British Medical Bulletin (1978) Vol. 34, No. 2, pp. 143-150

ANTITHROMBIN m AND HEPARIN

T W Barrowdiffe, E A Johnson & D Thomas

ANTTTHROMBIN m AND HEPARIN T W BARROWCLIFFE MA PhD E A JOHNSON MA DPhil DUNCAN THOMAS MD DPhil National Institute for Biological Standards and Control Hampstead, London

Antithrombins a Purification of antithrombin HI b Interaction of antrthrombin IK and thrombin c Inhibition of activated clotting factors by antithrombin in d Assays of antithrombin IQ e Antithrombin HI and Him^n« Heparin a Chemistry b Heparin assays c Mechanism of action d Heparin heterogeneity and fractfonation e The clinical use of heparin Summary References Heparin, discovered by a medical student in 1916 (McLean, 1959), has been in clinical use as an anticoagulant for over 40 years. Not only is heparin the oldest antithrombotic drug still in use, it also remains the most widely used agent for immediate anticoagulation. In recent years, there has been a considerable upsurge of interest in its chemistry and mode of action, and there have been symposia devoted solely to heparin (e.g. Bradshaw & Wessler, 1973; Kakkar & Thomas, 1976). Recognition of the importance of the interaction between heparin and antithrombin III (Atm) is of more recent origin, and this has led to a better understanding of how heparin works. An inherited deficiency of AtHI is one of the very few conditions in which a tendency to thrombosis can be clearly defined biochemically; furthermore, the interrelationship between At HI and heparin plays a key role in our understanding of the pathogenesis and management of thrombo-embolic disease. In this paper, recent advances in the field will be reviewed, with particular emphasis on biochemical developments. Despite recent progress, it is still true to say that the factors that promote coagulation are better characterized than those that inhibit it. It is clear that powerful mechanisms exist for neutralizing activated clotting factors, particularly thrombin. When blood dots in vitro, all the prothrombm is normally converted into thrombin and, were it not neutralized, the thrombin formed from 10ml of blood would be sufficient to clot all the fibrinogen in the body. The efficiency of the antithrombin "n^iflnism can be demonstrated by a simple

a Purification of Antithrombin III A t m has been purified by Abildgaard (1967a), using a combination of A1(OH)3 absorption, gel filtration and ionexchange chromatography. The best preparations had a specific activity 300 times that of plasma protein, but 143

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experiment, in which plasma is clotted by tissue factor and calcium chloride, and the clot removed. If the thrombin concentration is then measured at intervals by subsampling into fibrinogen, although 300 units of thrombin are generated per millilitre of plasma, the peak concentration rarely exceeds 10 units/ml and the total thrombin measured is only about 10 % of the potential amount. Six different types of antithrombin have been described, but only thefirstthree have been shown to play a physiological role (Lane & Biggs, 1977). The term "antithrombin HI" originally described the activity in plasma which causes progressive irreversible destruction of thrombin. However, it is now recognized that at least three proteins contribute towards this activity: aj antitrypsin, a 2 macroglobulm (these are general proteinase inhibitors) and an -4; (ii) that the uronic acid includes both D-glucuronic and L-iduronic acids; (iii) that the iduronic acid is normally sulphated in the 2 position and the glycuronic acid not sulphated; (iv) that the glycosamine is exclusively D-glucosamine; and (v) that the glycosamine is sulphated in the 6 position and also normally in the NA2) position, though in some heparins a small proportion of the amino groups is acetylated. The most obvious property of heparin that distinguishes it from other GAG is the much greater extent of sulphation, about 2-2.5 sulphate ions per disaccharide unit compared with one (or, in hyaluronic acid, none). However, it has long been known that laboratory sulphation of GAG or other polysaccharides never yields products with more than a small amount of the anticoagulant activity (by assay in vitro) of heparin, and it is now clear that the special properties which belong to heparin alone result from highly specific structural characteristics (Hopwood et al. 1976). These are almost certainly sequences in the polysaccharide chain which bind to protein cofactors and activate them (or, conceivably, inactivate them) by altering their conformation, and it is possible that a number of different sequences may all be able to mediate a particular conformational change, though with differing efficacy, i.e., with different binding constants. The fundamental heterogeneity of heparin is not an artefact of preparation, but appears to arise from a degree of randomness in the biosynthetic process (Cifonelli, 1974; Hook et al. 1974). There also seem to be real differences between heparins from different tissues (Cifonelli & King, 1970), though these may be statistical rather than absolute, in the sense that many individual molecular species may be present both in lung and in mucosal heparins. On the basis of differences observed in proton magnetic resonance spectra, Perlin and co-workers (Perlin et al. 1968; Perlin et al. 1970) divide commercial heparins into two classes, A and B, corresponding apparently to porcine mucosal heparin and bovine lung heparin, respectively. The principal difference is a much higher content of JV-acetyl groups in class A, but there are others not readily interpreted. The conformation of heparins has been investigated by optical methods in solution (Stone, 1977) and, in the solid state, by x-ray crystallography using orientated films (Atkins & Nieduszynski, 1977). In plasma, however, heparin is completely bound to proteins, either by direct electrostatic interaction with suitably orientated basic amino acid residues or by other non-covalent mechanisms (cf. Laurent, 1977), and its conformation will be controlled by such interactions.

