1 Mechanism of heparin action CRAIG

M. J A C K S O N

The current understanding of how heparin produces its anticoagulant action has evolved during the last 60 years as a result of several observations which may be marked as milestones. The discovery of heparin in 1916 by McLean (McLean, 1916), and its partial purification and characterization in the years immediately following (Howell, 1924; Jorpes, 1935; Jorpes and Bergstrom, 1937), resulted in identification of several aspects of heparin chemistry which remain as relevant to our understanding of heparin mechanism of action today as they did then. The compositional and structural heterogeneity (Brown, 1963) of heparin hampered the earliest investigations to establish its structure and continues to produce confusion both in terms of its effect on heparin activity, heparin binding to proteins and its role as a cell surface component. A particularly significant milestone for understanding heparin action was the observation that a protein in plasma (a 'cofactor') was required for heparin to express anticoagulant activity effectively (Brinkhous et al, 1939). (Although called a cofactor, this protein and another which is designated heparin cofactor II are actually reactants and are consumed during the inactivation of proteinases. Neither of the heparin cofactors are cofactors in the classical sense used for vitamins when they function as prosthetic groups for metabolic enzymes.) This discovery was followed by the demonstration (Monkhouse et al, 1955; Waugh and Fitzgerald, 1956) that the plasma cofactor was the same as antithrombin, the inhibitor responsible for the slow thrombin inhibiting capacity of plasma in the absence of heparin (Abildgaard, 1968). This observation set the stage for the investigations of the 1970s and 1980s upon which our current view of heparin action is based (Yin et al, 1971; Barton and Yin, 1973; Damus et al, 1973; Rosenberg, 1975, 1977; Bjork and Lindahl, 1982). The heparin cofactor was not only identified as antithrombin, but the two functional entities initially given the designations antithrombin II and antithrombin Ill were shown to be properties of this molecule, now called antithrombin III (Abildgaard, 1968), Moreover, antithrombin Ill not only inactivated thrombin (Abildgaard, 1969; Rosenberg and Damus, 1973; Seegers and Andary, 1974; Biggs et al, 1975), it also inactivated factor Xa (Yin and Wessler, 1969; Dombrose et al, 1971; Yin et al, 1971; Biggs et al, 1975; Rosenberg, 1977; Gitel et al, 1984), factor IXa (Rosenberg et al, 1975; Kurachi et al, 1976; Gitel et al, 1977; Holmer et al, 1981), factor XIa (Rosenberg, 1977; Holmer et al, 1981 ; Beeler et al, 1986; Tans et al, 1987) and factor XIIa (Rosenberg, Baillibre's Clinical Haematology-Vot. 3. No. 3. July 199(I ISBN 11-7020-1474--5

483 Copyright© |990, by Baillihre Tindall All rights of reproduction in any form reserved

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c.M. JACKSON

1977: Holmer et al, 1981 ; Pixley et al, 1985). Heparin enhances the rate of the inactivation of all of these proteinases (Rosenberg, 1977; Holmer et al, 1981) although, as will be noted below, not with equal effectiveness. More recent investigations (Briginshaw and Shanberge, 1974; Tollefsen and Blank, 1981; Tollefsen et al, 1982) have demonstrated that another thrombin inhibiting protein is also present in plasma, heparin cofactor II, and thus heparin action is now understood to involve both of these proteinase inhibitors. Some of the issues which were identified in the earliest studies which related heparin activity to its molecular structure remain as much a challenge to understand today as during past decades. For example, the consequences of molecular heterogeneity intrinsic to heparin preparations, in particular the influence of this heterogeneity on heparin anticoagulant activity, are still a topic of significant scientific controversy (Jaques, 1979; Barrowcliffe et al, t985; Fareed et al, 1988), albeit the discussion is at a different molecular level today. PROTEINASE INHIBITORS IN PLASMA AND THEIR RELATION TO THE CONCENTRATIONS OF PLASMA PROTEINASES Proteinases of the coagulation system are inactivated in plasma by four distinct active site-reactive proteinase inhibitors (Gitel et al, 1984; Harpel, 1987), although antithrombin III and heparin cofactor II almost certainly account for most of the inactivation of coagulation proteinases which occurs in plasma, particularly in the presence of heparin. Two of the inhibitors which can inactivate coagulation proteinases, cq-proteinase inhibitor and C1 inactivator, are insensitive to heparin. In addition to these inhibitors, oLz-macroglobulin also reacts with proteinases of the clotting system but does so by a mechanism entirely different from the irreversible active sitedirected inhibitors (Feinman, 1983). Knowledge of the concentrations of proteinase inhibitors relative to the proteinases which they inactivate provides a basis for obtaining some fundamental insights into what are critical characteristics of the inhibition process. A priori, it can be asked if proteinase inhibitors are present in plasma in stoichiometric amounts relative to the proteinases they inactivate, or in excess amounts. Interpretation of the importance of inhibitor concentrations in these two situations are different and are considered in the following section. The concentrations of the inhibitors in plasma and the maximum concentrations of the proteinases are given in Table 1. It is evident from these data that the concentration of antithrombin III exceeds the total possible concentration of proteinases which can be produced. More importantly, however, it has been known for many years that total conversion of proteinase precursors into active proteinases does n o t occur during in vitro clotting. This is evidenced by the observation that the concentration of plasma proteinase precursors measured immediately after clotting are the same as their starting concentrations (Gaston, 1964). Given these two limits for the concentrations of proteinases generated during clotting in plasma, and the much higher relative concentrations of inhibitors,

ACTION OF HEPARIN

485

Table 1. Concentrations of blood clotting proteinase precursors and proteinase inhibitors in plasma. Concentration (fxmol/litre) Proteinase precursors Prothrombin Factor X Factor IX Factor XI Factor XII Prekallikrein Proteinase inhibitors Antithrombin III Heparin cofactor II at-Proteinase inhibitor CI inactivator a2- Macroglobulin

1.5 0.15 0.07 0.04 0.38 0.4 2.7 1.5 24.5 1.7 3.5

Reference Suttie and Jackson (1977) Jackson and Hanahan (1968) Hedner and Davie (1987) Saito and Goldsmith (1977) Saito et at (1976) Scott and Colman (1980) Murano et al (1980); Conrad et al (1983) Griffith et al (1985) Jeppson et al (1978) Harpel (1987) Harpel (1987)

it appears virtually certain that antithrombin III+ heparin cofactor II and perhaps other inhibitors do not exist in their normal concentrations in order to be stoichiometric inactivators of the proteinases which are produced in such low concentrations. However, despite such excess of inhibitor over proteinase, a 50% decrease in the concentration of antithrombin III predisposes to thrombosis (Odegard and Abildgaard, 1977; Gram and Jespersen, 1985; Barrowcliffe and Thomas, 1987). Thus, it is necessary to question the reason or reasons for the high concentrations of the inhibitors which are maintained within relatively narrow ranges, particularly antithrombin III. When it is recalled that the rate of proteinase inactivation is directly proportional to the concentration of the inhibitor (Fersht, 1977), and thus a decrease in antithrombin III to 50% of its normal level would reduce the rate of proteinase inactivation twofold, a plausible reason is evident. (This is strictly true in the absence of heparin, but is only slightly more complicated when heparin is present.) It thus appears that reaction rates are of primary importance for understanding proteinase inactivation and its catalysis by heparin rather than stoichiometric considerations. This premise is implicit in the arguments which follow. STRUCTURE OF HEPARIN

