Anticoagulant activities of lung and mucous heparins

THROMBOSIS RESEARCH Printed in Great

Vol. 12, PP. 27-36, Pergamon Press,

Britain

1977 Ltd.

Trevor W. Barrowcliffe,Edward A. Johnson, C. Anne Eggleton and Duncan P. Thomas Divisionsof Blood Products and Chemistry, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NW3 6F!B,England. (Received 13.9.1977; in revised form 14.10.1977. Accepted by Editor G.P. McNicol)

ABSTRACT It is generally assumed that heparins prepared from different tissues have equivalent actions. However, significantdifferences were found between lung and mucosal heparins when they were examined in an anti-FactorXa assay, both in vivo and -in Vitro. These differenceswere not seen when the henzinsere assaved bv an APTT assay. With both types of heparin, anti-Xa activity-increased with low molecular weight fractions,while the APTT activity decreased. However, with lung heparin the specific activitiesby anti-Xa assay were much lower at all molecular weights examined. It is believed that these observationshave important implications for the assay and clinical use of heparin.

Until about 15 years ago, most heparin was prepared from ox lungs, but more recently, for largely commercial reasons, intestinalmucosa from various animals have become the predominant source of supply. It is generally assumed that there are no significantdifferencesbetween heparins prepared fram different tissues, either in texms of an in vitro clotting effect, or in clinical effectiveness(1). However, most ofihe previous work has not measured the specific effect of heparin on antithrombinIII (anti-Xa),and has assayed heparin solely by its effects on overall clotting. Pharmacopoeial assays, for example, used by manufacturersto standardizeproduction batches of heparin, bear little relation to present-day clotting methods, particularlyin their use of animal blood. There have been indicationsin the literaturethat lung and mucous 27

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heparin may not have identical actions. In a collaborative study carried out to replace the WHOSecond International Standard for heparin (which was of lung origin), it was noted that the British Phannacopoeial (BP) assay method gave a higher potency than the United States Pharmacopeial (LISP) assay for the new mucosal standard, when compared with the previous lung standard (2). More recently, in an experimental study, it was observed that the incidence of haemorrhage after systemic heparin was significantly higher following the use of heparin of intestinal mucosal origin compared to that of lung origin (3). The present study has examined several batches of lung and mucosal heparin, Significant both in vivo and in vitro, using more -modern clotting methods. diffesave Eei’i?&eerved between the two varieties of heparin, and it is believed that these observations have important implications for the assay and use of heparin. M0ERIAIS ANDMElRODS Heparins European PhamaSamples of bovine lung heparin were obtained from:copoeia Gmrnission @PC), Strasbourg, France, batch FF 783; Kobanyai Gyogyszerarugyar, Budapest, Hungary, batch 536920674; Upjohn Inc., Kalamazoo, Mich., USA, batches 193EC and 73lEH. Mucosal heparin was obtained from Weddel Pharmaceuticals Ltd., London, England, and Paines and Byrne Ltd., Greenford, Middlesex, England (both INORP, imported; from this material the 8 fractions referred to in references 4 and SA Barcelona S ain batch F4 EPC Strasbour iawer repared) ; Bioiberica I!F8634-2, and Leo Phannac~utical Pro&cl&, &allerup, bik, batch A%&. Clotting

Assays

All in vitro assays were carried out against the 3rd International Standard for Eps which is of porcine mucous origin. Pooled platelet-free hwnan plasma, spun at high speed (48,000g for 30 mins.) to remove lipid and stored at -3OoC for no longer than 3 months, was used as substrate. Activated partial thromboplastin times (APTI) and anti-Factor Xa (antiXa) assays were carried out as described previously (4); the bovine Factor Xa was an NIB.% reference preparation, 75/590, originally provided by Dr. Craig Jackson, St. Louis, MO. End-points were recorded with semi-automatic hook-type clot detectors (Depex Ltd., De Bilt, Holland). Calcium thrcmbin assays were performed with bovine thrombin (Diagnostic Reagents Ltd., Thame, Oxon., England), diluted to 10 u/ml. in 24 IM CaC12 0.1 ml. heparin dilution was added to 0.2 ml. plasma, /0.8% NaCl (1:l). A followed by 0.1 ml. thrombin; the end-point was measured visually. “best fit” curve was plotted from replicate readings of dilution of the standard in the range 0 to 0.25 IU/ml., and values for test dilutions were read directly from the curve. All heparin samples and fractions were assayed at a minimum of 3 dilutions, with replication wherever possible, and values quoted are from at least 2 independent sets of assays. In vivo assays were carried out using essentially the same techniques, but tliZ TTZidard curves were made with each subject’s pre-treatment plasma, and heparin levels in post-injection samples are from single-point estimates.

