In 1939, Brinkhous and coworkers discovered that the anticoagulant activity of heparin is mediated by an endogenous plasma component termed heparin cofactor.15 The nature of this component was obscure until 1968, when Abildgaard purified antithrombin (formerly called antithrombin III) from plasma and showed that this protein has heparin cofactor activity.16 Rosenberg and Damus later purified a sufficient amount of antithrombin for biochemical characterization.17
Structure and Biosynthesis
Antithrombin is a single-chain, 58-kDa glycoprotein that belongs to the serpin superfamily.18 The cDNA for antithrombin encodes a polypeptide that is 432 amino acids in length and that is preceded by a 32-residue signal peptide.19–21 Antithrombin contains three disulfide bonds, one of which is required for heparin cofactor activity.22,23 It also contains four biantennary asparagine-linked oligosaccharides in its fully glycosylated form.24 No other biosynthetic modifications have been reported.
Two forms of antithrombin that differ in their carbohydrate content were isolated from normal human plasma by heparin-agarose affinity chromatography.25 The major form (α-antithrombin), which comprises ~90 percent of the total antithrombin, is eluted from the affinity matrix with 1 M NaCl and is fully glycosylated. The minor form (β-antithrombin) is eluted at a higher salt concentration and lacks the oligosaccharide unit linked to Asn 135, which is near the heparin binding site.26 Both α- and β-antithrombin inhibit thrombin rapidly in the presence of heparin, but β-antithrombin requires a lower concentration of heparin for maximal activity, consistent with its higher affinity for heparin.
Nonenzymatic glycation of one or two lysine residues of antithrombin occurs in vitro in the presence of high concentrations of glucose. This modification, which may occur in some patients with diabetes mellitus, has been reported to decrease the heparin-dependent thrombin inhibitory activity ~10 to 20 percent.27
The antithrombin gene on human chromosome 1q23-2528 contains seven exons distributed over ~13.5 kb of DNA (GenBank X68793).29 The introns contain 10 Alu repeats, which are present at a greater density than average for the human genome. Homologous recombination among these repetitive elements appears to have been an important mechanism by which deletions producing type I deficiency occurred.29 Antithrombin mRNA is expressed in the liver, and synthesis of antithrombin has been demonstrated in cultured human hepatoma cells.30 Alternative splicing of the antithrombin mRNA has been demonstrated.31 The alternative splicing event introduces a 42-base segment between codons −19 and −18 of the signal peptide. This segment of mRNA contains an in-frame termination codon such that the predicted protein product encoded by the alternatively spliced mRNA is only 19 amino acids long. Although the alternatively spliced mRNA accounts for 20 to 40 percent of the antithrombin mRNA in human liver, it is not known whether translation of this mRNA occurs. In the adult rat, antithrombin mRNA was detected in the kidney at a level ~20 percent of that found in the liver.32
The 5′ flanking sequence of the antithrombin gene lacks a TATA-like sequence at the expected location 25 to 30 bases upstream from the transcription initiation site,31 but contains short sequences that are similar to an enhancer element found in the immunoglobulin Jκ-Cκ gene.33 When the antithrombin enhancer element was ligated to the chloramphenicol acetyltransferase gene and transfected into cells, expression of chloramphenicol acetyltransferase activity was increased preferentially in Alexander hepatoma (liver) and Cos-1 (kidney) cells. Thus, the enhancer may be involved in tissue-specific expression of the antithrombin gene. More recently, a 700-bp fragment of the human antithrombin promoter was shown to confer a high level of tissue-specific expression in the liver and kidney of transgenic mice,34 and several transcription factors that bind to regions flanking exon 1 of the antithrombin gene were identified.35 A DNA length polymorphism has been identified, resulting from insertion of either 32 or 108 bp of DNA at the position 345 bases upstream from the translation initiation codon.36 This polymorphism does not appear to affect the level of expression of antithrombin in human plasma.37
Little is known about the regulation of antithrombin biosynthesis. Biosynthesis of antithrombin by isolated rat hepatocytes is unaffected by the presence of protease-antithrombin complexes or by the supernatant medium of macrophages incubated with these complexes.38 However, antithrombin biosynthesis is stimulated by the supernatant medium of macrophages incubated with endotoxin or fibrinogen fragment D. Under these conditions, fibrinogen and α1-antitrypsin biosynthesis are also stimulated.