Heparin is a heteropolymer consisting of a repeating sequence of disaccharide units (Atkins and Nieduszynski, 1976; Horner, 1976; Kiss, 1976; Lindahl and Hook+ 1978; Bjork and Lindahl, 1982). The basic disaccharide contains one uronic acid residue, either glucuronic acid or iduronic acid, and a glucosamine residue (Figure 1). A variable number of sulphate groups are present which are esterified to the hydroxyls at positions 2, 3 and 6 of the uronic acid residues and the amino group at position 2 and the hydroxyl group at position 6 of the glucosamine residues (Kiss, 1976; Lindahl and Hook, 1978). Approximately 10% of the amino groups of the glucosamine residues may be acetylated

486

c.M. JACKSON

coo-

cH2osoz-

OH

[ (a)

CH2OSOz-

NH

0

s%

Disaccharide with G}ucuronic Acid

so;

I

NH

s%-

Disaccharide with Iduronic Acid

I

(b)

Figure 1. The structure of heparin. (a) The fundamental building block of heparin is a disaccharide consisting of one uronic acid and one glucosamine residue. The uronic acid is either glucuronic or iduronic acid. (b) The heparin molecule with activity in promoting proteinase inhibition by antithrombin III is a linear polymer of dissacharide units varying in molecular weight from 7000 to 30000. Heparin molecules in this molecular weight range contain from 10 to 40 disaccharides and vary from 8.9 nm to nearly 40 nm in length in their fully extended conformations.

rather than sulphated. Heparin preparations for clinical use have 2-3 sulphates per disaccharide leading to a net negative charge of - 3 to - 4 per disaccharide. Heparin is heterogeneous, not only with respect to the extent of sulphation of the individual monosaccharide residues, but also with respect to the molecular weight of the individual molecules (Lasker, 1977). The average molecular weight of clinically used heparin is 15 000-16 000 with molecules from as small as 7000 to some as large as 30 000 (Danielsson and Bjork, 1981). This molecular weight and charge (extent of sulphation) heterogeneity is significant in promoting proteinase inactivation by heparin and in its binding to a variety of other proteins (Laurent et al, 1978; Lane et al, 1979; Thunberg et al, 1979; Danielsson and Bjork, 1981; Holmer et al, 1981). Binding of heparin to most proteins depends on both the net charge and on the molecular weight of the heparin molecule (Scott, 1973). Antithrombin III is the known exception with an additional saccharide sequence specificity requirement (Hook et al, 1976; Rosenberg et al, 1978; Lindahl et al, 1979; Sinay et al, 1984). Because most of the force responsible for binding is due to electrostatic attraction between the negative charges on the heparin and the positive charges on the protein, binding is very dependent on the ionic strength, i.e. the salt concentration of the solution (Scott, 1973; Nordenman and Bjork, 1980, 1981). Although this is obviously not important in vivo, the osmolarity of plasma being constant, it is responsible for many of the differences observed by investigators studying the mechanism of heparin action in the inactivation of thrombin in vitro. Antithrombin III appears to be unique among the heparin binding proteins in that a specific sequence within a 5-monosaccharide segment of

ACTION OF HEPARIN

487 COO-

CH 20 S 0 3 0

CH2OSO 3 0

OH

OH

NH -

0

0

OSO-

OH

sos

CH20SO 3 -

CO(~

NH

0 o

0

so~

so~

NH

so£

Figure 2. The pentasaccharide sequence required to induce a conformation change in the antithrombin III molecule which enhances the affinity of the antithrombin for heparin. Groups identified as being critical for the activity of the pentasaccharide are the unique 3-O-sulphate on glucosamine (centre monosaccharide residue), the N-sulphates on the glucosamine residues and the 6-O-sulphate on the centre glucosamine residue (Bjork and Lindahl, 1982).

some heparin molecules produces specific, high affinity binding of the heparin to antithrombin III (Rosenberg et al, 1978; Lindahl et al, 1979, 1984; Sinay et al, 1984). The sequence of this pentasaccharide (Figure 2) binds to antithrombin Ill in a two-step process (Olson et al, 1981). In contrast to the specific binding which is expressed in the second step, heparin molecules which do not possess the pentasaccharide sequence, and thus interact only in the first binding step, have much lower affinity for antithrombin Ill. They are classified as low affinity heparins. Practically, high and low affinity heparins are determined by the salt concentration required to elute them from antithrombin III (Andersson et al, 1976; Hook et al, 1976; Lam et al, 1976; Jordan et al, 1979). Both low and high affinity heparins interact with amino acid residues in the same region of the antithrombin Ill molecule (Nordenman et al, 1978; Nordenman and Bjork, 1981). Low affinity heparin has been shown to compete with high affinity heparin for binding to antithrombin III (Danielsson and Bjork, 1978). Chemical equations which summarize the binding of heparin to antithrombin are shown in Figure 3. The second step of high affinity heparin binding to antithrombin III produces a conformational change in the antithrombin III molecule which

K! H

+

AT

K2 ~ H~AT

""

H~AT

KHAT---- !/,leek 2, where KHATiS the overall dissociation constant for high affinity heparin binding to antithrombin Ill Figure 3. The two steps in the binding of heparin with high affinity for antithrombin Ill to the antithrombin molecule. The two binding steps are described by two equilibrium constants, Kj and K> Individually K~ = 4.3 x 10 5M and K_, = 3 x 10 3 M. H is heparin; A T is antithrombin [II; H * AT is the first, weak complex; H * A T is the complex with tightly bound heparin. The independently measured overall dissociation constant is 7.8 x Ill s M (Olson and Shore, 1982). All values are for pH 7.4, 0.15 M ionic strength and 25 °C.

488

C . M . JACKSON

makes the binding of heparin tighter (Olson et al, 1981). Associated with this conformational change is an alteration in the environment of a tryptophan residue in the antithrombin molecule which provides an easily observed spectral change. This change is monitored by either fluorescence spectroscopy or ultraviolet difference spectrophotometry (Danielsson and Bjork, 1978; Bjork and Nordling, 1979; Villanueva and Danishefskv, 1979; Villanueva et al, 1980; Olson and Shore, 1981; Blackburn et al, 198"4; Karp et al, 1984). Although it was initially argued that the spectral change in antithrombin III alone implied a conformational change within the protein (Nordenman et al, 1978; Blackburn et al, 1984; Karp et al, 1984), it was actually a detailed kinetic study which identified the two steps in the binding of high affinity heparin to antithrombin III that proved it (Olson et al, 1981). The tightened binding which this conformation change produces is extremely important for the mechanism of high affinity heparin action. As noted above, low affinity heparin only interacts with antithrombin III in the first step and thus the binding constants for low affinity heparin are essentially the same as those for high affinity heparin in this first step (Nordenman et al, 1978). It is this first step which is predicted to be dependent on net charge, molecular weight and ionic strength (Nordenman and Bjork, 1981). PROTEINASE INACTIVATION The fundamental chemical reaction through which heparin activity is expressed is inactivation of a proteinase with an active site-reactive irreversible inhibitor, either antithrombin III or heparin cofactor II. Based on the data in Table 1 and the arguments about the relative inhibitor and proteinase concentrations, it is the kinetic behaviour of this reaction and its modification by heparin that will be the focus for the analysis which follows. Reaction of a proteinase with an irreversible inhibitor can be viewed from several perspectives. This reaction is perhaps most readily understood, however, by viewing it as a proteolytic reaction which stops at an intermediate step in the process. This will be discussed first for the case in which no heparin is present. From an enzyme action perspective, the proteinase (E) reacts first with the inhibitor (I) to form a reversible or Michaelis complex (EI), similar to the reaction between an enzyme (E) and substrate (S) to form (ES), the enzyme-substrate complex in an enzyme-catalysed reaction (Figure 4). For proteinases, (ES) changes to enzyme product (EP) in a subsequent step and an enzyme-product complex is formed in which the product, a peptide, is covalently linked to an active site residue of the proteinase. When this step in the reaction involves (EI), an enzymeinhibitor rather than an enzyme-substrate, the reaction stops and an active site blocked proteolytic enzyme is produced. If the reaction were between (E) and (S), then a water molecule would be available which would hydrolyse the bond with the active site residue, e.g. serine. Thus the peptide product would be split out and the enzyme regenerated so that it could act on other substrate molecules. (This mechanism is essentially the same as that