146 Br. Med. Bull. 1978

ANTTTHROMBIN HI AND HEPARIN T W Barrowdiffe, E A Johnson & D Thomas FIG. I. The effect of heparin on the destruction of bovine thrombln ( x , O) and of bovine factor Xa ( + , D) by antlthrombln III (Adapted from Biggs wt ml. 1970) 100-**--+

30D)

'5

£

10-

ID

X o

t»

CO «•-

o c

3-

!5

e 1-

0.3 30

60

Time (minutes) Thrombln or factor Xa was Incubated at 37°C In diluted plasma with (Interrupted lines) or without (solid lines) addition of heparin (0.025unlts/mn. The destruction of human factor Xa takes a similar course but Is significantly slower

d Heparin Heterogeneity and Fractionation

Although Silverglade (1975) regarded bovine lung and porcine mucosal heparins as being biologically equivalent, Ganesan & Bass (1976) reported significant differences in plasma lipolytic activity mediated by lung and mucosal heparins. There had been clear indications from Bangham & Woodward (1970) that, when tested by various coagulation assays, lung and mucosal heparins were not equivalent. Consistent differences can be demonstrated between the two, both in regard to their absolute activity by anti-factor Xa and, more significantly, in the ratio of activity by anti-factor Xa to activity by activated partial thromboplastin time (APTT), which was nearly twice as high for mucosal as for lung samples (Barrowcliffe et al. 1978). This difference also manifested itself in anti-factor-Xa blood levels after intravenous injection in human subjects. A consequence of the heterogeneity of heparin is that there is no sharp boundary between purification and fractionation; the procedures that have been the most often used on commercial heparin samples are those summarized by Rodin et al. (1972). The relation of the variables which control such separations to those required for heparin to perform its specific functions is likely to be very generalized and non-

heparin and thrombin complicates the study of the heparinthrombin-Atin interaction, and is responsible for an alternative hypothesis—that heparin binds to thrombin, thereby making it more accessible to neutralization by At III. However, neither thrombin nor factor Xa is present in normal plasma, and introduced heparin is almost certainly bound exclusively to A t m , since there seems to be no other protein that can compete effectively with it in terms of plasma concentration and binding constant At a heparin concentration of 1 unit/ml, there are still about three molecules of A t m for each one of heparin. According to Machovich (1975), blocking a thrombin arginine site which is responsible for heparin binding has no effect on its fibrinogenic activity either alone or with excess A t m (slow inactivation), but it very largely abolishes the accelerating effect of heparin on the A t m inactivation. Smith (1977) found that, in systems using physiological concentrations, preincubation with A t m had no effect when a mixture of heparin and a synthetic substrate was subsequently mixed with thrombin. However, in contrast 147

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with most other workers, including Machovich, he found no inhibition of thrombin nmidnw activity by A t m alone. Both Lie/a/.(1974)andSmith(1977)found higher binding constants for the heparin-thrombin interaction than those reported elsewhere for the heparin-Atm system, agreeing in this with Machovich et al. (1975); so it is possible that, even though A t m may be the heparin carrier in plasma, the initial consequence of an encounter with thrombin may be heparin transfer, effecting thereby activation of thrombin rather than A t m . A difficulty with such a mechanism is that heparin activation of thrombin should activate it also for its normal substrate, if A t m is regarded as a substrate-competitive inhibitor (cf. Seegers, 1975); most workers, however, find that heparin alone inhibits the action of thrombin on fibrinogen. There is also nothing to support a heparin transfer mechanism in the work of Li et al. (1976). It is possible that the true situation may be a combination of these concepts, with heparin taking place in a reaction with both thrombin and A t m . This could explain the increase in specific activity (per mole) with increase in molecular weight (Barrowdiffe et al. 1978), the larger molecules being more capable of binding both thrombin and Atm. The amount of heparin needed to give an accelerating effect is remarkably small. An increase in the rate of thrombin neutralization can be demonstrated with as little as 0.02 units/ml of heparin (Biggs et al. 1970). At this concentration, there are 150 molecules of A t m for each one of heparin, which suggests a catalytic effect. Gitel (1975) has postulated that heparin is released for further binding once the thrombin— A t m complex has formed, and it has been shown (Andersson et al. 1977) that the thrombin-Atm complex binds heparin only weakly. By accelerating the inhibition of factor Xa, heparin is capable of preventing the development of much larger amounts of thrombin than could be neutralized directly (Wessler & Yin, 1974). This effect of very small amounts of heparin on factor-Xa neutralization has given a rational theoretical basis for the success of heparin in the prophylaxis of venous thrombo-embolism.

