American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 129-132, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.F258
Building A Better Heparin
George H. Caughey
Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, San Francisco, California
Address correspondence to: George H. Caughey, UCSF, Box 0911, San Francisco, CA 94143-0911. E-mail: ghc{at}itsa.ucsf.edu
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Heparin: A Work in Progress
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Heparin is the polyanion of the body's strongest organic acid. As such, it has extraordinary chemical properties and has long interested pulmonologists as an injected drug to prevent and treat thromboembolism (1). Inhaled, it also has been explored as an anti-inflammatory and antiasthmatic agent (2, 3). For most of the years since its discovery early in the 20th century, pharmaceutical heparin has been used in an "unfractionated" preparation heterogeneous in size and chemical properties. This standard heparin is superb at blocking clot formation, and is used to initiate therapeutic anticoagulation because its actions begin promptly and can be reversed quickly. However, the dose needed to produce a given level of anticoagulation is difficult to predict and control, has a relatively narrow therapeutic index, and is associated with risks of bleeding, thrombocytopenia, and osteoporosis. The past two decades have seen introductions of more uniform, "fractionated" heparins with advantageous features, including easier administration, and more prolonged and predictable effects and half-lives, with less need for monitoring—and possibly improved safety profiles (4). Anticoagulation remains risky business, however. And so the search continues for better drugs, including chemically modified heparins, such as the heparin–serpin complexes described by Berry and coworkers (5) in this issue of the AJRCMB and elsewhere (6); selective, nonheparin inhibitors of thrombin and other procoagulant peptidases (7); and pharmaceutical anticoagulant peptidases, such as activated protein C (8).
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Alveolar Fibrin: Heparin's Next Frontier?
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Most of the effort to develop better heparins or heparin substitutes emphasizes prevention of clotting in blood vessels. However, the fruit of this effort may also apply to prevention of alveolar and interstitial fibrin polymerization that is widespread in respiratory distress syndrome and other types of severe lung injury. Fibrin may accumulate in injured lung because of the combined consequences of increased formation (i.e., heightened procoagulant activity) and tardy resorption (i.e., diminished fibrinolysis). To a large extent, fibrin polymerization is a consistent response to microvascular injury and can be an early—and likely essential–step in the healing of wounds in diverse tissues. In the lung, persistent thrombin is a major component of provisional matrix that serves as a template for young granulation tissue preceding formation of more mature scars that can compromise alveolar gas transfer severely and irreversibly. The evidence that fibrin deposition occurs and persists in the injured lung—and that reducing such deposits is beneficial—has been reviewed recently (9, 10), and will be mentioned here only briefly. Clearly, clinical strategies to reduce alveolar fibrin potentially can benefit from the development of improved versions of heparin, such as inhalable forms with prolonged half-life and limited systemic absorption (6).
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Heparin Is What, Exactly?
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To make a better heparin, one first must understand what heparin is in relation to what it does. And one must separate heparin pharmacology from physiology, for as a drug, heparin is used in unnatural amounts and places. For a medication so long in clinical use, heparin is more challenging to define and describe than one might think. In part this is because "heparin" is not one substance but a mixture of complex, natural polymers varying in size, charge, and chemical configuration, a challenge heightened by the fact that many of these polymers, to varying degrees, bind to and alter the function of dozens of proteins (1, 4). The extensive scientific literature describing these interactions will not be reviewed here. Some of these effects relate to desirable pharmacologic properties; others are irrelevant or contribute to unwanted side effects. Moreover, heparins are difficult to purify to chemical homogeneity from natural sources in useful amounts, and also are hard to synthesize to a uniform standard in the organic chemistry laboratory for the purpose of establishing relationships between structure and activity (11, 12). All pharmaceutical heparins are linear chains of polyanionic saccharides. These chains can exceed 100 sugar units in length. Typical unfractionated heparins range from 3–30 kD, with a mean of  15 kD, corresponding to polymers of 40–50 monosaccharides. Fractionated, "low-molecular-weight" heparins are derived from unfractionated heparins by controlled depolymerization followed by chromatography to achieve a target size, typically 5 kD. Low-molecular-weight heparins remain heterogeneous, however, with a range of 2–10 kD. The fundamental repeating unit is a disaccharide consisting of an amino sugar (glucosamine), and an acid sugar (mainly iduronate, which is derived from epimerization of glucuronate) joined in ether linkage. This unit is heavily but variably derivatized, with the amino group being acetylated or sulfated (more often the latter) and with one or more alcohol groups on each monosaccharide being sulfated. At least two dozen disaccharide structures can occur in heparin. As shown in Figure 1, the final product can achieve an extremely dense distribution of negative charge, which nearly surrounds the strand of the polymer, with the majority of charges contributed by sulfate groups (which cannot be neutralized at physiologic pH), and the remainder by free carboxylates (13). The spacing of charges varies along different faces of the somewhat flexible heparin strand, which allows heparin to bind to proteins with varying topographies of surface cations. Heparan sulfate, a proteoglycan-associated glycosaminoglycan of cell surfaces and extracellular matrix (14), is similar to heparin in basic design but features less epimerization to iduronate, lower density of N- and O-sulfation, higher N-acetylation, and greater asymmetry of sulfation and charge density along the polymer.