Protease Inhibition by Antithrombin
Antithrombin inhibits several of the proteases involved in blood coagulation, including thrombin, factor Xa, factor IXa, and factor XIa.39 Antithrombin also inhibits factor VIIa bound to tissue factor.40,41 In vitro experiments in which 125I-labeled proteases were incubated with plasma in the absence of heparin suggest that antithrombin is the major inhibitor of factor IXa, factor Xa, and thrombin, although α1-antitrypsin and α2-macroglobulin also contribute to inhibition of the latter two proteases.42–44 In the presence of heparin at concentrations likely to be achieved therapeutically, factor IXa, factor Xa, and thrombin are inhibited almost exclusively by antithrombin. In the presence of higher concentrations of heparin or dermatan sulfate, thrombin is inhibited primarily by heparin cofactor II.45,46 Factor Xa is also inhibited by tissue factor pathway inhibitor, which is present in plasma bound to lipoproteins at about one-thousandth the concentration of antithrombin,47 and it is the factor Xa-tissue factor pathway inhibitor complex that inhibits factor VIIa-tissue factor.48 Factor XIa is inhibited primarily by α1-antitrypsin in plasma49 or by protease nexin-2, a form of the amyloid precursor protein released from platelets.50,51 Antithrombin does not inhibit activated protein C.52
Antithrombin may regulate several proteases in addition to those involved in blood coagulation. In the presence of heparin, the streptokinase-plasminogen complex (which has plasmin-like activity) is inhibited more rapidly by antithrombin than by α2-antiplasmin,53 whereas plasmin itself is inhibited more rapidly by α2-antiplasmin.54,55 Antithrombin also inhibits granzyme A (tryptase), a protease from cytotoxic T lymphocytes,56,57 and inhibits one or more of the steps in complement activation.58
Antithrombin forms an essentially irreversible, equimolar complex with each of its target proteases.17 The serine residue at the active site of the protease is required for complex formation but thereafter becomes inaccessible to substrates. Furthermore, a small peptide is cleaved from the C-terminus of antithrombin during complex formation.59 Thrombin, factor Xa, and factor IXa cleave the same peptide bond in antithrombin (Arg 393 to Ser 394)
, which is termed the reactive site.60
The antithrombin-protease complex resists dissociation in denaturing agents, suggesting that a covalent bond is formed between the two proteins. The complex can be dissociated by treatment with nucleophiles, which release the protease along with the cleaved form of antithrombin from the complex.59,61
These properties are consistent with the presence of an ester linkage between the active center serine hydroxyl group of thrombin and the α-carbonyl group of Arg 393 in the reactive site of antithrombin. Currently, it is unknown whether the reactive site peptide bond is cleaved in the native antithrombin-protease complex or whether cleavage occurs on exposure to a denaturing agent.