ANTITHROMBIN DI AND HEPARIN

T W Barrowcliffe, E A Johnson & D Thomas e The Clinical Use of Heparin Heparin is used clinically in three main ways: (i) in low dosage, by subcutaneous injection, for the prophylaxis of venous thrombosis; (ii) in standard dosage, intravenously, for the treatment of overt thrombosis; and (iii) as a general anticoagulant for maintaining the fluidity of the blood in extracorporeal circulations, such as heart-lung machines, or in renal dialysis. It is important to differentiate the purposes for which heparin is used therapeutically; and the differing role of heparin in prophylaxis, as opposed to treatment, of disease can best be understood in relation to the central role of thrombin. When heparin is used for prophylaxis, thrombin generation is checked by the marked heparin-induced increase in At HI activity. The action of heparin-Atm in inhibiting factor Xa is likely to be more important in the context of prophylaxis than is the ability of At HI to neutralize thrombin, if only because factor Xa is potentially more thrombogenic than thrombin itself (Yin et al. 1971b). However, in overt thrombosis, there is the additional requirement of neutralizing thrombin that has already been formed, as well as of preventing further generation of thrombin. The evidence from both laboratory and clinical studies suggests that at least twice as much heparin is required to treat thrombosis as is required for prophylaxis. For example, while the effective dose for preventing DVT is 10000-15000Lu. given subcutaneously daily in divided doses, the dose required to control established venous thrombo-embolism is at least 25000-30000 i.u. given intravenously daily (Thomas, 1976). One of the main drawbacks in the clinical use of heparin is the attendant risk of haemorrhage. Even when heparin is given in low dosage to prevent post-operative DVT, there is an increased incidence of wound haematoma (International Multicentre Trial, 1975). When administered intravenously in full dosage, the incidence of major haemorrhage is of the order of 10% (Salzman et al. 1975; Mant et al. 1977). There is, therefore, considerable interest in any development which, while leaving the therapeutic value of heparin unimpaired, would diminish the hazard of haemorrhage. The fractionation of heparin may lead to such an improvement, at least in the field of prophylaxis. It has been shown in vitro, and also in human pharmacology experiments, that low-molecularweight heparin fractions specifically enhance the neutralization of factor Xa and have relatively much less effect on over-all clotting than whole heparin (Andersson et al. 1976; Barrowcliffe et al. 1978). It has been suggested that the effectiveness of low-dose heparin therapy results from the enhancement of the body's own defence mechanism against thrombin generation by markedly increasing the rate of neutralization of factor Xa (Wessler, 1974). It should, therefore, be advantageous to employ those heparin fractions that have the most effect on anti-factor Xa and least effect on over-all clotting. These considerations may not be valid when thrombin generation has already occurred, as in overt thrombosis. The effectiveness of low-molecular-weight heparin fractions in preventing venous thrombosis without haemorrhagic side-effects has, however, yet to be demonstrated by clinical trials.