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Figure 1. Models of heparin and antithrombin. (A) Three views of a short heparin glycosaminoglycan. These models are based on the coordinates of 1HPN, a dodecasaccharide copolymer with six repeating disaccharide units, whose structure was deduced from nuclear magnetic resonance analysis of bovine lung heparin in solution (13). The model was generated using RasMac v. 2.6. The linear extent of one disaccharide unit is shown by the bracket. In these space-filling models, carbon atoms are black, hydrogens white, nitrogens green, oxygens red, and sulfurs yellow. The net charge (-24) of this representative heparin, at neutral pH, is very high for an organic molecule of this size, with charges contributed by 6 carboxylates and 18 sulfates (some -NH-SO3-, others -O-SO3-). The upper left model reveals irregular spacing of carboxylates and sulfates on opposing sides of the length of the chain. The right model, which is rotated 90 degrees relative to the first model, reveals an additional set of opposed rows of sulfates, which are more regularly but widely spaced. Between the two views, all of the carboxylates and sulfates contributing to heparin's extraordinary electronegativity can be seen, as indicated by the minus signs. As further shown in the third view, heparin approximates a flexible, grooved cylinder equipped with four longitudinally arrayed rows of charged functional groups. The spacing of these groups varies in different parts of the cylinder, allowing for interactions with variously spaced cationic amino acid side chains on the surface of the sizable number of proteins with which heparin forms strong electrostatic contacts. (B) A model of a heparin pentasaccharide co-crystallized with antithrombin, revealing the heparin's principal binding site. This model is based on the X-ray diffraction-derived coordinates of 1E03 (15). To effectively inhibit thrombin, the heparin chain needs to be three to four times longer than shown here, so that the rest of the chain can engage thrombin.
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What Is Heparin's Target?
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Surprisingly, it is not completely clear which of heparin's several effects on coagulation pathways—famously complex and rife with feedback loops—are critical for its clinical benefits. Traditionally, inhibition of thrombin is considered to be paramount, but heparin by itself has little effect on thrombin. Its influence in this regard is mediated by the serpin-class peptidase inhibitor antithrombin, a close relative of  1-antitrypsin, which can convert to a thrombin inhibitor with a single amino acid substitution. By binding simultaneously to thrombin and antithrombin, heparins form an enzymatically inactive, "ternary" complex. However, heparin-bound antithrombin also inhibits activated factor X (Xa), which also may be an important aspect of its antithrombotic activity. From endothelium, heparins also liberate an Xa inactivator known as tissue-factor-pathway inhibitor. Thus, heparin inhibits Xa (which itself promotes clotting by activating thrombin) by more than one mechanism. A specific pentasaccharide in heparin binds to antithrombin, thereby inducing a conformational change that accelerates the pace of its inhibition of thrombin and factor Xa 1,000-fold (15). This pentasaccharide alone induces antithrombin to inhibit Xa; however, to inhibit thrombin, the chain must be over three times longer—at least 18 saccharide units—so that the heparin chain can also engage thrombin (4). Only about a third of unfractionated heparin chains—and an even smaller proportion of chains in low-molecular-weight formulations—contain this pentasaccharide. Thus, the great majority of glycosaminoglycan chains in pharmaceutical-grade heparins are inactive toward the proposed major coagulant protein targets. On this basis alone, there is room for the development of heparins of improved purity, potency, and target selectivity. In connection with the heparin complexes proposed by Berry and colleagues, another potential target is heparin cofactor II, which, like antithrombin (a.k.a. heparin cofactor I), is a circulating serpin activated by heparin in vitro to inhibit thrombin. This effect does not require the specific pentasaccharide sequence (and in fact can be catalyzed by nonheparin glycosaminoglycans), but it does call for concentrations of heparin considerably higher than those activating antithrombin. Therefore, in patients receiving heparin, most thrombin inhibition is mediated by antithrombin. However, heparin cofactor II has the distinction of being selective for thrombin, i.e., of not inhibiting Xa.