Antithrombin and other serpins undergo a striking conformational change after proteolytic cleavage at the reactive site (Fig. 176-2). X-ray crystallography of intact serpins, such as ovalbumin, suggests that the P1 to P12 residues N-terminal to the cleavage site form an exposed loop on the surface of the molecule.62 This structure is consistent with the finding that the region immediately upstream from the reactive site in many serpins is susceptible to proteolytic cleavage by enzymes other than the target protease, resulting in loss of the serpin's inhibitory activity.63,64 In α1-antitrypsin cleaved at P1-P1′, movement of the exposed loop about a “hinge” located near P12 allows residues P1 to P12 to become the fourth strand of a six-membered β-sheet (termed β-sheet A), separating the P1 and P1′ amino acids by 69Å.18 This conformational change results in greater thermal stability of the cleaved (“relaxed”) form in comparison with the intact (“stressed”) form of the serpin. Mutations of the “hinge” region of antithrombin (e.g., P10 Ala→Pro or P12 Ala→Thr) appear to interfere with insertion of the exposed reactive site loop into β-sheet A, prevent the “stressed” to “relaxed” conformational change, and convert antithrombin from an inhibitor to a substrate for thrombin and factor Xa.65,66 Antithrombin is also converted from an inhibitor to a substrate by a monoclonal antibody that recognizes the P8–P12 sequence67 or by a synthetic peptide that corresponds to P1–P14;68,69 these reagents are assumed to prevent insertion of the reactive site loop into β-sheet A. Thus, the ability of the reactive site loop to become inserted into β-sheet A appears to be critical for the inhibitory activity of a serpin. Recent x-ray crystallographic studies demonstrate partial insertion of the reactive site loop into β-sheet A of the uncleaved, inhibitory form of antithrombin.70– 73
Typical structures and native and cleaved serpins. The structures of native ovalbumin62 and cleaved α1-antitrypsin309 determined by x-ray crystallography are shown. The α-carbon tracing of the polypeptide backbone is indicated for each protein. The P1 to P12 residues of ovalbumin form an exposed loop on the surface of the protein that is susceptible to proteolytic attack. After cleavage of the reactive site peptide bond (P1-P1′) of α1-antitrypsin, residues P1 to P12 become incorporated into a β-sheet structure (thick lines; strands numbered according to α1-antitrypsin), and the P1 and P1′ amino acid residues are separated by 69Å.
Neutrophil elastase inactivates antithrombin by proteolytic cleavage in the reactive site loop to yield the “relaxed” conformation of the inhibitor.74 Inactivation by this protease may explain the decreased antithrombin activity observed in patients with sepsis.75 Other proteases involved in inflammation, including matrix metalloproteinases -1, -2, and -3, inactivate antithrombin very slowly or not at all.76 Antithrombin is also inactivated by certain snake venom proteases.77
Proteolytic inactivation of antithrombin by target proteases such as thrombin and factor Xa occurs in the presence of heparin at low ionic strength.78,79 Thus, the stoichiometry of inhibition of thrombin in the presence of heparin decreases from ~0.9 to ~0.1 mol of thrombin per mol of antithrombin as the ionic strength is lowered from 0.3 to 0.01.78 The mechanism by which heparin at low ionic strength favors cleavage of the reactive site rather than stable complex formation is unclear.
Stimulation of Antithrombin by Heparin
The concentration of antithrombin in plasma (2 to 3 μM) greatly exceeds that of any of the target proteases generated during coagulation. Under these conditions, protease inhibition follows pseudo-first-order kinetics. In the absence of heparin, thrombin and factor Xa are inhibited by antithrombin in plasma with t½'s of 0.5 to 1.5 min, while factor IXa is inhibited about 10 times more slowly.80 Addition of heparin to plasma increases the rate of inhibition of all three proteases ~1000 times. As a result, inhibition of thrombin, factor Xa, and factor IXa by antithrombin becomes essentially instantaneous (t½ = 10 to 60 ms).80 Heparin also stimulates inhibition of XIa, XIIa, kallikrein, and plasmin, but the magnitude of the effect is much less.
The anticoagulant effect produced by an IV infusion of heparin is thought to be caused mainly by stimulation of antithrombin-protease reactions, although inhibition of thrombin by heparin cofactor II45 and factor Xa by tissue factor pathway inhibitor may also contribute.81 The major effect of heparin is apparently to blunt the positive feedback reactions of thrombin on activation of factors V and VIII, thus decreasing the rate of generation of thrombin.82–84
Structure of Heparin and Heparan Sulfate.
