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

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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.

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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

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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.

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Study of anticoagulant mechanism of low molecular weight heparin.

The binding ability of low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) to factor Xa (F.Xa), thrombin and A...
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