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Behavior of Wild-Type Heparin in the Lung: Field Observations
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The lung is a rich native source of heparin. Bovine lung is an important source of scientific and pharmaceutical-grade heparin. Heparin's abundance may be explained by the fact that the lung is rich in mast cells, which may be heparin's sole cell of origin (16). Mast cell heparin resides in secretory granules, where most of the glycosaminoglycan chains are linked to a core protein (serglycin), forming macromolecular proteoglycans much larger than commercial heparin, which is processed to glycosaminoglycan chains with little if any residual proteoglycan. Very little heparin is incorporated into cell surface proteoglycan of epithelial and endothelial cells, which are more likely to contain heparan sulfate (14), which (as noted) is under-sulfated compared with heparin. Some heparan sulfate chains of vascular endothelium contain short heparin-like sequences (17). Nonetheless, most native lung heparin is locked up in mast cells as large proteoglycans. This does not necessarily mean that heparin's physiologic realm of action is exclusively within cells, because stimulated mast cells secrete heparin proteoglycan outside of the cell along with granule-associated mediators, such as histamine, chymase, and tryptase (18). In the mast cell secretory granule, the highly polyanionic heparin allows high-density packing of cationic amines (e.g., histamine) and proteases (e.g., chymase), which can be highly positively charged in the acidic environment of the granule. Thus, in one view, heparin serves mainly as granular packing material or scaffolding for a collection of positively-charged mediators.
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Insights from Heparin Knockout Mice
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Supporting the view that heparin is important in the packaging of mast cell mediators is the phenotype of "heparin knockout" mice, which lack the enzyme N-deacetylase/N-sulphotransferase-2 (19, 20). These animals cannot fully sulfate glycosaminoglycans, which therefore cannot mature into heparin. These animals appear to develop and reproduce normally, but their mast cells are deficient in cationic mediators (e.g., histamine and chymases) normally packed densely with heparin in the granule. Whether heparin plays an extracellular role—especially as a natural anticoagulant allied with its main use as a drug—is an unresolved question. Heparin-deficient mice do not seem more prone to clotting. Because mast cells reside almost exclusively in extravascular compartments, it is unlikely that mast cell heparin regulates clotting in vessels. Possibly, mast cell–secreted heparin proteoglycan maintains a zone protected from fibrin deposition in the immediate proximity of a degranulating mast cell. Such a zone could improve egress of plasma-derived immunoproteins and effector cells to sites of parasite invasion detected by mast cell–bound IgE. Because heparin stabilizes some mast cell tryptases and modulates the activity of various mast cell chymases after release, heparin may play an important extracellular role in this regard—but this remains to be shown in vivo.
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Newer Heparins and Heparin Substitutes
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There has been widespread effort to increase anticoagulant drug safety, ease of administration and monitoring, and efficacy—at this point it isn't clear that heparin–serpin complexes meet any of these goals, although some of them may be more potent and selective as inhibitors of thrombin than heparin formulations now in use. Success in developing fractionated heparins (e.g., enoxaparin) with improved properties was noted above. The expense of these low-molecular-weight heparins is offset in many situations by less need for hospital admission, shorter inpatient stays, and lowered frequency of side effects. Another effort led to development of heparinoids, e.g., danaparoid sodium, an Xa inhibitor composed of mixed porcine heparan sulfates and other nonheparin glycosaminoglycans, which can substitute for heparin in the setting of heparin-induced thrombocytopenia (21). Development of modified forms of heparin, such as covalent complexes with antithrombin or heparin cofactor II, is at a fairly early stage (6, 22). Although these approaches can improve potency and half-life, it remains to be seen whether they are therapeutic advances. Other innovators have discarded the heparin template to create more selective, direct inactivators of thrombin or Xa (7, 21). Among these are lepirudin and bivalirudin, which are recombinant polypeptides based on a leech inhibitor of thrombin, and argatroban, a small, arginine-related, active-site inhibitor of thrombin. These are parenteral drugs approved mainly for patients with heparin-induced thrombocytopenia, or at risk of it. In other settings, there is little evidence of advantage over heparins. One of the newer entries into the anticoagulant scene is recombinant activated protein C, an anticoagulant protease which opposes clot formation by disabling procoagulant proteins (especially Factors V and VIII) and encourages clot lysis by inactivating plasminogen activator inhibitor. Like heparin (3), activated protein C has anti-inflammatory properties, which in part may underlie its ability to reduce mortality in humans with severe sepsis (8). It is not yet clear the extent to which the anticoagulant properties of heparins and protein C can be teased apart from their anticoagulant actions. It is also unknown how activated protein C would perform relative to heparins if used in settings of ordinary thromboembolic disease.
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Summary
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One may well ask: do we need a better heparin? The answer, based on current evidence, is a resounding maybe. Heparins are chemically crude, even when "fractionated," but are highly effective at preventing and treating clots in vessels. Low-molecular-weight preparations have improved properties. Still, heparins are neither safe nor particularly easy to use. So there is room for improvement—in therapeutic index, side effects, and ease of administration. One might especially wish for an oral drug for prevention and initial treatment of acute thromboembolism, for which warfarin sodium is ill-suited. Given what is known of the essential structural features of heparins, it seems unlikely that a heparin could metamorphose into a pill. Improvement in this direction is more likely to come from small-molecule inhibitors of Xa or thrombin. Nonetheless, inhaled heparins for airway and alveolar indications can be readily envisioned and hold promise.
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Acknowledgments
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This work is supported by NIH grant HL-24136.
Received in original form December 17, 2002
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