Heparin occurs in the secretory granules of mast cells. A closely related glycosaminoglycan, heparan sulfate, is found on the surface of most eukaryotic cells and in the extracellular matrix. Heparin and heparan sulfate are synthesized from UDP-sugar precursors as linear polymers of alternating D-glucuronic acid and N-acetyl-D-glucosamine.14,85,86 Each glycosaminoglycan chain is built on a core structure consisting of one xylose and two galactose residues covalently attached to serine in a polypeptide backbone. About 10 to 15 glycosaminoglycan chains, each containing 200 to 300 monosaccharide units, are attached to the core protein serglycin to yield the heparin proteoglycan, which has a molecular mass of 750 to 1000 kDa. By contrast, heparan sulfate proteoglycans vary considerably in structure. They are generally smaller than the heparin proteoglycan and contain fewer glycosaminoglycan chains linked to one of several core proteins (e.g., syndecan, glypican, perlecan). In some cases, the core protein has a hydrophobic domain that anchors the proteoglycan to a cell membrane.
As the glycosaminoglycan chains are being synthesized, they undergo a series of modification reactions that include:14,85,86 (a) N-deacetylation of glucosamine residues, followed by sulfation of the free amino groups to yield N-sulfated glucosamine; (b) epimerization at the C5 position of D-glucuronic acid to yield L-iduronic acid; (c) O-sulfation of iduronic acid residues at the C2 position; and (d) O-sulfation of glucosamine residues at the C6 position. In addition, several minor but important reactions occur, including O-sulfation of glucuronic acid at C2 and C3 and glucosamine at C3. The reactions that modify the glycosaminoglycan chain appear to be catalyzed by membrane-bound enzymes in the Golgi apparatus and are completed within minutes of synthesis of the core protein. Many of these reactions are regulated by modifications that have occurred on neighboring sugar residues. Furthermore, all the reactions, with the exception of N-sulfation, are incomplete, yielding heterogeneous oligosaccharide structures within the glycosaminoglycan chain. Heparan sulfate generally undergoes less polymer modification than heparin and, therefore, contains higher proportions of glucuronic acid and N-acetylglucosamine and fewer sulfate groups.
Binding Site in Heparin for Antithrombin.
Heparin can be fractionated according to its ability to bind to antithrombin.87–89 The high affinity molecules account for virtually all the anticoagulant activity of the starting material, while the low affinity molecules are inactive. Heparin binds to antithrombin with a dissociation constant of ~20 nM.90,91 Binding is disrupted at high ionic strength, and therefore appears to result primarily from electrostatic interactions between sulfate or carboxylate groups in heparin and basic amino acid residues in antithrombin. The smallest fragment of heparin that binds to antithrombin with high affinity is the pentasaccharide shown in Fig. 176-3.92–94 This structure contains a 3-O-sulfate group that occurs predominantly in the high affinity binding site. Several of the sulfate groups within the pentasaccharide are essential for binding to antithrombin, while others do not appear to be required. In commercial heparin preparations, ~30 percent of the molecules contain this structure and bind to antithrombin with high affinity. A similar pentasaccharide structure can arise during the biosynthesis of heparan sulfate, although usually at a much lower frequency in comparison with mast cell heparin. Other types of glycosaminoglycans (e.g., dermatan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate) do not interact with antithrombin.46
Structure of the antithrombin binding pentasaccharide of heparin. Sulfate groups marked with asterisks are essential for high-affinity binding to antithrombin. The first residue may be either N-sulfated or N-acetylated, and the C6 position of the third residue may or may not be sulfated.
About 1 to 10 percent of the heparan sulfate chains synthesized by vascular endothelial cells contain antithrombin binding sites and stimulate protease inhibition by antithrombin.95 The high affinity heparan sulfate chains appear to be segregated from the low affinity chains on different subpopulations of endothelial cell core proteins.96 A variety of other cells, including fibroblasts and melanoma cells, also synthesize high affinity heparan sulfate chains.97 Antithrombin binding sites are particularly abundant in heparan sulfate isolated from mouse Reichert's membrane (an extraembryonic uterine basement membrane)98 and the basement membrane of mouse mammary epithelial cells.99 There is good evidence that antithrombin interacts with endothelial heparan sulfate (see “Activation of Antithrombin by Vascular Heparan Sulfate” below), but the physiological significance of antithrombin binding sites in other tissues is unknown.
Binding Site in Antithrombin for Heparin.