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Gel Filtration Separation of heparin fractions of different molecular weights on Five fractions Ultrogel AC44 was carried out as described previously (5). in the range 6,000 to 3O,C!COwere collected, and their heparin concentrations measured by planimetry of the refractive index curve (4). In Vivo Studies On separate occasions, 1,OUU IU of mucous (Leo) and lung (Upjohn) heparin were administered to six normal male volunteers, aged 21 to 54 years. Venous blood samples were obtained at 12, 24, 48 and 96 mins. after IV heparin injection. After centrifugation of the titrated blood samples (48,ooO g for 5 mins.), the platelet-free plasma was kept at WC until the heparin assays were performed; assays were carried out within 4 hours of collection of the blood. All volunteers gave informed consent for the study. RESULTS In Vitro Assays Table 1 shows the anticoagulant activities by the various methods of two batches of commercial heparin, one lung and one mucosal. both batches were labelled 1,ooO IU/ml. by pharmacopoeia1 assay, and our AF’IT assay results agreed with this figure for both heparins. However, the anti-Xa assay results show a substantial discrepancy, the mucosal material being about 70% more potent than the lung. Calcium thrombin assays show a much smaller difference, the mucosal material having 14% less activity than the lung. TABLE1 In Vitro Anticoagulant Activities of Lung and ticosal _Heparins by Three Assay Methods activities are given in III/ml. both batches were labelled Heparin Upjohn 193EC (Lung) Leo A6OA (Mucosal)

1,000 IU/ml.)

Anti-Xa

Ca/Thrombin

982

797

1003

1064

1380

860

Assays on several other batches of lung and mucosal heparins showed substantially similar results, with a relatively low anti-Xa activity in all samples of lung heparin. Table 2 gives ratios of anti-Xa to APTT activity for four batches each of mucosal and lung heparin. The mean ratio was 1.24 for mucosal and 0.68 for lung, and there was no overlap in the ratios for the two types of heparin. Gel Filtration The lung heparins examined were found to have a somewhat flatter molecular weight distribution curve than the nnrcosal samples, particularly towards the low end (see Table 2), though covering the same total range (cf. Sugisaka

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TABLE2 Molecular

Weight Distributions and Anti-Xa/APlT of Lung and Mucosal Heparins % m-w. < 7,ooO

Batch S?!X Upjohn 193EC EPC FF 783 KG 536920674 Upjohn 731 EH

% maw. ’ 23,ooO

Ratios Anti-Ka/ART

19.6 17.2 22.8 18.4

11.7 11.3 7.8 13.8

0.81 0.58 0.78 0.55

10.4 6.2

20.9 12.8 9.9 18.5

1.33 1.28 1.30 1.04

Micosal IAORP Bioiberica F4 Leo A6Q4 EPC IPF8634-2

::3”

and Petracek (6)), except for the EPC sample, which had a sharper cut-off the low molecular weight end. Anticoagulant

