The Structure of Heparin and Some Aspects

of Its Biologic Activity*

LEON FREEMAN, PH.D.

Northridge, California

D ESPITE the fact that heparin was discovered nearly 50 years ago,’ its complete struc-

ture still remains an enigma for the chemist and the biologist. This is due in part to the difficulties inherent in polysaccharide chemis- try, which has not reached the same state of sophistication as has the chemistry of other important biologic macromolecules such as proteins and nucleic acids. Another important reason for our limited knowledge is that the absolute purity of heparin has not yet been established. Without a truly homogeneous material free of contaminants, the possibility of determining the complete chemical and physical structure appears to be remote.

CHEMISTRY AND STRUCTURE

A substantial body of chemical information does exist, and many reports on the structure of heparin have appearedP-7 From this evi- dence it is possible to define heparin in terms which are meaningful but not yet completely definitive. Heparin is a member of a family of compounds of animal and bacterial origin. These are mucopolysaccharides which are large molecules made up of simple sugar units containing nitrogen.

Heparin consists of an alternating sequence of a uranic acid and hexosamine. Figure 1 shows a proposed structure of heparin.Q Recent evidence has been reported in which a 1-4 and l-6 bond from the uranic acid to the glucosamine has been shown.+l1 The evidence is very good that the hexosamine is glucosamine.7u For many years, it has been assumed that the uranic acid moiety was glucuronic acid. Despite the fact that the bulk of the evidence favors glucuronic acid as the uranic acid moiety, reports have appeared that heparin has a different uranic acid. Cifonelli and Dorfmans have reported the

presence of iduronic acid. Browni has pre- sented indirect evidence that a ketouronic acid is part of heparin. It is also possible that there may be more than one uranic acid.

Other mucopolysaccharides include chondroitin sulfate A, chondroitin sulfate B or dermatan sulfate, chondroitin sulfate C, keratosulfate, hyaluronic acid and heparitin sulfate (or heparin monosulfate) (Fig. 2 and 3). Of these substances, heparitin sulfate is the most closely related to heparin.

The sulfate groups in heparin constitute a third component, which is important from the point of view of its biologic activity. Heparin is the most highly sulfated substance known in mammalian tissue. It is also the molecule with the highest anionic charge-density of any natural substance known in animals (Fig. 1). It is the salt of an acid as strong as sulfuric acid. Furthermore, heparin is unique among the mucopolysaccharides in that essentially all the amino groups are sulfated. Heparitin sulfate differs from heparin in that part of its amino groups are sulfated and part of them are acetylated,14,i5 while in all other mucopoly- saccharides the amino groups are fully acet- ylated.

The best data available16,17 indicate that heparin has a molecular weight of between 10,000 and 12,000. It is, therefore, a molecule consisting of some 40 to 50 monosaccharide units plus 1.25 sulfate residues per monosac- charide. Very little is known about the ar- chitecture of the molecule. Because of the high charge-density, it has been assumed that it is an extended, linear molecule. It is pos- sible that branching might exist, and there are some data to support such a concept. These physicochemical data are subject to the limitation that the purity of any heparin sample is in doubt.

* From the Research and Development Division, Riker Laboratories, Northridge, Calif.

JULY 1964 3

4 Freeman

FIG. 1. Proposed structure of heparin.

OH ChS-8 bH !.e

FIG. 2. Structure of chondroitin sulfate A, B and C; and of hyaluronic acid.

Thus far, the elegant methodology of x-ray or electron diffraction has not been applied to the examination of the macromolecular configuration of this molecule. Molecular bi- ologists have not yet devoted much attention to the study of this important biologic substance.

BIOLOGIC ACTIVITY

The main biologic activities which help define and distinguish heparin from related substances are its ability to inhibit the clotting of blood or plasma, and its ability to cause the release of lipemia-clearing factor when injected into animals.18 This factor is an enzyme in the hydrolysis of triglycerides in lipoproteins.*gv20 These biologic properties will be discussed in detail in other papers of this symposium.

The ability of heparin to inhibit the clotting of recalcified sheep plasma is the basis for the USP assay for potency. The USP minimum is 120 USP units per milligram of dry heparin. Most commercial heparin is of higher activity, ranging from 130 to 170 units/mg. Prepara- tions with activities in excess of 200 units/mg. have been reported.

Relation of Structure to Biologic Activity: The biologist’s main concern with the structure of heparin is its relation to biologic activity. The large number of sulfate groups appear to be important both for the antithrombic and lipoprotein lipase-activating properties of heparin. Its role as a biologic ion exchanger also depends on this high degree of sulfation.