The heparin binding site in antithrombin has been studied by chemical modification, analysis of natural and site-directed mutants, and x-ray crystallography. The following observations suggest that amino acid residues between positions 107 and 145 are involved in binding to heparin: (a) Heparin blocks the chemical modification of Lys 107, Lys 114, Lys 125, Arg 129, Lys 136, and Arg 145, and these modifications decrease the heparin cofactor activity of antithrombin without affecting its ability to inhibit thrombin in the absence of heparin.100–103 (b) An antibody against residues 124 to 145 blocks heparin binding and partially mimics the ability of heparin to stimulate formation of the thrombin-antithrombin complex.104 (c) The synthetic peptide corresponding to residues 123 to 139, but not a random peptide of the same composition, competes with antithrombin for binding to heparin.105 (d) The presence of an oligosaccharide linked to Asn 135 decreases the affinity of α-antithrombin for heparin relative to that of β-antithrombin.26 (e) The disulfide bond between Cys 8 and Cys 128 is required for the integrity of the heparin binding site.22
Other studies suggest that the heparin-binding site includes residues in the N-terminal portion of antithrombin. For example, natural mutations of Ile 7, Arg 24, Pro 41, and Arg 47,106–110 as well as chemical modification of Trp 49,111 decrease the affinity of antithrombin for heparin. In addition, proton NMR experiments implicate His 1 and possibly His 65 in heparin binding.112 Based on molecular modeling, the suggestion was made that Arg 47 and the basic amino acid residues near Lys 125 are clustered on the surface of antithrombin to form a single heparin-binding site.18
Estimates of the number of ion pairs (four or five) that exist in the antithrombin-heparin complex imply that not all of the basic amino acid residues identified in the experiments mentioned above participate directly in heparin binding.79 Recently, Carrell and coworkers crystallized antithrombin complexed with a synthetic analog of the high affinity heparin pentasaccharide.72 X-ray diffraction analysis suggested that hydrogen bonds occur between sulfate and carboxylate groups on the pentasaccharide and the side chains of Arg 46, Arg 47, Lys 114, Lys 125, and Arg 129, in general agreement with the previous studies.
Allosteric and Template Effects of Heparin.
Rapid kinetic analyses indicate that heparin binding induces a conformational change in antithrombin that locks the glycosaminoglycan into place on the surface of the inhibitor.91 The heparin-antithrombin complex then reacts rapidly with a target protease. Formation of the antithrombin-protease complex reduces the affinity of antithrombin for heparin, allowing the complex to dissociate from the heparin molecule.113 Thus a single heparin molecule can catalyze the formation of many antithrombin-protease complexes.
The mechanism by which heparin catalyzes the inhibition of proteases by antithrombin involves both allosteric and template effects. Heparin binding induces a conformational change that affects the reactive site of antithrombin, which permits target proteases to interact more rapidly with this site. This model is supported by the fact that changes in antithrombin as a consequence of heparin binding can be detected by UV absorbance,114 fluorescence,115,116 circular dichroism,114,117 and proton NMR.118 Additional evidence for a conformational linkage between the heparin-binding site and the reactive site was obtained using a fluorescence probe covalently attached to the reactive site P1 residue119 and a monoclonal antibody that binds to the 1C/4B region adjacent to the reactive site loop.120 Comparison of the structures of antithrombin crystallized with and without the heparin pentasaccharide suggests that binding of heparin induces elongation of α-helix D, which, in turn, causes closure of β-sheet A with expulsion of the partially inserted reactive site loop (Fig. 176-4).72,121 As a result, the orientation of the P1 arginine residue may change so that it becomes accessible to attack by a proteolytic enzyme.122
Conformational changes in antithrombin induced by heparin. Shown are the structures of native inhibitory antithrombin73 and the antithrombin-pentasaccharide complex72 determined by x-ray crystallography. Binding of heparin (H) is associated with elongation of α-helix D (D), closure of the upper portion of β-sheet A (A), and expulsion of residues P14 and P15 of the reactive site loop (P14/15). As a result of heparin binding, the orientation of the P1 arginine residue (P1) may change so that it becomes accessible to attack by a target protease. However, this change is not apparent on comparison of the two structures, because the P1 residue in both crystals was constrained by contact with an adjacent antithrombin molecule.