ACTION OF HEPARIN

489

k 2 /

Kd P

+

i . ....

PII

proteinase Inactivation

"Pel Proteolytic

P "Michaelis Complex"

÷

Im

Cleavage

Products

Figure 4. The chemical equations describing the pathway for inactivation of the coagulation proteinases in the absence of heparin. Kd is the dissociation constant for formation of the non-covalent, reversible Michaelis complex, k2 is the rate constant for formation of the inactive, covalent proteinase inhibitor (antithrombin III or heparin cofactor II) complex (P * I), k~.is the catalytic constant which here describes the formation of the modified inhibitor, lm. The rate constant used to describe the efficiency of the inactivation reaction, k*. is equal to kz/Kd.

for other irreversible inhibitors of enzymes and is an example of the now classical mechanism proposed by Kitz and Wilson (1962).) It is important to reiterate that the fundamental process is proteinase inactivation by the mechanism shown in Figure 4 and thus much of what is known about other irreversible inhibitors of other enzymes can be drawn upon when trying to understand heparin-enhanced proteinase inactivation. Reaction of proteinases with antithrombin does not always result in quantitative inactivation of each proteinase molecule which forms an (El) complex (Fish et al, 1979; Marciniak, 1981; Olson, 1985). The antithrombin in a few (El) complexes is 'treated' by the enzyme as a substrate and is cleaved to form an inactive antithrombin molecule, designated ATm (Fish et al, 1979; Jornvall et al, 1979; Marciniak, 1981). This reaction occurs to a very limited extent unless heparin is present. Cleavage of antithrombin to form ATm occurs with all of the proteinases examined (Fish et al, 1979; Marciniak, 1981; Bjork et al, 1982) and can confound interpretation of in vitro experimental results. In order to compare the inhibitors listed in Table 1 with respect to their efficiency in inactivating the different proteinases of the clotting system, and, more importantly, to compare heparins with different molecular weights with respect to the efficiency with which they promote proteinase inactivation, a brief description is necessary of how the fundamental kinetic information from measurement of proteinase inactivation can be interpreted. When an excess of inhibitor relative to proteinase is present in the reaction, which was noted above to be the situation in vivo, the logarithm of the proteinase concentration changes linearly with time. The slope of this line is equal to the rate constant for the reaction multiplied by the inhibitor concentration and thus the rate constant is easily calculated. The rate constant (k*) can be viewed as equal to the velocity of the inactivation reaction when the concentrations of all reactants are 1 M. Clearly concentrations of the reactants are not 1 M; however, this example points out the important fact that the rate constant does not depend on the concentrations

490

c. M, JACKSON

of the reactants. A rate constant is thus the simplest single number to use for making comparisons of efficiency of different heparins in enhancing proteinase inactivation. Although this way of understanding k* values is somewhat oversimplified, it provides an accurate indicator of the efficiency with which inhibitors act and the efficiency with which different heparins increase the rate of proteinase inactivation. In subsequent sections of this chapter proteinase inactivation efficiencies will be compared using the rate constants, abbreviated k*. The efficiency of proteinase inactivation in the presence of heparin can be readily compared, both to the inactivation process when heparin is absent and, for heparins of different affinity for antithrombin III and molecular weight, with each other. Rate constants for the inactivation of thrombin and factor Xa by antithrombin III in the absence of heparin differ by no more than twofold; factor IXa is significantly slower (Jordan et al, 1980). Inactivation of thrombin by heparin cofactor II, also in the absence of heparin or other glycosaminoglycans, occurs with a rate constant which is similar to the rate constant for thrombin inactivation by antithrombin III. Thus, the general conclusion can be drawn, from comparisons of rate constants for proteinase inactivation in the absence of heparin, that all of the proteinases are inactivated by antithrombin III with similar, although not the same, efficiency. In vivo and in plasma samples ex vivo or in vitro all of the proteinase inhibitors listed in Table 1 contribute to the inactivation of the clotting proteinases. Reasonable estimates of the contribution of each can be made using the available values for the inactivation rate constants and the inhibitor concentrations, Thrombin and factor Xa are inactivated by eqproteinase inhibitor with 1% and 5% respectively of the efficiency of antithrombin Ill (Downing et al, 1978; Ellis et al, 1982) in the absence of heparin. The quantitative contribution of each inhibitor to the inactivation of the proteinase is determined by the reaction velocity, V = k* [P] [I], not by k* alone. Thus, when the inhibitor concentrations are taken into account, the differences between the contributions of antithrombin III and eqproteinase inhibitor are smaller, because of the high oq-antiproteinase inhibitor concentration. In the absence of any heparin, cq-proteinase inhibitor accounts for 10% of the thrombin inhibition and 30% of the factor Xa inhibition. These calculations are part of the basis for the conclusion, which was stated earlier, that antithrombin is the main inhibitor of the coagulation proteinases. HEPARIN CATALYSIS OF PROTEINASE INACTIVATION The catalytic action of heparin in proteinase inactivation was firmly established by experiments which demonstrated that a single heparin molecule could increase the rate at which many proteinase molecules, thrombin or factor Xa, were inactivated (Markwardt and Walsman, 1959; Gitel, 1975; Bjork and Nordenman, 197& Kowalski and Finlay, 1979; Biork and Lindahl, 1982). Based on the demonstrated tight binding of high affinity

491

ACTION OF HEPARIN

k 2

Pel

Proteinase Inactivation

P

+

HI~

>Pel

+

H Proteolytic

P "Michaelis or Ternary Complex"

÷

]m

Cleavage

Products

Figure 5. The chemicalequations describing the pathway for inactivationof the coagulation

proteinases during the heparin catalysed process. The heparin-inhibitor (antithrombin III) complex(HI) is formedas describedin Figure3. Kj is the dissociationconstantfor formationof the noncovalent,P , I complex, the reversible, Michaeliscomplex, k2 is the rate constant for