Activities

at

of Fractions

Figure l(a)* shows the anticoagulant activities of five fractions of a the activities in the other three batches were single batch of lung heparin; As we have found previously for mucosal heparin, the anti-Xa quite similar. assays showed an increase towards low molecular weight, whereas the APTI actIn all fractions, the activities decreased, so that the two curves cross. ivities by calcium thrombin assays agreed closely with those by APR. The increase in anti-Xa activity is much less marked than with mucosal heparin; comparative data for a batch of Leo (mucosal) heparin are given in Figure l(b). The crossover point of the anti-Xa and APlT curves is at a much lower molecular weight for lung than mucosal, and all fractions of lung heparin have intrinsically less anti-Xa activity than the corresponding fractions of mucosal hepar in. Figure 2 shows the specific activities by each assay method calcuThis shows that, by anti-Xa assays, the “antilated in terms of IU/nmole. coagulant efficiency” of each molecule does not vary very much with molecular weight for either lung or mucosal heparin, even though lung has a much lower intrinsic activity. APlT activities are markedly dependent on molecular weight, the most active molecules being those of highest molecular weight. In Figure 3, the ratios of the activities by anti-Xa and APTI are plotted against molecular weight for four batches each of lung and of mucosal heparills. The preparations from the tklo tissues are clearly differentiated; had a lung heparin been used as a standard throughout instead of a mucosal, the differentiation would have been maintained, but all curves would have passed through higher ratios in the middle of the molecular weight range. In Vivo Studies Figure 4 shows the heparin levels obtained after intravenous injection of 1,ooO nominal units of lung (Upjohn 193EC) and mucosal (Leo A6OA) preparations into 6 healthy volunteers.

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8

10

12

Anti-Xa APTT Cd-Th

31

HEPARINS

15

20

25

30

25

30

n

I

6

8

10

12 15 mol. WI

20 x 10‘3

FIG. 1 Anticoagulant activities in vitro (a) lung (Upjohn 19?JT.~@)

of fractionated heparins: mucosal (Leo A6CW)

The peak heparin level measured by anti-Xa was 50% higher with the mucosal preparation than with the lung; this difference is highly significant (P < 0.001). In contrast, the APTT assays showed no difference between mucosal and lung heparin. However, the peak levels measured by APTI were less than half the anti-Xa values for either heparin, and the decay rate was also faster (Fig. 4).

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Vof.12,No.l

*-

I-

Specificanticoagulant activitiesexpressedin IU/nmole (preparations as in Fig. 1). !Or

10-

\

f-

2.

l-

b.6 -

0.2 -

A

5

6

8

12

15

mo~wt.

r1Q3

10

20

25

30

35

FIG. 3 Anti-Xa/ARTratiosof molecularweightfractions. The INORFfractionsare thosedescribedin Refs.4 and 5.

LUNG

vo1.12,No.l

AND

MUCOUS

33

HEPARINS

03

02

01 $. 4 T 5

0.0

f

01 12

2L

LB

96

Minutespost lnjectlon FIG. 4 Assay results (mean L s.e.m.) on plasma after intravenous injection of lumg and mucosal heparins (1,ooO IU) in 6 volunteers. DISCUSSION In 1970, Banghamand Woodward (2) commented that while the differences they detected between lung and mucosal heparins were not so consistent as to necessitate a separate international standard for each, the imprecision of the pharmacopoeia1 assays they employed may have concealed real differences between the preparations. The present study, using more specific clotting assays, confirms that this is indeed the case. The difficulty of defining the anticoagulant potency of heparin by a single figure is illustrated by the results in Table 1. ‘Ike commercial heparins, one of lung and the other of mucosal origin, and both labelled 1,ooO IV/ml., differed by as much as 70% in an anti-Xa assay. That this is a real difference, and not an -in vitro artefact, is shown by the heparin levels obtained after intravenous inJection. The mucosal heparin gave a peak level 50% higher than the lung heparin by anti-Xa assays (P < O.oOl), whereas the levels were not significantly different when measured by APTT. However, peak levels measured by AP’IT were much lower than the levels measured by anti-Xa for either heparin. The rate of decay was also faster when measured by APlT (Fig. 4), so that 50 minutes post-injection virtually no heparin was detectable by APTT, whereas anti-Xa levels were still around 0.1 Ill/ml. It is important to emphasize that this