Heparin can also function as a template to which both antithrombin and the target protease bind. Thus, catalysis occurs by an approximation effect. This model is supported by the fact that heparin molecules containing ≥18 sugar residues are required to catalyze the reaction of antithrombin with thrombin, even though smaller molecules bind with high affinity and induce a conformational change.93,123 Physical evidence also indicates formation of ternary complexes containing antithrombin, heparin, and thrombin.124,125
The balance between the allosteric and template effects appears to explain differences in the rate enhancement for inhibition of thrombin and factor Xa produced by heparin chains of varying length (Table 176-1). For example, the synthetic pentasaccharide that contains only the antithrombin binding site of heparin increases the rate of inhibition of factor Xa ~270 times, but has relatively little effect on the rate of inhibition of thrombin.79 Because an oligosaccharide of this size is unlikely to function as a template, induction of a conformational change in the reactive site of antithrombin may be sufficient to promote inhibition of factor Xa. Longer heparin chains produce only an additional twofold increase in the rate of factor Xa inhibition. Stimulation of the thrombin-antithrombin reaction requires heparin molecules that contain at least 18 sugar residues, which are the smallest chains capable of forming a ternary complex with antithrombin and active site-blocked thrombin.125 The factor IXa-antithrombin reaction has a similar requirement for longer heparin chains. Therefore, inhibition of thrombin and factor IXa appear to depend primarily on the template effect.
Table 176-1: Second-Order Rate Constants for Inhibition of Proteases by Antithrombin-Heparin Complexes |Favorite Table|Download (.pdf) Table 176-1: Second-Order Rate Constants for Inhibition of Proteases by Antithrombin-Heparin Complexes
| ||Thrombin ||Factor Xa |
| ||× 105 M–1s–1 (Increase) |
| || |
|Antithrombin ||0.0087 ||0.0023 |
|Antithrombin + pentasaccharide ||0.0146 (1.7) ||0.61 (270) |
|Antithrombin + full-length heparin* ||37 (4300) ||1.3 (570) |
At low heparin concentrations, the rate of inhibition of thrombin or factor Xa is proportional to the concentration of heparin-antithrombin complexes present in the incubation.80,90 The rate of inhibition plateaus at a concentration of heparin that is sufficient to saturate the antithrombin. Higher concentrations of heparin decrease the rate of inhibition of thrombin, presumably by favoring the binding of thrombin and antithrombin to separate heparin chains, but do not decrease the rate of inhibition of factor Xa.80,90 These observations are consistent with predominance of the template effect in catalysis of the thrombin-antithrombin reaction.126
Thrombin binds to heparin with a dissociation constant of 6 to 10 μM under physiologic conditions.127 An increase in the NaCl concentration from 0.15 to 0.30 M causes parallel reductions of 20 to 30 times in the affinity of thrombin for heparin and in the rate of inhibition of thrombin by antithrombin in the presence of full-length heparin.79,126 By contrast, the thrombin-antithrombin reaction in the absence of heparin is much less dependent on the ionic strength. Chemical modifications of thrombin that decrease its affinity for heparin greatly reduce the ability of heparin to stimulate the thrombin-antithrombin reaction.124 Factor Xa also binds to heparin, but apparently with a much lower affinity in comparison to thrombin. Inhibition of factor Xa by antithrombin in the presence or absence of heparin is essentially unaffected by changes in ionic strength or by chemical modification of factor Xa to reduce its affinity for heparin.79,128 These observations suggest that binding of thrombin, but not factor Xa, to heparin is required for catalysis of the antithrombin-protease reaction.
Modulators of Heparin Catalysis.