Two other mucopolysaccharides, heparitin sul- fate and chondroitin sulfate B, share some of the activity of heparin but with substantially less activity per unit weight. Both have ap- proximately the same sulfate content as chon- droitin sulfate A or C, which are biologically inactive. Chondroitin sulfate B, which con- tains iduronic acid, apparently differs from chondroitin sulfate A, which contains glucur- onic acid, only in configuration of the carboxyl group on the uranic acid (Fig. 4). This sug- gests that the nature of the uranic acid may be important for biologic activity.

&j&e& Heparinoid: The importance of the large number of sulfate groups for biologic activity is demonstrated by the fact that all synthetic heparin-like substances have been highly sulfated polymers, usually carbohydrate in nature. It has not been possible to prepare a synthetic heparinoid with clot-inhibiting activity as great as commercial heparin. How- ever, it has been possible to prepare heparinoids with activity exceeding that of heparin in their ability to stimulate lipoprotein lipase. Sul- fopolyglucin, a synthetic heparinoid prepared in our laboratories, is an example of these variations of potency with structure. It is a polyglucose molecule of 12 to 15 residues and is fully sulfated.21 It has about 20 units of anticoagulant activity per milligram by the U.S.P. assay but produces five to six times the lipoprotein lipase response as does an equivalent weight of heparin. Data have accumulated in our laboratories which suggest that the mode of action of this synthetic heparinoid may be different from that of heparin. The biologic response in terms of released lipoprotein lipase differs in time from heparin, particularly at high doses. Levy and Cronheim2* have shown that when sulfopolyglucin is administered to animals following heparin, there is inhibition of the heparin response.

Distribution in Animal Bodv: Heparin is widely distributed in the animal body. It is present in essentially every tissue that has been carefully examined. For many years it has

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Structure of Heparin 5

been assumed that heparin occurs either en- tirely or primarily in the mast cell granules in association with histamine.3 To establish the relation of mast cell count to the amount of heparin in the same tissue, we performed a study of several organs of rats and rabbits. We failed to find any consistent relation between the mast cell count and the heparin content of a variety of tissues as measured by its ability to inhibit clotting under conditions of the USP assay.23 While other mucopolysaccharides might very well have contributed to the ob- served activity, both heparitin sulfate and chondroitin sulfate B have very low activity in this test system.

The question of whether heparin is intracel- lular only, or both an intracellular and extracel- lular component of tissue, has not been estab- lished. There is certainly intracellular heparin in the granules of cells like the mast cell. How- ever, it is not possible to account for all the heparin in those tissues which have a very low mast cell count unless part is extracellular. This, again, remains another area of explora- tion for the biologist.

Distribution in Human Body: A careful and systematic examination of the heparin content of the human body has never been conducted. However, several human tissues have been examined and found to contain heparin.24 The quantities present appear to be greater per unit weight of tissues than those of cor- responding tissues from experimental animals,

COIH

I OH H

NHAC

FIG. 3. A, heparitin sulfate (possible structure). B, keratosulfate.

such as rats and rabbits, but less than that contained in some of the large meat animals, such as hogs and steers. The total amount of heparin in the human body is not known, but it may be greater than the usual dose of 20,000 to 40,000 units administered daily in clinical practice.

The presence or absence of heparin in human blood has been the subject of some controversy. A number of reports’ state that there is no free heparin in the blood. The methods used for these studies detect heparin only in free form. Freeman and associatesz5 have shown that there is a heparin-like activity in the blood but that it is intimately associated with proteins. The exact nature of the substance giving rise to this activity has not been elucidated as yet. Studies performed by the authorz4 produced evidence that heparin-like activity may arise from heparitin sulfate-like materials and pos- sibly from chondroitin sulfate B. Further investigation is under way to attempt to isolate

H

H OH

FIG. 4. Left, structure of glucuronic acid, and right, of iduronic acid.

JULY 1964

6 Freeman

sufhcient quantities of the mucopolysaccharides from human serum to fully characterize them and to determine both their biologic and chem- ical nature.

Biosynthesis and Metabolic Pathways of Hekarin: At the present time, little is known about the mechanism of biosynthesis of heparin and about the catabolic pathways of either endogenous or exogenous heparin. Biosynthetic studies have shown that glucose is incorporated into the carbon chain of heparin, as might be expected,26 and that inorganic sulfate is incorporated into the molecule.27 Mast cell tumors have been used in most of these studies. The biosynthetic pathways in normal mammalian tissue are undetermined.