\H/

formationof the inactive, covalent proteinase-inhibitor(antithrombinIII or heparin cofactor II) complex, kc is the catalytic constant which here dissolves the formation of the modified inhibitor Im. P: proteinase; I: inhibitor. heparin to antithrombin III and the high antithrombin III concentration relative to proteinase concentration, it is reasonable to propose that when heparin with high affinity for antithrombin III is present, the inactivation reaction occurs primarily between the heparin-antithrombin complex and the proteinase (Figure 5). The presence of the heparin on the product side of the chemical equation indicates that it is released after the proteinase is inactivated. The heparin molecule is then free to bind to another antithrombin molecule (see Figure 3) and catalyse the inactivation of another proteinase molecule. Under physiological conditions (pH 7.4) and ionic strength 0.15, the dissociation constant (Kd) for binding of high affinity heparin (molecular weight 7000) to antithrombin III is 7 x 10-~M (Olson and Shore, 1982, 1986). At an antithrombin III concentration of 2.7 ~M (Murano et al, 1980; Conard et al, 1983), equivalent to its concentration in plasma, greater than 80% of the high affinity heparin is bound to the antithrombin III in purified, in vitro experimental systems. A 15000 molecular weight heparin molecule will bind approximately six times more tightly and thus essentially all high affinity heparin will be bound to antithrombin III. In blood and plasma the concentration of heparin-antithrombin III complex will be less than this because of heparin binding to the other plasma proteins which are present. The low affinity heparin in standard therapeutic heparin preparations also binds to these other proteins and thus spares some of the high affinity heparin for its selective binding to antithrombin III (Barrowcliffe et al, 1984). The extent to which heparin partitions among the various proteins to which it binds in plasma is not easy to determine. This uncertainty in the actual concentration of reactive heparin-antithrombin III complex is one important source of differences in results and conclusions obtained from investigations of heparin action using purified components and those which use plasma. It is very likely that a significant number of

492

c.M. JACKSON

discrepancies currently in the literature will disappear once this confounding situation can be quantitatively taken into account. As noted above for reactions in the absence of heparin, antithrombin I II is processed as a substrate as well as an inhibitor in the presence of heparin (Fish et al, 1979) (Figure 5). With thrombin, the extent to which cleavage to form ATm occurs relative to inactivation depends very strongly on ionic strength (Olson, 1985). Because this ionic strength dependence has only recently been described, some early studies of thrombin inactivation at low ionic strength may require re-evaluation or reinterpretation (Aranyi et al, 1977; Borsodi and Machovich, 1979; Machovich et al, 1979). Formation of ATm may also be responsible for a decreased antithrombin concentration after administration of heparin to patients (Marciniak and Gockerman, 1977). Measurement of the inactivation of the proteinases, thrombin or factor Xa in the presence of heparin is performed in the same way as described above for the inactivation of these proteinases by antithrombin III in the absence of heparin. However, because heparin is so effective in increasing the rate of inactivation, the concentration of antithrombin lII in in vitro experiments is rarely as high as in plasma. As a consequence, less of the heparin is bound to the antithrombin III than indicated above and thus estimates for the rate constants from experiments which include heparin are frequently lower than the true values. In the situations in which all high affinity heparin is not bound to antithrombin III, the rate constant cannot be simply obtained by dividing the slope of the inactivation curve by the heparin concentration or the antithrombin concentration. Instead, what must be done is to solve the algebraic equation which describes the concentration of heparin-antithrombin III complex as a function of heparin and antithrombin concentrations and the binding constant for these conditions. The concentration of the heparin-antithrombin III complex must then be used for calculating the rate constant from the inactivation curve slope. The magnitude by which an individual heparin molecule increases the rate constant for proteinase inactivation by antithrombin III depends on the molecular weight of the heparin (Laurent et al, 1978; Lane et al, 1979; Thunberg et al, 1979; Danielsson and Bjork, 1981; Holmer et al, 1981). Data showing the efficiencies as k* values for high affinity heparins for thrombin and factor Xa inactivation from experiments in which all heparin is present as heparin-antithrombin III complex are shown in Table 2. Rates for the situation in which antithrombin concentration is equal to its concentration in plasma and the proteinases are at the same relative concentration as their precursors are also given in Table 2. (It must be noted that these numbers cannot be applied without qualification to in vivo situations or in plasma samples because of a difference in the pH, because bovine factor Xa was employed in these experiments, and because they apply to a solution which does not contain other proteins to which heparin binds.) Several conclusions can be drawn from these data in addition to the dependence of efficiency on molecular weight already stated. First, although thrombin and factor Xa are inactivated with rate constants which differ by a factor of only two in the absence of heparin, these two proteinases are inactivated with

493

ACTION OF HEPARIN Table 2. Rate constants for proteinase inactivation by antithrombin III and heparin.

Calculated inactivation velocities*

k* (M-'S-~) No heparin Plus heparin

(M.s-~)

Thrombin

Factor Xa

Thrombin

Factor Xa

1.0 x 104

5.8 x 103

2.7 x 10 -11

1.6 x 10 -11

1.6 × -1.2 x 1.2 x 1.5 x 2.0 x

2.7 1.9 2.7 3.2

3.2 3.2 4.1 5.4

(molecular weight) 1720 3000 4500 6000 15400 20300

1.0 2.2 6.9 9.9 1.2

-× x x x x

107 107 107 108 109

106 106 106 107 107

x x x x

10 - s 10 7 10 .6 10 -6

x x x x

10 lo 10 -1° 10 -9 10 -9

¢ Thrombin, 1 riM; factor Xa, 0,1 riM; antithrombin III, 2.7 tXM.

very different efficiencies when heparin is present to catalyse the reaction. Moreover, the efficiencies, given by k* values in the table, are different for thrombin and factor Xa for each of the different molecular weight heparins. Heparin molecules with molecular weights greater than 15 000 enhance the rate of thrombin inactivation by a factor of 120 000, whereas they enhance factor Xa inactivation by a factor of only 3500, a more than thirtyfold greater efficiency for thrombin than factor Xa inactivation. Heparin molecules with molecular weights of less than 6000 enhance the rate of thrombin inactivation by a factor of as much as 2000, whereas they enhance factor Xa inactivation by only 200-300 relative to the uncatalysed reaction. (This enhancement occurs in vitro with purified components under conditions where all the heparin is bound to antithrombin III and none is bound to thrombin. In situations in which heparin is bound to thrombin, the enhancement by these low molecular weight heparins will be very substantially less.) Even these low molecular weight heparins exhibit a tenfold greater enhancement of thrombin than factor Xa inactivation. Efficiencies of high molecular weight heparins relative to low molecular weight heparins in catalysing thrombin inactivation are clearly much greater than they are for factor Xa. The ratio of k* values for thrombin inactivation by high versus low molecular weight heparins is 100; for factor Xa it is only 10. Viewed from another perspective, low molecular weight heparins are only 1% as effective as high molecular weight heparins in promoting thrombin inactivation, but are 10% as effective in promoting factor Xa inactivation. This difference in relative efficiency may explain in part why low molecular weight heparins are generally concluded to be inactive in thrombin inactivation; a level of 1% would certainly appear inactive in most traditional assay procedures, whereas a 10% level would be detected and considered significant in most factor Xa based assay procedures. Another process which contributes to the observed difference in relative ability of low and high molecular weight heparins to catalyse the inactivation of these two proteinases is the binding of heparin directly to thrombin, which is discussed in more detail in the sections below. Heparin binding to thrombin interferes with the reaction of thrombin with heparin-antithrombin III

