Two heparin families (SM- and FM- heparins) have been fractionated with barium by selective precipitation at different temperatures. Fragments were obtained ...
THROMBOSIS RESEARCH 40; 49-58, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.
STRUCTURAL STUDIES AND "IN VIVO" AND "IN VITRO" PHARMACOLOGICAL ACTIVITIES OF HEPARIN FRACTIONS AND FRAGMENTS PREPARED BY CHEMICAL AND ENZYMIC DEPOLIMERIZATION.
B. Osima, B. Parma, C.P. Dietrich*, H.K. Takahashi* and H.B. Nader*. Research Laboratories, Corlo, Modena, Italy, and Opocrin Paulista de Medicins, Departamento de Bioquimica, Escola Sgo Paulo, C.P.20372, S.P., Brazil. P.
Bianchini,
(Received 5.12.1984; Accepted in revised form 10.5.1985 by Editor S.E. Lasker) (Received in final form by the Executive Editorial Office 15.7.1985)
ABSTRACT Two heparin families (SM- and FM- heparins) have been fractionated with barium by selective precipitation at different Fragments were obtained from temperatures. these two fractions by enzymic and chemical degradation. Some structural features and pharmacological activities of FM-and SM-heparins as well as the fragments are now reported. The fragments, prepared by ascorbate/ H702 oxidation (M.W. 4,500) or heparitinase II (M.W. 5,500), are enriched with trisulfated disaccharide units whereas heparin (M.W.12,500) and FMheparin (M.W. 10,200) contain also N-sulfated and Nacetylated disaccharides. The chemical and enzymic fragments show l/3 to l/5 of the anticoagulant activity of heparin by the USP and APTT methods. The LPL releasing activity is also low in the fragments as well as the AXa activity measured by the chromogenic method. On the other hand the AXa activity measured by the Yin and Wessler procedure is two times higher in the fragments when compared to heparin.The ,fragments resulting from heparitinase II heparin after degradation and ascorbate/H202 oxidation were rich in trisulfated disaccharide units whereas heparin and FMheparin contained also measurable amounts of monosulfated and N-acetylated disaccharide units. The mechanism of ascorbate oxidation and the structural activities of requirements for the pharmacological heparin is discussed in view of these and other findings. depolymerization,cheKey words: Henarin fractionation,enzymic mica1 degradat?on,anticoagulant,antilypemic. 49
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INTRODUCTION seems The anticoagulant and antithrombotic action of heparin to be related with its accelerating effect on the interaction of antithrombin III with a number of serine proteases of the coagulation cascade. Its activity in preventing blood coagulation is mainly due to the binding of the heparin-antithrombin complex with thrombin whereas its action as an antithrombotic at least in part, with the binding agent seems to be related, activity a with factor Xa. For the first of the complex molecule is required, length of the heparin certain chain whereas for the latter, it does not seem to be an important factor(l-5). We have recently reported the fractionation of 15 faheparin preparations of different tissues in two main significant milies(6). Studies of these fractions have shown to variations of the pharmacological activities in relation weight of the fractions. This implies that a the molecular is required for a certain chain length besides the structure, given pharmacological activity. This paper reports the pharmacological activities and some structural features of the two heparin families after chemical and enzymic A preliminary communication of fragmentation. these findings has appeared(7). MATERIALS AND METHODS commercial heparin Fractionation of heparin. Different preparations fzm bovine and hog mucosa were fractionated by selective barium precipitation(8) as follows: 10 g of heparin were dissolved in 400 ml of water. To the solution 20 g of barium acetate were added slowly under stirring and the pH adjusted to 6.5 . The mixture was heated at 60°C and then left at room temperature for 24 hours. The precipitate formed was collected by centrifugation and ressuspended in 250 ml of O.lM disodium hydrogen phosphate and the pH adjusted to 8.8. The mixture was heated to 60% and filtered at this temperature. 2 volumes of methanol were added To the filtrate and the mixture maintained in the cold for 2 hours. The precipitate formed was washed twice with methanol and dried under vacuum. This precipitate contains a heparin with high molecular weight with a slow migration in the discontinuous system barium/diaminopropane(see SM-heparin. The below), and was labelled supernatant.was maintained at 5% for 24 hours. The precipitate formed was collected by centrifugation at 50 c. The barium was then removed with sodium EiS phosphate described above. This fraction contained another heparin with lower molecular weight and a fast migration in rate the discontinuous system and was labelled FM-heparin. Fragmentation of heparin a)Enzymic degradation. 1 g of heparin, FM- and 2M- heparins were incubated with 1,000 u of heparitinase II prepared from Flavobacterium heparinum as previously described (9) in 0.05M ethylenedismine acetate