Several proteins competitively inhibit binding of antithrombin to heparin. They include histidine-rich glycoprotein129 and vitronectin (complement S protein),130 both of which are present in plasma at micromolar concentrations. Whether these proteins regulate hemostasis remains to be determined. In this regard, histidine-rich glycoprotein does not inhibit the interaction of antithrombin with heparan sulfate on the surface of cultured aortic endothelial cells.131 Platelet factor 4 is released from the α-granules during platelet aggregation and binds tightly to heparin.129 It may promote local clot formation at the site of hemostasis by blocking the binding of antithrombin to heparan sulfate. Soluble fibrin monomers decrease the rate of inhibition of thrombin by antithrombin in the presence or absence of heparin,132 and thrombin bound to a fibrin clot is protected from inhibition by antithrombin in the presence of heparin.133
Activity of Antithrombin in Vivo
Radiolabeled thrombin rapidly forms complexes with antithrombin after IV injection in rabbits.134 Furthermore, low concentrations of thrombin-antithrombin complex (20 to 100 pM) can be detected in plasma from healthy human subjects and may reflect the basal rate of generation of thrombin under normal circumstances.135 The concentration of the thrombin-antithrombin complex is increased in certain pathological conditions such as disseminated intravascular coagulation.135,136
Formation of factor IXa-antithrombin complexes also occurs rapidly in vivo.42 By contrast, factor Xa mainly forms complexes with α2-macroglobulin after IV injection in the mouse, although the major inhibitors of factor Xa incubated with murine plasma in vitro are α1-antitrypsin and antithrombin.137 Factor Xa is protected from inhibition by antithrombin in vitro when the protease is bound to platelets138 or to the prothrombinase complex which contains factor Va, prothrombin, and phospholipids.139 It is uncertain whether these mechanisms also protect factor Xa from inhibition by antithrombin in vivo. The factor Xa-antithrombin complex can be detected at low concentrations (20 to 50 pM) in normal subjects.140
Clearance of Antithrombin-Protease Complexes.
Antithrombin-protease complexes are cleared from the circulation by hepatocytes with a half-life of 2 to 3 min,42,141 which is considerably more rapid than the rate of clearance of free antithrombin (t½ ~3 days).142 The hepatocyte uptake mechanism is saturable both in vivo and in vitro.143 Cross-competition experiments suggest that the receptor for hepatic uptake also recognizes complexes of proteases with α1-antitrypsin, α1-antichymotrypsin, and heparin cofactor II.144 The low density lipoprotein receptor-related protein (LRP) is responsible for endocytosis and degradation of thrombin-antithrombin, thrombin-heparin cofactor II, and trypsin-α1-antitrypsin complexes by human hepatoma (HepG2) cells and for the in vivo clearance of 125I-thrombin-antithrombin complexes in rats.145 In human serum, the thrombin-antithrombin complex is associated with vitronectin.146 Vitronectin mediates binding of the thrombin-antithrombin complex to endothelial cells in vitro,147 and internalization of the thrombin-antithrombin complex by cultured human umbilical vein endothelial cells has been demonstrated.148 Whether these processes contribute to the clearance of thrombin-antithrombin complexes from the circulation or serve some other function remains to be determined.
Activation of Antithrombin by Vascular Heparan Sulfate
Because of the dramatic effect of heparin on the activity of antithrombin in vitro, it has long been assumed that an endogenous heparin-like substance must stimulate antithrombin in vivo. Under normal circumstances, heparin is not released from mast cells into the circulation and cannot be detected in plasma. However, a small amount of heparin may appear in the circulation of patients with systemic mastocytosis and produce a mild prolongation of the activated partial thromboplastin time.149 Circulating heparan sulfate, apparently released from damaged tissues, has been reported to cause marked prolongation of the activated partial thromboplastin time and bleeding in a few severely ill patients.150– 153
Interaction of Antithrombin with Endothelial Cells.