494

c.M. JACKSON

complex. The low molecular weight heparins, which will be assa~ced at higher concentrations in thrombin inactivation assays because the k-values, i.e. their efficiencies, are lower, will produce more heparin-thrombin complex and thus appear even poorer catalysts of thrombin inactivation than they actually are under ideal conditions. The hundredfold greater efficiency of high molecular weight heparin in catalysing thrombin inactivation over factor Xa also implies that, on the basis of the number of proteinase molecules inactivated per heparin molecule in any time interval, thrombin is the preferred target proteinase for inactivation by a factor of 100. MOLECULAR MECHANISM OF HEPARIN CATALYSIS The large rate enhancements shown by the data in Table 2 are indicative of the efficiency by which heparin catalyses proteinase inactivation, not how it might be doing it. Referring to Figure 5, two places in the inactivation reaction can be identified as potential candidates for the step at which heparin acts. The first is the step in which the proteinase-heparinantithrombin complex is formed; the second is the step in which the antithrombin III in this complex reacts with the active site of the proteinase to produce the inactive proteinase. An early suggestion was that the conformation change in antithrombin III, which occurs as a result of high affinity heparin binding, made the antithrombin III to which the heparin was bound more reactive (Rosenberg and Damus, 1973). In this case, the effect of heparin would be on the second step, i.e. the step in which the irreversible, covalent protein-antithrombin complex is formed. A detailed investigation of the reaction of thrombin with antithrombin III and with the heparinantithrombin complex using rapid kinetic techniques indicates, however, that this step is not a good candidate, something which could not have been known at the time of the original proposal. (The studies which have definitively established the mechanism of prothrombin inactivation by high affinity heparin and antithrombin III were designed to eliminate the complication of direct binding of heparin to thrombin. This was done by carrying out the reactions in 0.3 Msodium chloride. Although there is no reason either to suspect or question the conclusions of these studies, the consequences of direct interaction of heparin with thrombin which occur at lower ionic strengths are not included in the results of this study and thus must be considered separately and in addition to that.) The rate constant (k2) in Figures 4 and 5 for the step involving direct formation of the irreversible complex is not significantly different for the uncatalysed or the heparin catalysed reactions (Olson and Shore, 1982, 1986). If anything, the rate constant for the second step is smaller in the presence of heparin rather than larger (Olson and Shore, 1982, 1986). Unless future investigations indicate an error in this work, the mechanism which was originally proposed (Rosenberg and Damus, 1973) does not appear to be applicable to thrombin inactivation. The other alternative, an effect on the formation of the heparin-thrombin-antithrombin complex (the Michaelis complex) has direct evidence to support it. The dissociation constant for formation of this

ACTION OF HEPARIN

495

ternary complex with a 7000 molecular weight heparin has been determined to be 4 ~M, whereas the dissociation constant for the formation of the thrombin-antithrombin non-covalent complex in the absence of heparin is 1.4 mM (Olson and Shore, 1982, 1986). Heparin thus causes the formation of the reactive intermediate complex to occur at approximately 350 times lower concentration. Stated otherwise, 350 times more complex is formed at the same thrombin and antithrombin III concentrations when the heparin is present than would be formed if heparin were not present. This evidence indicates that heparin action and thrombin inactivation occur by enhancing binding of the reactants to form the Michaelis complex, an effect sometimes called a 'proximity effect' in chemical kinetics. No published experimental data exist which provide the same kind of detailed information about factor Xa inactivation, and thus both mechanisms must still be considered as plausible for the inactivation of this proteinase. PROTEINASE INACTIVATION AT HIGH HEPARIN CONCENTRATIONS

Although heparin action as a catalyst of proteinase inactivation by antithrombin III generally implies low concentrations of heparin relative to the antithrombin or proteinase concentrations, investigations of thrombin inactivation at heparin concentrations in excess of the antithrombin concentration have provided insight into the mechanism of heparin action and some additional explanations for discordant results from studies in which plasma is the source of antithrombin. Investigations of the behaviour of thrombin inactivation in systems containing only thrombin, antithrombin III and heparin indicate that such increases in heparin concentration lead to the formation of a significant concentration of thrombin-heparin complex as well as heparin-antithrombin complex (Machovich, 1975; Jordan et al, 1980; Griffith, 1982; Pletcher and Nelsestuen, 1982; Nesheim, 1983: Pletcher et al, 1985). In vitro studies reflect formation of the thrombinheparin complex by a maximum in the plot of the thrombin inactivation rate or rate constant versus heparin concentration (or log of heparin concentration when the heparin concentration range is very wide) (Jordan et al, 1980; Griffith, 1982; Pletcher and Nelsestuen, 1982; Nesheim, 1983; Pletcher et al, 1985). The presence of such a maximum in this graph is evidence for a thrombin-heparin-antithrombin ternary complex as the productive complex in the proteinase inactivation process because it indicates that interaction of thrombin-heparin with heparin-antithrombin, the two binary complexes which will be present under these conditions, do not form a productive reaction intermediate such as that shown in Figure 5. The maximum in the dependence of the rate constant or rate on heparin concentration also produces values for k*, the intrinsic rate constant, which are less than what would be determined with catalytic heparin. These reactions, i.e. pathways by which both the productive ternary complexes can form and non-productive formation of both binary complexes interfere, are shown in Figure 6. The formation of substantial concentrations of both binary

496

c . M . JACKSON

When K(HAT) Pe/~T

PAT + H

', H ,' When

K(pH)

PeH+AT


> PAT + H

't H /

When

H is in e x c e s s ,

P+H+AT~

&H>K

Pert+HeAT

/

(HAT) \

& H>K

(PH)

peAT

'H'

Figure 6. Preferred pathway determination from relative magnitudes of the H-AT and H-P dissociation constants and the H concentration. 1. When Kd(HAT)"~Kd(PVI),binding of the limited number of H molecules will preferentially be to AT thus producing primarily, if not exclusively, H.AT as the heparin-containing reactant. In this situation P reacts with H.AT to produce the P- AT intermediate complex. This is the usual situation when all H has high

\H/

affinity for AT. 2. When Kd(pH) ~ Ka(HAT),binding of the limited number of H molecules will preferentially be to P thus producing primarily, if not exclusively, PH. In this situation PH will react with free AT, i.e. AT without a heparin molecule bound to produce the P - AT complex.

\H/

This is a plausible route for forming the P - AT complex when the H does not possess the pentasaccharide which conveys high affinity for AT. 3. When excess H is present, particularly when both high and low affinity molecules are contained in the heparin mixture, both PH and HAT complexes will form. The poor or non-reactivityof these two species results in a decreased proteinase inactivation rate. c o m p l e x e s l e a d s to t h e d e c r e a s e in r e a c t i o n r a t e a n d is r e s p o n s i b l e for the d e s c e n d i n g l i m b o f the g r a p h o f r a t e v e r s u s h e p a r i n c o n c e n t r a t i o n w h i c h is s e e n at high h e p a r i n c o n c e n t r a t i o n s . A s n o t e d a b o v e , in t h e s e c t i o n on t h e m o l e c u l a r m e c h a n i s m o f h e p a r i n action, t h e p r i m a r y p a t h w a y for p r o t e i n a s e i n a c t i v a t i o n in t h e p r e s e n c e of high affinity h e p a r i n at c a t a l y t i c c o n c e n t r a t i o n s involves r e a c t i o n o f free t h r o m b i n o r f a c t o r X a with h e p a r i n - a n t i t h r o m b i n c o m p l e x , t h e first r e a c t i o n s h o w n in F i g u r e 6. T h e e x t e n t to which e a c h o f the b i n a r y c o m p l e x e s , h e p a r i n - a n t i t h r o m b i n a n d h e p a r i n - t h r o m b i n , c o n t r i b u t e to t h e f o r m a t i o n o f t h e r e a c t i v e t e r n a r y c o m p l e x d e p e n d s on the e q u i l i b r i u m c o n s t a n t s for t h e i r f o r m a t i o n a n d the c o n c e n t r a t i o n s o f h e p a r i n , t h r o m b i n a n d antit h r o m b i n III. D i s s o c i a t i o n c o n s t a n t s w h i c h q u a n t i t a t i v e l y d e s c r i b e t h e b i n d i n g o f h e p a r i n to t h r o m b i n s p a n a v e r y w i d e r a n g e of v a l u e s , f r o m as low as 10 nM (Griffith, 1982) to as high as 1 ~M ( E v i n g t o n et al, 1986a,b). T h e b i n d i n g c o n s t a n t at t h e l o w e r limit i m p l i e s that h e p a r i n b i n d s at least as

