determined using Student’s t-test for unpaired data

each

experiment

were

RESULTS

The affinity of F.Xa, thrombin and ATliI to FR-860- and BF-heparin-Sepharoses Fig. 1 shows the typical fractionated pattern by FR-WO-Sepharose chromatography. F.Xa was rinsed out by the 50 mMTris-EC1 buffer. But, both thrombin and ATlU remained on the FR-860Sepharose column. In the elution performed by NaCl linear gradient, thrombin was eluted before ATlU. On the other hand, the typical fractionated pattern by UF-heparin-Sepharose chromatography is shown in Fig. 2. After binding to BF-heparin-Sepharose, F.Xa. thrombin and ATlil were eluted in the same order, i.e. F.Xa followed by thrombin and ATliI.

0.5

1.0

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0 b

%

%

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1.0

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M M 0.5 dl

2.5;

0.2

B

x" Ii C % a"

2 2z

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0.1

C

2

0

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0

10

20

30

40

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0

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60

70

80

90

100

FractionNo.

FIG. 1. Typical fractionated pattern by FR-860Sepharose chromatography. Rabbit’s F.Xa, thrombin or ATl8 was applied. After rinsing with 50 BJ@I Tris-EC1 (PH 7.4) up to fraction No. 50. elution was performed using a 0 to 1 M NaCl linear gradient. The activities of F.Xa, thrombin and ATl8 were measured using S-2222 and S-2238. Each mark indicates F.Xa (0). thrombin (0) and ATI (A). Effect of ATI on the reaction between coagulation factors UF-heparin-Sepharose The effect of ATI on the binding between F.Xa and FR-860- or

and FR-860- or

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I

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I

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I

I

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0

10

20

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70

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1

100

FractionNo.

FIG. 2. Typical fractionated pattern by UF-heparin-Sepharose chromatography. Rabbit’s F.Xa, thrombiu or ATIll was applied. After rinsing with 50 mMTris-EC1 (pH 7.4) up to fraction No. 50. elution was performed using a 0 to 1 M NaCl linear gradient. The activities of F.Xa, thrombin and ATH were measured using S-2222 and S-2238. Each mark indicates F.Xa (0). thrombin (0) and ATlU (A). UF-heparin-Sepharose was estimated by measuring the remaining activity of F.Xa in the supemate. As shown in Fig. 3, in the absence of ATlli (column A, B), non-binding F.Xa activity did not decrease at all. Namely, there is no complex between F.Xa and FR-860-Sepharose. On the other hand, F.Xa directly bound to UF-heparin-Sepharose without ATIU. and consequently non-binding F.Xa activity decreased to half. In the presence of ATiU (column C, D). however, non-binding F.Xa activity remarkably decreased. Therefore, AT Xi mediated the binding between F.Xa and FR-860Sepharose and accelerated the reaction between F.Xa and UF-heparin-Sepharose. Fig. 4 shows the effect of ATlU on the reaction between thrombin and FR-860or UF-heparin-Sepharose by measuring the non-binding activity of thrombin in the supemate. The non-binding thrombin activity significantly reduced in the presence of only FR-860- or only UF-heparin-Sepharoses (column A, B). thrombin directly bound to FR-860- or UF-heparin-Sepharose. Therefore, However, the binding abilities between thrombin and both heparins were not affected by the presence of ATliI (column C. D). Effect of DFP-thrombin on the reaction between ATJii and FR-860- or UF-heparinSepharose We investigated the possibility to form ternary complex of FR-860 or UF-heparin. ATliI and thrombin. Fig. 5 shows the effect of DFP-thrombin which has no binding site with ATlU on the reaction between ATlIi and FR-860- or UFheparin-Sepharose by measuring the remaining ATiU activity in the supemate.

805

MECHANISM OF LMW-HEPARIN

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FR-860

UF-heparin pco.oo1 1 1

L

p<0.001

p
1.0

1. 0.5

-

-

A

0

B

C

A

D

FIG. 3. Effectof ATIR on the reactionbetween F.Xa and FR-860-or IJF-heparinSepharose. F.Xa activitywas measured using the syntheticsubstrate S-2222. Each column indicatesthe mean ($-SS.E.) of 3 experiments.A: F.Xa only,B : F.Xa + FR-860-or IJF-heparin-Sepharose. C : F.Xa + ATIU.D: F.Xa + ATlU + FR-860or DF-heparin-Sepharose.