Current evidence suggests that heparan sulfate proteoglycans anchored in the vessel wall interact with circulating antithrombin to produce an antithrombotic effect. Glycosaminoglycans extracted from cloned endothelial cells possess anticoagulant activity.154 Treatment of the extracts with heparinase abolishes the activity, indicating that the active moiety is heparin-like. De novo biosynthesis of heparan sulfate proteoglycans has been demonstrated by culturing endothelial cells in the presence of [35S]sulfate.95 Approximately 1 to 10 percent of the labeled heparan sulfate from endothelial cells binds to immobilized antithrombin with high affinity, and this fraction possesses essentially all the anticoagulant activity of the cell extract. Structural analysis of the high-affinity heparan sulfate has revealed the presence of the 3-O-sulfated glucosamine residue that is characteristic of the antithrombin-binding structure of heparin.95
Direct binding of antithrombin to endothelial cells cloned from bovine aorta has been demonstrated. The inhibitor binds to approximately 60,000 sites per cell with a dissociation constant of 12 nM.95 Binding is diminished by pretreatment of the cells with heparinase. Similar results were obtained with intact segments of bovine aorta.155 However, the binding of antithrombin to intact rabbit aortic endothelium is weak, whereas antithrombin appears to bind more avidly to heparinase-sensitive components beneath the endothelial cell layer.156 Electron microscopic autoradiography of 125I-labeled antithrombin bound to endothelial cells in culture or after perfusion of segments of rat aorta ex vivo indicates that >90 percent of the antithrombin is associated with the extracellular matrix located in the subendothelium.157 Binding to the subendothelial matrix is greatly increased after crush injury of the aorta, which removes most of the endothelial cells. Because the intact endothelium appears to be permeable to proteins, coagulation proteases may interact with antithrombin bound to subendothelial heparan sulfate proteoglycans.157 Inhibition of thrombin in the subendothelium appears to be mediated primarily by β-antithrombin.158
Interleukin-1 and tumor necrosis factor decrease heparan sulfate biosynthesis in cultured endothelial cells and reduce the amount of antithrombin that can be bound per cell by ~50 percent.159 This mechanism could contribute to the increased thrombogenicity of the endothelium induced by cytokines.
Stimulation of Antithrombin Activity in Vivo.
Evidence for the stimulation of antithrombin by vascular heparan sulfate in vivo was obtained using a rodent hind limb preparation.160 The hind limb was first perfused with thrombin to saturate thrombin binding sites (most likely thrombomodulin) present in the microvasculature. When the concentration of thrombin present in the venous effluent had reached a steady state, antithrombin was perfused through the preparation, and the amount of thrombin-antithrombin complex recovered in the effluent was determined. Complex formation occurred fifteenfold to nineteenfold more rapidly within the microvasculature as compared with in vitro incubations in the absence of heparin. The rate enhancement was diminished by prior perfusion of the hind limb preparation with heparinase or by chemical modification of the antithrombin at Trp 49 to decrease the affinity for heparin, suggesting that interaction of antithrombin with microvascular heparan sulfate was responsible for the enhanced rate of inhibition.
When a trace amount of thrombin is injected into the circulation, the thrombin appears to become bound initially to thrombomodulin on the endothelial cell surface.134 Thrombomodulin mediates internalization of thrombin by endothelial cells in vitro, but the importance of this pathway for the clearance of thrombin in vivo is uncertain.161 In comparison with free thrombin, thrombin bound to bovine lung thrombomodulin reacts less rapidly with fibrinogen and heparin cofactor II, more rapidly with protein C, and at about the same rate with antithrombin.162 The net effect of these changes in substrate specificity may be a small increase (approximately threefold) in the rate of the thrombin-antithrombin reaction because of diminished competition from other substrates. Accordingly, only when thrombomodulin becomes saturated with thrombin is the excess thrombin inhibited rapidly by antithrombin bound to heparan sulfate proteoglycans.
Thrombomodulin may have different effects on the thrombin-antithrombin reaction depending on the tissue or species of origin. Rabbit lung thrombomodulin is a proteoglycan that bears a single chondroitin sulfate chain. It accelerates the thrombin-antithrombin reaction fourfold to eightfold by a mechanism that depends on the presence of both the protein and glycosaminoglycan components.163,164 Expression of recombinant human thrombomodulin in human embryonal kidney cells yields two forms of the protein; the higher molecular weight form contains a chondroitin sulfate chain and stimulates the thrombin-antithrombin reaction.165 By contrast, thrombomodulin purified from human placenta or bovine lung does not have these properties.162,166