ACTION OF H E P A R I N

497

tightly, if not more so, to thrombin as to antithrombin III, whereas at the high limit, binding of high affinity heparin to antithrombin III is 30-100 times tighter than to thrombin. Two underlying causes for this uncertainty in the values for heparin-antithrombin dissociation constants have been identified: (1) the strong dependence of binding to thrombin on pH and ionic strength or salt concentration in the solution (these dissociation constants are from solutions of different composition); and (2) the binding of more than one thrombin molecule to a high molecular weight heparin molecule (Evington et al, 1986a). A predictable consequence of this variation in binding constant is that, under different reaction conditions, thrombin inactivation will proceed to variable extents via the thrombin-heparin and the heparin-antithrombin binary complexes seen in Figure 6. Considerable confusion has resulted from this strong dependence of the dissociation constant for thrombin-heparin on ionic strength, particularly in early studies of the mechanism of heparin action in the inactivation of thrombin (Machovich, 1975; Sturzebecher and Markwardt, 1977; Machovich et al, 1979). In plasma, heparin concentrations are increased to overcome the decrease in the extent of heparin-antithrombin complex formation which results from heparin binding to other plasma proteins. Inactivation of factor Xa in the presence of heparin is simpler than that of thrombin (Jordan et al, 1980). Binding of heparin to factor Xa is very weak compared to its binding to thrombin, at least × 1000 times weaker (Jordan et al, 1980), and thus no significant formation of a factor Xa-heparin complex occurs at heparin concentrations generally used in laboratory investigations. The other clearly important target proteinase for inactivation by antithrombin III in the presence of heparin, factor IXa, may be more like thrombin than it is like factor Xa. This tentative proposal is based on inferences which can be made from the heparin molecular weight dependence of factor IXa inactivation (Holmer et al, 1981). No other direct evidence exists by which one may be more certain about the inactivation characteristics of factor IXa. INACTIVATION REACTIONS INVOLVING ANTITHROMBIN III IN THE PRESENCE OF LOW AFFINITY HEPARIN Heparins and heparin-like glycosaminoglycans which do not contain the pentasaccharide which confers high affinity for antithrombin have become interesting because of their demonstrable anticoagulant and antithrombotic effects in vivo (Buchanan et al, 1985; Ofosu et al, 1985). Studies of heparin action in vitro have frequently produced inconsistent results which, however, have chemical and physical explanations. If a heparin sample is devoid of heparin with high affinity for antithrombin III, then binding of heparin will only occur to thrombin. This occurs because the dissociation constant for the formation of the heparin-thrombin complex under most conditions is much lower than that for binding of low affinity heparin to antithrombin III. As a consequence, only the pathway shown at the centre of Figure 6 is available for formation of the ternary thrombin-heparin-

498

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antithrombin complex, the complex through which thrombin is efficiently inactivated. Hogg and Jackson (1989) indicate that k* values for thrombin inactivation with low affinity heparin are significantly smaller than those for high affinity heparin. However, because the contribution of a particular pathway depends on k* values and reactant concentrations, i.e. thrombin, heparin and antithrombin concentrations, conditions may exist for which this low affinity heparin-catalysed pathway is quantitatively important in thrombin inactivation and to exclude it a priori does not appear to be justified. One situation in which low affinity heparin may be particularly important is on the surface of the endothelial cell, where only small fractions of the surface glycosaminoglycans are high affinity heparins (Marcum and Rosenberg, 1987). HEPARIN COFACTOR Ih THROMBIN INHIBITION CATALYSED BY HEPARIN AND OTHER HIGHLY NEGATIVELY CHARGED POLYMERS

Inactivation of thrombin by heparin cofactor II is also catalysed by heparin, although, in contrast to inactivation by antithrombin III, no heparin structural specificity appears to exist for heparin binding to heparin cofactor II (Briginshaw and Shanberge, 1974; Tollefsen and Blank, 1981; Tollefsen et al, 1982). Not only are heparins effective in promoting the inactivation of thrombin by heparin cofactor II, but so are dermatan sulphates and various other sulphated polysaccharides (Tollefsen et al, 1983; Sie et al, 1986; Church et al, 1987). Heparin cofactor II is not effective in the inactivation of factor Xa, although it is effective in the inactivation of chymotrypsin and cathepsin B (Parker and Tollefsen, 1985)= Inactivation of these latter proteinases is not accelerated by heparin, k values for heparin cofactor II inactivation of thrombin are lower than those for the heparin-antithrombin complex with values of 2 × 108M-~s -~ for heparin cofactor II under conditions where heparin-antithrombin inactivation with the same heparin is described by a k* value of 1.2× 109M-Is-1. No values for dissociation constants for heparin binding to heparin cofactor II have been reported; however, from the relative affinity of heparin cofactor II and antithrombin for heparin linked to agarose, it is clear that the affinity of heparin cofactor II for heparin is lower than that of antithrombin for heparin. No mechanistic studies comparable to those for heparin catalysed inactivation of thrombin by antithrombin III have been reported for heparin cofactor II. Although the primary mechanism of proteinase inactivation by antithrombin III in the presence of heparin is via the heparin-antithrombin complex, thrombin inactivation via the thrombin heparin complex with heparin cofactor II cannot be excluded under some conditions, perhaps for example, at celt surfaces. Although the complexity of the interactions of heparin with proteins present in plasma cannot be eliminated, quantitative descriptions of these interactions and reactions can provide a clearer picture which can make

ACTION OF HEPARIN

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future evolution of understanding possible. It is virtually certain that compensation for the effects of change and molecular weight heterogeneity can be explicitly taken into account and thus the confusion they produce reduced to a negligible level. It can be predicted with considerable confidence that the next decade will produce as dramatic advances as the last, and thus the new heparin derivations currently being investigated will join the conventional heparin of the past as effective and well understood antithrombotic agents.

SUMMARY Heparin catalysis of clotting proteinase inactivation occurs most efficiently through the reaction of the proteinase with the antithrombin-heparin complex. The efficiency of a heparin molecule in this reaction depends on the presence of a specific pentasaccharide sequence in it, and its molecular weight. The mechanism by which such high affinity heparin acts when antithrombin III is the inhibitor is p r o m o t i o n of the formation of an intermediate p r o t e i n a s e - h e p a r i n - a n t i t h r o m b i n complex. Heparin p r o m o t i o n of thrombin inactivation by heparin cofactor II may occur by a similar mechanism. The requirement for a specific oligosaccharide sequence within the heparin molecule does not, however, exist for heparin cofactor II. Binding of heparin to both thrombin and antithrombin I I I interferes with thrombin inactivation. This binding is very dependent on the ionic strength of the reaction mixture and may explain some of the discordant results and interpretations f r o m early studies on the mechanism of heparin action. Low ionic strength in in vitro reactions also results in cleavage of antithrombin III by thrombin in the presence of heparin and effectively converts antithrombin III from an inhibitor to a substrate.