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specific. Nevertheless, fractions obtained by these methods do show consistent variations in anticoagulant activity when pharmacopoeial assays are employed (Laurent, 1961; Lasker & Stivala, 1966; Walton et al. 1966). Other properties such as sulphur content and mean molecular weight also change consistently over a range of fractions from a particular heparin and, although correlations between such properties and anticoagulant activity have been observed, Kavanagh & Jaques (1973) have shown that no such correlations can be found for commercial heparin samples in general. Gel filtration can be used to fractionate heparin on a molecular-weight basis, with very little effect on other properties (Walton et al. 1966; Wasteson, 1969). Cifonelli (1974) found that when either bovine lung or porcine mucosal heparin, of a quality equivalent to good commercial products, was subjected to such fractionation there was little change in anticoagulant activity (presumably using the method in the United States Pharmacopeia) with molecular weights above 7000-8000, but below this the activity dropped sharply. Generally similar results were obtained by Johnson & Mulloy (1976) for commercial mucosal heparin by APTT assay, but the same fractions gave very different results (Andersson et al. 1976) when assayed by the more specific anti-factor-Xa procedure. The low-molecular-weight fractions then had a high activity which decreased with increasing molecular weight. The ratio of activity by anti-factor Xa to activity by APTT changes dramatically with molecular weight for both lung and mucosal heparins, though the two can be distinguished by the actual ratios obtained, particularly in the middle of the molecular-weight range (Barrowcliffe et al. 1978). Subcutaneous injection of a low-molecular-weight fraction of mucosal heparin in human subjects was found to give considerably increased blood levels by anti-factor Xa when compared with a high-molecular-weight fraction (Johnson et al. 1976). An informative fractionation procedure recently devised for heparin is affinity chromatography, using a matrix-bound protein known to interact with it electrostatically. As yet, published work has been concerned principally with A t m as adsorbent (Andersson et al. 1976; Hook et al. 1976; for a related technique see l a m et al. 1976). Heparin has been separated in this way into active and inactive fractions which, to date, have been found to be physically and chemically indistinguishable, although it is possible that the inactivity may result from monosaccharide sequence differences like those observed by Cifonelli (1974) in relatively inactive heparins obtained by more conventional fractionation procedures. The terms "active" and "inactive" seem to apply, though perhaps in varying degree, to all the anticoagulant assay procedures that have been applied to these fractions, and this provides strong support for the view that the action of heparin as an anticoagulant depends primarily on its binding to, and activation of, At i n . Active fractions of mucosal heparin give the highest assay results so far obtained; further fractionation by gel filtration has yielded low-molecular-weight material with activity by anti-factor Xa of about lOOOi.u./mg (Andersson et al. 1976). However, by APTT assays, and hence probably also by'pharmacopoeial assays, its activity is lower by an order of magnitude. Bengtsson et al. (1977) have shown that there is no difference between active and inactive heparins in lipase release systems. Different sequences in the heparin family are therefore involved in lipase release.

A recent surprising observation by Marciniak & Gockerman (1977) was the reduction in circulating A t m levels that they found in 26 patients given intravenous heparin. In all the patients studied, heparin therapy was associated with a progressive reduction in both Atm-binding capacity and 148 Br. Med. Bull. 1978

ANTITHROMBIN m AND HEPARIN

T W Barrowcliffe, E A Johnson & D Thomas A t m was significantly shortened and the catabolic rate increased. Their tentative conclusion was that there was a higher physiological turnover of the Atm-heparin complex. 3

Summary

The importance of antithrombin m (Atm) as a natural defence mechanism against thrombosis is indicated by the relative frequency of venous thrombo-embolism in patients who have an acquired or congenital deficiency of A t m . The effect of heparin in potentiating the action of A t m , and the ability of Atm-heparin to inactivate rapidly most of the serine proteases of the coagulation system, is now believed to be its primary mechanism of action. The biological consequences of the heterogeneity of heparin are being increasingly recognized, and it is now apparent that significant differences can be demonstrated in the effect on A t m of heparins prepared from different tissues and of heparin fractions of varying molecular weights. It may be possible to exploit these differences to improve the therapeutic efficiency of heparin.

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antigenic protein. Plasma A t i n concentration returned to normal within a few days after heparin was stopped. Their findings, for which they offer no explanation, do not relate to patients receiving low-dose heparin and, indeed, they comment that heparin must be present in blood for long periods to reduce plasma At HE concentration. They discount the possibility that the underlying thrombo-embolic disease could have contributed to the fall in At i n level, primarily because of the consistent appearance and size of At ID reducion in all their patients receiving standard heparin therapy, which was unrelated to the location and extent of the thrombotic manifestations. If these observations are confirmed, it may become necessary to modify the standard heparin regimen used in thrombo-embolic disease, with a view to minimizing A t m depletion. The findings of Marciniak & Gockerman are also in keeping with the observation of Collen etal. (1977), who found that whereas the turnover of labelled A t m was normal in three patients with venous thrombosis not given heparin, in three further patients with venous thrombosis who were treated with heparin, the plasma radioactivity half-life of

ANTTTHROMBIN HI AND HEPARIN

T W Barrowcl\ffe, E A Johnson & D Thomas

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ISO Br. Med. Butt. 1978