FR-860 r

UF-heparin pcO.col I

r=%

pcO.DOl 1

r

pc0.001 I

I

T

3.0

2.0

1.0

0 A

B

C

D

A

B

C

D

FIG. 4. Effect of ATIU on the reaction between thrombin and FR-866- or DF-heparin-Sepharose. Thrombin activity was measured using synthetic substrate S-2238.Each column indicatesthe mean (*SE.) of 3 experiments.A: thrombin only, B : tbrombin+FR-860- or DF-heparfn-Sepharose.C : thrombin+ ATIU.D : thrombin+ATllI+FR-860-or DF-heparin-Sepharose.

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Non-binding AT Ill in the supernate significantly increased in the presence of DFP-thrombin at 0.25-o. 5 U and FR-860~Sepharose. Therefore, DFP-thrombin inhibited the binding between ATllI and FR-86OSepharose. But, DFP-thrombin did not affect the reaction between ATHI and UF-heparin-Sepharose.

0.6 0.5 0.4 0.3 0.2 0.1 0’ AT III 0%

AT III 100%

0

0.25

0.5

Amount of DFP-thrombin

0.75

1

(U)

FIG. 5. Effect of DFP-thrombin on the reaction between ATlU and FR-860- or BF-heparin-Sepharose. Amount of DFP-thrombln represents as the unit equivalent to thrombin. AT JR activity was measured as the antithrombln activity. Each column and each point indicates the mean ( + SE.) of 3-4 experiments. Each mark indicates FR-860-Sepharose ( 0 1 and BF-heparinSepharose (0). ATlU 100 % : added ATIU to the supernate. ATlU 0 % : without ATIU in the supernate. + : significant difference from the point without DFPthrombin (0) at p-&05.

DISCUSSION By FR-860- and BF-heparin-Sepharose chromatography in this study, ATlU showed the highest affinity to FR-860 and BF-heparin compared with thrombin and F.Xa. Therefore, it is concluded that ATIU strongly forms complexes with FR-860 and BF-heparin. It has been reported that the anti-F.Xa activity is dependent on the binding capability between ATlU and heparins (8. 14). As shown in Fig. 3, F.Xa bound to FR-860- or DF-heparin-Sepharose in the presence of ATIll. Namely, ATIU mediated the binding of F.Xa to FR-860 and accelerated the reaction between F.Xa and BF-heparin. BF-heparin but not FR-860 directly bound to F.Xa. It appears that the ternary complex of ATIU - BF-heparin - F.Xa are formed. But, kinetic analysis has suggested that the ternary complex formation is only of minor importance for the acceleration of anti-F.Xa activity (15. 16). Furthermore in our previous study, FR-860 shows an anti-F.Xa activity comparable to BF-heparin (6, 7). These results support that the

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anti-F&r activity of FR-860 and UF-heparin depends on the binding with ATlK, but not on forming ternary complex. Reportedly, UF-heparin shows antithrombin activity by accelerating the binding between ATM and thrombin (1). In our previous study, UF-heparin but not FR-860 shows obvious autithrombin activity (6, 7). It has been reported that the formation of ternary complex is required for the acceleration of the binding between ATM and thrombin (14. 17). As shown in Fig. 4. FR-860 and UF-heparin bound to thrombin directly and in the presence of AT III. But, DFP-thrombin which. has no binding site for ATIll (13) inhibited the binding between ATM and FR-860. but not between ATM and UF-heparin (Fig. 5). These results suggest that UF-heparin is able to form a ternary complex with ATM and thrombin. On the other hand, although FR-860 individually bind to ATIII or thrombin, FR-860 hardly forms the ternary complex with ATI and thrombin because of the size of its low molecular weight. FR-860 may binds to thrombin through ATM without forming the ternary complex. In conclusion, the anti-F.Xa activity of FR-860 is dependent on the binding with AT III, and it is hardly to fom the ternary complex of FR-860. ATM and thrombin. On the other hand, the antithrombin activity of UF-heparin is exhibited by forming the ternary complex.

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