REFERENCES Abildgaard U (1968) Highly purified antithrombin III with heparin cofactor activity prepared by disc electrophoresis. Scandinavian Journal of Clinical and Laboratory Investigation 21: 89-91. Abildgaard U (1969) Binding of thrombin to antithrombin III. Scandinavian JournalofClinical and Laboratory Investigation 24: 23-27. Andersson L-O, BarrowcliffeTW, Hotmer E, Johnson EA & Sims GEC (1976) Anticoagulant properties of heparin ffactionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration. Thrombosis Research 9: 575-583. Aranyi P, Batke J & MachovichR (1977) Remarks on the interaction of thrombin and heparin. Archives of Biochemistry and Biophysics 181: 678-679. Atkins EDT & Nieduszynski IA (1976) Heparin: crystalline structures of the sodium and calcium salts. In Kakkar VV & Thomas DP (eds) Heparin: Chemistry and Clinical Usage, pp 21-35. London: Academic Press. BarrowcliffeTW & Thomas DP (1987) Antithrombin III and heparin. In Bloom AL & Thomas DP (eds) Haemostasis and Thrombosis, pp 849-869. London: Churchill Livingstone. Barrowcliffe TW, Merton RE, Havercroft SJ et al (1984) Low-affinityheparin potentiates with action of high-affinityheparin oligosaccharides. Thrombosis Research 34. 125-134. Barrowcliffe TW, Curtis AD, Tomlinson TP et al (1985) Standardization of low molecular weight heparins: a collaborative study. Thrombosis and Haemostasis 54: 675-679.

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Barton PG & Yin ET (1973) Inhibitors of blood-clotting mechanisms. Metabol&mlnhibitors 4: 215-310. Beeler DL, Marcum JA, Schiffman S & Rosenberg RD (1986) Interaction of Factor XIa and antithrombin in the presence and absence of heparin. Blood 67: 1488-1497. Biggs R, Denson KWE, Akman N, Borrett R & Hadden M (1975) Antithrombin III, antifactor Xa and heparin. British Journal of Haematology 19: 283-305. Bjork I & Lindahl U (1982) Mechanism of the anticoagulant action of heparin. Molecular and Cellular Biochemistry 48: 161-182. Bjork I & Nordling K (1979) Evidence by chemical modification for the involvement of one or more tryptophanyl residues of bovine antithrombin in the binding of high-affinity heparin. European Journal of Biochemistry 102: 497-502. Bjork I & Nordenman B (1976) Acceleration of the reaction between thrombin and antithrombin III by non-stoichiometric amounts of heparin. European Journal of Biochemistry 68: 507-511. Bjork I, Jackson CM, Jornvall H, Lavine KK & Nordling K (1982) The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa and thrombin. Journal of Biological Chemistry 2575: 2406-2411. Blackburn MN, Smith RL, Carson J & Sibley CC (1984) The heparin-binding site of antithrombin III: identification of a critical tryptophan in the amino acid sequence. Journal of Biological Chemistry 259: 939-941. Borsodi A & Machovich R (1979) Inhibition of esterase and amidase activities of ~x- and 13-thrombin in the presence of antithrombin III and heparin. Biochimica et BiophysicaActa 566: 385-389. Briginshaw GF & Shanberge JN (I974) Identification of two distinct heparin cofactors in human plasma separation and partial purification. Archives of Biochemistry and Biophysics 161: 683-690. Brinkhous KM, Smith HP, Warner ED & Seegers WH (1939) The inhibition of blood clotting: an unidentified substance which acts in conjunction with heparin to prevent the conversion of prothrombin into thrombin. American Journal of Physiology 125: 683-687. Brown KD (1963) Chemistry of heparin. In Engelberg H & Brown KD (eds) Heparin: Metabolism, Physiology and ClinicalApplication, pp 5-19. Springfield: CC Thomas. Buchanan MR, Boneu B, Ofosu F & Hirsh J (1985) The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin. Blood 65: 198-201. Church FC, Treanor RE, Sherrill GB & Whinna HC (1987) Carboxylate polyanions accelerate inhibition of thrombin by heparin cofactor II. Biochemistry and Biophysical Research Communications 148: 362-368. Conard J, Brosstad F, Larsen ML, Samama M & Abildgaard U (1983) Molar antithrombin concentration in normal human plasma. Haemostasis 13: 363-368. Damus PS, Hicks M & Rosenberg RD (1973) Anticoagulant action of heparin. Nature 246: 355-357. Danielsson A & Bjork I (1978) The binding of low-affinity and high-affinity heparin to antithrombin. European Journal of Biochemistry 90: 7-12. Danielsson A & Bjork I (1981) Binding to antithrombin of heparin fractions with different molecular weights. Biochemical Journal 193: 427-433. Dombrose FA, Seegers WH & Sedensky JA (1971) Antithrombin: inhibition of thrombin and autoprothrombin C (F-Xa) as a mutual depletion system. Thrombosis et Diathesis Haemorrhagica 26: 103-123. Downing MR, Blood JW & Mann KC (1978) Comparison of the inhibition of thrombin by three plasma protease inhibitors. Biochemistry 17: 2649-2653. Ellis V, Scully M, MacGregor I & Kakkar V (1982) Inhibition of human factor Xa by various plasma protease inhibitors, Biochimica et Biophysica Acta 701: 24-31, Evington JRN, Feldman PA, Luscombe M & Holbrook JJ (1986a) Multiple complexes of thrombin and heparin, Biochimica et Biophysica Acta 871: 85-92, Evington JRN, Feldman PA, Luscombe M & Holbrook JJ (1986b) The catalysis by heparin of the reaction between thrombin and antithrombin. Biochimica et Biophysica Acta 870: 92-101. Fareed J, Walenga JM, Racanelli A, Hoppensteadt D, Huan X & Messmore HL Jr (1988)