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difference is not due to lack of sensitivity of the APTT assay, which can detect as little as 0.02 N/ml of heparin. Clearly, one of these assays is The giving a false impression of the amount of heparin that is present. high incidence of haemorrhage reported when heparin therapy is controlled by APTI (7) suggests that this method may indeed underestimate the amount of anticoagulant present in the blood. Relatively low anti-Xa activity has been found in every batch of lung This difference between lung and mucous heparin may be heparin examined. relevant to the use of heparin for the prevention of venous thrombosis (8). If, as is sometimes assumed (9), the anti-Xa potentiating effect most closely reflects the ability of low-dose heparin to prevent venous thrombosis, lung heparin would be less effective than mucosal heparin at the same dose level. Although most heparin now used is of mucosal origin, the changeover from lung was made for commercial rather than scientific reasons, and it is perhaps fortunate that the change seems to have been in the right direction for this Cur data suggest that the anti-Xa/APTI ratio could in fact particular use. be used to distinguish between heparins of lung and mucosal origin (Table 2 and Figure 3). Since all these assays were carried out using the Third International Standard Heparin, a porcine mucosal preparation, it was anticipated that the average anti-Xa/AP’IT ratio for the other mucosal preparations would have been It may be that the International Standard is not nearer to unity than 1.24. the purified material supan entirely characteristic mucosal preparation; plied by the European Pharmacopoeia Coaunission is closest to it in assay behaviour, and this may be the consequence of additional purification. These large differences in anti-Xa activity highlight the inadequacies of pharmacopoeia1 assays, at least in the context of the effect of heparin on antiXa. However, it is difficult to reccennend a single assay as an alternative, since anti-Xa assays could give a false estimate of dosage when heparin is used as a general anticoagulant. A possible approach muld be to use the APTI as the best overall indicator of anticoagulant activity, whereas a high anti-Xa/APTI ratio would indicate a heparin suitable for prophylaxis. In previous work (4) we have shown that molecular weight fractions of mucosal heparin differ markedly in anticoagulant activities, the anti-Xa activity The increasing towards low molecular weight, and the APTI activity decreasing. same trends were observed for all batches of lung heparin, but the specific activities by anti-Xa were much lower at all molecular weights (Fig.1). It is clear from Table 2 that the higher anti-Xa/AP’IT ratios for the mucosal heparins cannot be due to a preponderance of low molecular weight material, since the proportion of material with a molecular weight less than 7,000 was in fact higher in all batches of lung heparin. Thus the lower anti-Xa activity of lung heparin is an intrinsic property of its chemical structure, and not related to its molecular weight distribution. The anti-Xa/APTI ratios remain lower for lung than for mucosal heparin except at the extreme low molecular weight end, where they tend to converge. The most likely explanation of the increase in anti-Xa activity with decreasing molecular weight is in terms of binding of heparin to antithrombin III. Recent work by Einarsson and Andersson (10,ll) has indicated that there is only one significant binding site on the antithrombin III molecule, and it seems likely that heparin-activated At III consists of a 1:l molecular complex irresIf the anti-Xa activity is detpective of the molecular size of the heparin. ermined by the strength of the antithrombin III binding, one would expect the

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r molecule

to remain the same throughout the molecular weight range fractions of lung and mucosal suggest that this may be so. The brizontal nature of the graph of anti-Xa activity, in IU/IPnole, against molecular weight, shows that the increase in specific activity towards low molecular weight is largely a reflection of the increase in number of molecules This concept of anticoagulant activity “per molecule” is per unit weight. most useful in studying fractions of different molecular weight, and clearly emphasizes the different nature of the anti-Xa and APIT activities. The activities measured by calcium thrombin time closely follow the APIT data. It seems clear that the different anticoagulant effect in these assays cannot be explained solely in terms of differences in At III binding. Results for the zi~p!zEmmz~rin. heparh that we have studied (shown in Fig.2)