Functional or metabolic relationships be- tween different mucopolysaccharides have not been established, although it has been suggested that heparitin sulfate might be a precursor of heparin. Data are not yet available to es- tablish this as a valid assumption.

Studies of the catabolism of heparin have been made.28J9 Little is known outside the fact that in some manner the heparin is modi- fied, since only a small portion of the original activity can be recovered in the urine. The tissue site where the catabolism of heparin occurs has not been established. There are published data that would indicate desul- fation is one of the actions which occurs in vivo.30 In studies of the excretion products of human subjects receiving heparin, we deter- mined that there is a large amount of material excreted in the urine which differs from both the normal mucopolysaccharides of urine and the original heparin, and it has lost most of its biologic activity.24 Work is under way to establish further the nature of the metabolism of exogenous heparin in both animals and man.

Inhibitions of Various Enzymes: Another in- triguing aspect of heparin activity is its apparent ability to inhibit a rather large number of enzyme systems.31 This may be due, in part, to the fact that heparin, because of its acidic nature, will combine rather tightly with almost any basic protein. However, the levels at which heparin is capable of inhibiting some enzymes would suggest a mechanism different from simple combination. Very few studies to ascertain its mode of inhibiting various enzymes have been reported. The importance of this ability and its relation to biologic proc- esses in vivo have not been established.

Problems concerning elucidation of the struc- ture of heparin are intimately related to the problems of determining the natural phys- iologic role of this important substance. It is hoped that studies now under way in a number of laboratories will uncover some of the un- known aspects of heparin structure and biology.

SUMMARY The structure of heparin is not known in

detail as yet. Its normal biologic role and its biosynthetic and metabolic pathways remain to be determined. In spite of this, heparin has become an important therapeutic agent be- cause of its antithrombotic activity and effect upon serum lipids.

1.

2.

MCLEAN, J. The thromboplastic action of cephahn. Am. J. Physiol., 41: 250, 1916.

JORPES, J. E. Heparin in the Treatment of Throm- bosis, ed. 2, p. 24. London, 1946. Oxford Uni- versity Press.

3.

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JORPES, J. E. Heparin, its chemistry, pharmacology and chemical use. Am. J. Med., 33: 692,1962.

FOSTER, A. B. and HUGGARD, A. J. The chemistry of heparin. Advances Carbohydrate Chem., 10: 335, 1955.

WALTON, K. W. Chemistry and mode of action of heparin and related compounds. &it. M. Bull., 11: 62, 1955.

6.

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WOLFROM, M. L. Heparin and related substances. In: Polysaccharides in Biology, Transactions of the Fourth Conference, p. 115. New York, 1958. Josiah Macy, Jr., Foundation.

JEANLOZ, R. W. Structure of heparin. Fed. Proc., 17: 1082, 1958.

9.

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WOLFROM, M. L., MONTGOMERY, R., KARABINOS, J. V. and RATHGEB, F. The structure of heparin. J. Am. Chem. SOG., 72: 5796, 1950.

CIPONELLI, J. A. and DORFMAN, A. Structural studies on heparin and heparitin sulfate. Biochem. @ Biophys. Res. Commun., 4: 328, 1961.

DANISHEFSKY, I., EIBER, H. B. and LANGHOLTZ, E. Investigations on the chemistry of heparin Biochem. & Biophys. Res. Commun., 3: 571, 1960.

DANISHEFSKY, I., EIBER, H. B. and WILLIAMS, A. H. Investigations on the chemistry of heparin. IV. The glucosidic linkages. J. Biol. Chem., 238: 2895, 1963.

12. JORPES, J. E. and BERGSTROM, S. Der Aminozucher des Heparins. Ztschr. Physiol. Chem., 244: 253, 1936.

13. BROWN, K. D. Chemistry of heparin. In: ENGEL- BERG, H. Heparin: Metabolism, Physiology and Clinical Application, p. 5. Springfield, Ill., 1963. Charles C Thomas.

14.

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JORPES, J. E. and GARDELL, S. On heparin mono- sulfuric acid. J. Biol. Chem., 176: 267, 1948.

LINKER, A., HOFFMAN, P., SAMPSON, P. and MEYER, K. Heparitin sulfate. Biochim. et Biophys. acta, 29: 443, 1957.

16. FRIEDEN, E. Personal communication, 1961. 17. BARLOW, G. H., SANDERSON, N. D. and MCNEILL,

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Structure of Heparin 7

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