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buffer,pH 7.0 at 400C for 18 hours in a final volume of 200 ml. After incubation the oligosaccharides resulting from heparin and heparin fractions (FM- and SM- oligos) were precipiwith two volumes of ethanol in the presence of 2M tated NaCl (final concentration) at -2OoC for 24 hours. The precipitate was collected by centrifugation at 3,000 r.p.m. for 30 minutes, washed once with 80% ethanol and dried. The yield was g in different experiments. b)Chemical degradation. 0.5-0.7 The procedure used was that previously described(l0) with the following modifications: 10 g of heparin and heparin fractions dissolved at 5'C in 1 liter solutions containing were 0.5M 0.35M sodium acetate, 0.35% ascorbic acid, 0.045% copNaCl, at the pH of 7.8. The per acetate and 1.8 volumes of H202 were heated for 20 hours at 500 c. The fragments mixtures resulting from these incubations( FM- and SM- LMW) were precipitated with 2 volumes of methanol, washed twice with 80% methanol and dried. Pharmacological assays. Anticoagulant activity was assayed in whole plasma according to the United States Pharmacopeia (USP) the Third International Standard of heparin from the against International Laboratory of Biological Standards. LPLWHO activity (LPL) was determined in postheparin rat releasing Different dilutions prepared in saline of the samples plasma. to be tested were administered to groups of Sprague-Dawley rats by i.v. injection through the tail vein and, exactly 10 min after, under ether anesthesia, the blood was collected by puncture with a syringe bathed in 20% sodium citrate cardiac solution. The blood was drawn into a graduate tube containing sodium citrate to a final concentration of 1.2% and the plasma separated by centrifugation. LPL activity was assayed in was The LPL and activated this plasma using EDIOL as substrate. partial thromboplastin time (APTT) units determined "in viva" were obtained from the average of three different doseresponse curves corresponding to three different doses of the heparin samples using six individual rats per dose compared with a reference standard simultaneously according assayed values to the "six point method" are (11). Experimental reported as the mean +S.E.M. APTT "in vitro" was determined according to Basu et al. (12) with a kit obtained from Hoffman LaRoche Diagnostics (Basel, Switzerland). Inactivation of Factor Xa (AXa) was determined according to Teien and Lie (13) with the chromogenic substrate S-2222 (Bz-Ile-Glu-Glysupplied by Kabi Diagnostica and by coagulation Arg-p-NA) according to Yin et al. (14) with a kit obtained from the Sigma Chemical Co. (St. Louis, MO, USA). Other methods. The amount of disaccharide products formed from FM- and SM-heparins and the chemical and enzymic fragments were estimated after degradation by heparinase and heparitinase II as previously described (9,151. Electrophoresis was performed in the discontinuous system barium/diaminopropane as previously described (16). Molecular weight was estimated by polyacrylamide gel electrophoresis (17).
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RESULTS Fig. 1 Electrophoretic behaviour _-of the two heparin families. heparin shows the agarose gel electrophoresis of different system barium/diaminopreparaticns in the discontinuous propane. In this system heparin migrates as two main components a slow moving hepsrin (EM) and a fast moving heparin (FM). +he two components were fractionated by selective barium precipitation as described in Methods. The SM-heparin precipitates at room temperature whereas the FM-heparin precipitates at 5'C. Fig.1 shows that this fractionation is quite effective since the two heparins prepared by the barium precipitation migrations in the discontinuous have distinct electrophoretic system without noticeable cross contamination.
ORIGIN
I
I i
/\ I
,_-,’
;
. II
\
:n : t
_ .’
- SF!-HEPARIN
FIGURE 1 Agarose gel electrophoresis of heparin fractions in the discontinuous system.About 10 pg of different heparin prepand purified arationscl-3) FM- and SM-heparins were subjected to electrophoresis in the discontinuous system and stained with toluidine blue in ref.16.Tne as described figure shows the densitolnetric tracings of the differerlt heparins. CHS,cho,ldroitin sulfate; DS, dermatan sulfate; HTS, heparan sulfate.
-
FN-HEPARIN
“l_____-
Molecular weight and structural features Lf heparin fractions and fragments. The - scheme of fragmentation of the FM- and SMhyarins by heoaritinase II and chemical the degradation. nomenclature of the fragments as well as their average molecular weights are shown below. A two to three times reduction of molecular weight is observed for heparin, FM- and SMheparins after either enzymic or chemical degradation. The
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53
main disaccharide units present in each one of the fractions and fragments as well in the unfractionated heparin are shown amounts of Nin Table I. The FM-heparin exhibits higher disaccharides when compared with and N-acetylated sulfated Heparitinase II degradation
1_
SM-Oligo < (5,700)
Ascorbate/H202 degradation
SM-heparin (17,800)
'
>
SM-LMW (4,900)
>
(i;k::,
? H-Oligo< (5,800)
(;E2T2:iy
\1 FE;I;;e;;;;n .->
;:-;;;foc----_. ,
;T-;;;,
9
,
SCHEME OF HEPARIN DEGRADATION the unfractionated heparin whereas the SM-heparin is enriched of trisulfated disaccharide units. The oligosaccharides resulting from the FM- and SM-heparins as well as from the unfractionated heparin have similar molecular weights and are TABLE I MAIN DISACCHARIDE
UNITS IN HEPARIN FRACTIONS AND FRAGMENTS
Disaccharide
Units(%)
Compound
Heparin FM-heparin SM-heparin FM-oligo SM-oligo FM-LMW SM-LMW
Trisulfated disaccharide
Disulfated disaccharide
N-sulfated disaccharide
N-acetylated disaccharide
43 15 60 70 70 65 68
42 50 35 30 30 27 28
10 20 5 2 2 8 4
5 15