It has been suggested by Laurent et al. (12) that the increase with molecular weight in overall anticoagulant activity, when measured by the BP method, A~TT or thrombin inhibition, can be explained by differences in antithrcmbin It is postulated that the probability of finding the right conIII binding. figuration for At III binding in any given heparin chain increases with molecular weight, and thus the high molecular weight fractions have the highest This does not explain the high antiproportions of At III binding material. Also from this hypothesis one Xa activity of low molecular weight heparin. might expect that absorption of heparin by matrix-bound antithrombin III would result in enrichment of the high molecular weight material. In fact, the At III absorbed material displays virtually the same molecular weight distribution as ordinary heparin and, if anything, there is a slight shift towards low molecular weight when excess heparin is applied to the column (4). An alternative hypothesis which would explain the data in Figures 1 and 2 is that, for any given heparin, there are essentially no differences in antithrcmbin III binding strengths among fractions of different molecular weights, above about 5,000 Mv. If the anti-Xa potentiating effect is directly related to At III binding, differences in anti-Xa activity between lung and mucosal heparins could be due to differences in At III binding strengths. In support of this hypothesis, it has been found that antithrombin III elutes at a lower salt concentration from matrix-bound lung heparin than from mucosal (13)) suggesting a lower binding constant. The At III binding properties of molecular weight fractions are presently being studied by crossed innnmoelectrophoresis (14); preliminary results indicate that the low molecular weight material is bound to at least the same extent as the high molecular weight. If this alternative hypothesis is correct, clearly the variation of AFlT and calcium thrombin activities with molecular weight do not relate directly to At III binding. In other words, although binding to At III is a necessary prerequisite for anticoagulant activity, as expressed by APTT or Ca-thrumbin time, it is not the sole determinant of the degree of activity.

We would like to thank the camaercial firms, and the European Pharmacopoeia Commission, for supplying us with various batches of heparin. REFERENCES 1.

SILVERGL4DE,A. Biological equivalence of beef lung and hog mucosal heparins. C’urr.Ther.Research, 18, 91, 1975

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2.

BANGHAM,D.R. and WOODWARD,P.M. differentsources. -Org.,

3.

ABBOTT,W.M.,WARNOCK,D.F. and AUSTEN,W.G. The relationship of heparin sourceto the incidenceof delayedhemorrhage. J.Surg.Res., 22, 593, 1977

4.

ANERSSON, L.-o.,BARR(XLIFFE,T.W.,HOIMER,E., JOHNSON,E.A. and SIMS,

A collaborative studyof heparinsfrom 42, 129, 1970

G.E.C. Anticoagulant propertiesof heparinfractionated by affinity chrcmmtography on matrix-bound antithrombin III and by gel filtration. ThrombosisRes., 9, 575, 1976 5.

JMSCN, E.A. and MULIOY,B. The molecularweightrangeof mucosal heparinpreparations. Carbohyd.Res., 51, 119, 1976

6.

SLIGISAKA, N. and PETRACEK,F.J. Rapidmolecularsize characterisation ;;7eparins by high pressureliquidchromatography.Fed.Proc.,36, 89,

7.

MANI', M.J., THONG,K.L.,BIRTWHISTLE, R.V.,O'BRIEN,B.D.,m, G.W. and GRACE,M.G. Hemorrhagiccomplications of heparintherapy. Lancet, 1, 1133,1977

8.

THCMAS,D.P. Heparinin the prophylaxisand treatmentof venous thromboembolism.Semin.Hematol., January1978

9.

WESSLER,s. Small dosesof heparinand a new conceptof hypercoagolability. Thxsnb.Diath.Haemorrh., 33, 81, 1974

heparin, 10. EINARSSON,R. The bindingof 1-anilino-8_naphthalenesulfonate, salicylateand caprylateby humanantithrombin III. Biochim.Biophy. g, 446, 124, 1976 antiL.-O. Bindingof heparinto htsnan 11. EINARSSON,R. and w, thrombinIII as studiedby measurements of tryptophanfluorescence. Biochim.Biophy.Acta, 490, 104, 1977 M. and LINDAHL,U. 12. l&JRENT,T.C.,TENGBLAD,A., THDNBERG,L., HtjiJK, (inpress) 13. PEPPER,D.S.

Personalconummication

on antithrombin 14. SAS, G., PEPPER,D.S. and CASH,J.D. Investigations 30, 265, 1975 III in normalplasmaand serum. Brit.J.Haemat.,