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Validity of the newly established low-molecular-weight heparin standard in crossreferencing low-molecular-weight heparins. Haemostasis 18: 33--47. Feinman RD (1983) Chemistry and biology of alpha-2-macroglobulin. Annals of the New York Academy of Sciences 421: 1-478. Fersht AR (1977) Enzyme Structure and Mechanism. San Francisco: WH Freeman. Fish WW, Orre K & Bjork I (1979) The production of an inactive form of antithrombin through limited proteolysis by thrombin. FEBS Letters 98: 103-106. Gaston LW (1964) The blood-clotting factors. New England Journal of Medicine 270: 236242. Gitel SN (1975) In Bradshaw RA & Wessler S (eds) Heparin: Structure, Function and Clinical Implications, pp 243-247. New York: Plenum Press. Gitel SN, Stephenson RC & Wessler S (1977) In vitro and in vivo correlation of clotting protease activity: effect of heparin. Proceedings of the National Academy of Sciences USA 74: 3028-3032. Gitel SN, Medina VM & Wessler S (1984) Inhibition of human activated factor X by antithrombin III and alpha-1-proteinase inhibitor. Journal of Biological Chemistry 259: 68906895. Gram J & Jespersen J (1985) On the significance of antithrombin-IlI, alpha-2-macroglobulin, alpha-2-antiplasmin, histidine-rich glycoprotein, and protein C in patients with acute myocardial infarction and deep vein thrombosis. Thrombosis and Haemostasis 54: 503505. Griffith MJ (1982) Kinetics of the heparin-enhanced antithrombin III/thrombin reaction. Evidence for a template model for the mechanism of action of heparin. Journal of Biological Chemistry 257: 7360-7365. Griffith M J, Noyes CM & Church FC (1985) Reactive site peptide structural similarity between heparin cofactor II and antithrombin III, Journal of Biological Chemistry 260: 2218-2225. Harpe! PC (1987) Blood proteolytic enzyme inhibitors: their role in modulating blood coagulation and fibrinolytic enzyme pathways. In Colman RW, Hirsh J, Marder VJ & Salzman EW (eds) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, pp 219-234. Philadelphia: JB Lippincott. Hedner U & Davie EW (1987) Factor IX. In Colman RW, Hirsh J, Marder VJ & Salzman EW (eds) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, pp 39-47. Philadelphia: JB Lippincott. Hogg PJ & Jackson CM (1989) Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III: implications for heparin efficacy, Proceedings of the National Academy of Sciences of the USA 86: 3619-3623. Holmer E, Kurachi K & Soderstrom G (1981) The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor IIa and kallikrein by antithrombin. Biochemical Journal 193: 395-400. Hook M, Bjork I, Hopwood H & Lindahl U (1976) Anticoagulant activity of heparin: separation of high-activity and low-activity heparin species by affinity chromatography on immobilized antithrombin. FEBS Letters 66: 90-93. Horner AA (1976) High and low molecular weight forms of heparin. In Kakkar VV & Thomas DP (eds) Heparin: Chemistry and Clinical Usage, pp 37--47. London: Academic Press. Howell WH (1924) The purification of heparin and its presence in blood. American Journal of Physiology 71: 553-562. Jackson CM & Hanahan DJ (1968) Studies on bovine factor X: I. Large scale purification of the bovine plasma protein possessing factor X activity. Biochemistry 7: 4492-4505. Jaques LB (1979) Heparin: an old drug with a new paradigm. Science 206: 528-533. Jeppson JO, Laurell CB & Fagerhol M (1978) European Journal of Biochemistry 83: 143-153. Jordan R, Beeler D & Rosenberg R (1979) Fractionation of low molecular weight heparin species and their interaction with antithrombin. Journal of Biological Chemistry 254: 2902-2913. Jordan RE, Oosta GM, Gardner WT & Rosenberg RD (1980) The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin. Journal of Biological Chemistry 255: 10081-10090, Jornvall H, Fish WW & Bjork I (1979) The thrombin cleavage site in bovine antithrombin. FEBS Letters 106: 358-362. Jorpes E (1935) Heparin, a chondroitin polysulfuric acid. Naturwissenschaften 23: 196-197.

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Jorpes E & Bergstrom S (1937) Heparin, a mucoitin polysulfuric acid, Journal of Biological Chemistry 118: 447-457. Karp GI, Marcum JA & Rosenberg RD (1984) The role of tryptophan residues in heparinantithrombin interactions. Archives of Biochemistry and Biophysics 233:712-720. Kiss J (1976) Chemical structure of heparin. In Kakkar VV & Thomas DP (eds) Heparin: Chemistry and Clinical Usage, pp 3-20. London: Academic Press. Kitz R & Wilson IB (1962) Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. Journal of Biological Chemistry 237: 3245-3249. Kowalski S & Finlay TH (1979) Heparin and the inactivation of thrombin by antithrombin III. Thrombosis Research 14: 387-397. Kurachi K, Fujikawa K, Schmer G & Davie E (1976) Inhibition of bovine factor IXa and factor Xa beta by antithrombin III. Biochemistry 15: 373-377. Lam LH, Silbert JE & Rosenberg RD (1976) The separation of active and inactive forms of heparin. Biochemistry and Biophysical Research Communications 69: 570-577. Lane DA, MacGregor IR, VanRoss M, Celia G & Kakkar VV (1979) Molecular weight dependence of the anticoagulant properties of heparin: intravenous and subcutaneous administration of fractionated heparins to man. Thrombosis Research 16: 651-662. Lasker SE (1977) The heterogeneity of heparins. Federation Proceedings 36: 92-97. Laurent TC, Tengblad A, Thunberg L, Hook M & Lindahl U (1978) The molecular-weightdependence of the anti-coagulant activity of heparin. Biochemical Journal 175: 691-701. Lindahl U & Hook M (1978) Glycosaminoglycans and their binding to biological macromolecules. Annual Review of Biochemistry 47: 385~117. Lindahl U, Backstrom G, Hook M, Thunberg L, Fransson LA & Linker A (1979) Structure of the antithrombin-binding site in heparin. Proceedings of the NationaI Academy of Sciences of the USA 76: 3198-3202. Lindahl U, Thunberg L, Backstrom G, Riesenfeld J, Nordling K & Bjork I (1984) Extension and structural variability of the antithrombin-binding sequence in heparin. Journal of Biological Chemistry 259: 12368-12376. Machovich R (1975) Mechanism of action of heparin through thrombin on blood coagulation. Biochimica et Biophysica Acta 412: 13-17. Machovich R, Regoeczi E & Hatton MWC (1979) The influence of heparin, NaCI and CaCI2 on the rate of the thrombin-antithrombin III reaction. Thrombosis Research 15: 821-834. McLean J (1916) The thromboplastic action of cephalin. American Journal of Physiology 41: 250-257. Marciniak E (1981) Thrombin-induced proteolysis of human antithrombin III: an outstanding contribution of heparin. British Journal of Haematology 48: 325-336. Marciniak E & Gockerman JP (1977) Heparin-induced decrease in circulating antithrombin III. Lancet ii: 581-584. Marcum JA & Rosenberg RD (1987) Anticoagulantly active heparan sulfate proteoglycan and the vascular endothelium. Seminars in Thrombosis and Hemostasis 13: 464-474. Markwardt F & Walsman P (1959) The mechanism of the antithrombin effect of heparin. Hoppe-Seyler's Zeitschrift fiir Physiologische Chemie 317: 64--77. Monkhouse FC, France ES & Seegers WH (1955) Studies on the antithrombin and heparin cofactor activities of a fraction adsorbed from plasma by aluminum hydroxide. Circulation Research IIh 397-402. Murano G, Williams L, Miller-Andersson M, Aronson DL & King C (1980) Some properties of antithrombin III and its concentration in human plasma. Thrombosis Research 18: 259262. Nesheim ME (1983) A simple rate law that describes the kinetics of the heparin-catalyzed reaction between antithrombin III and thrombin. Journal of Biological Chemistry 258: 14708-14719. Nordenman B & Bjork I (1980) Fractionation of heparin by chromatography in immobilized thrombin correlation between the anticoagulant activity of the fractions and their content of heparin with high affinity for antithrombin. Thrombosis Research 19: 711-718. Nordenman B & Bjork I (1981) Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin. Biochimica et Biophysica Acta 672: 227-238. Nordenman B~ Danielsson A & Bjork I (1978) The binding of low-affinity and high-affinity heparin to antithrombin: fluorescence studies. European Journal of Biochemistry 90: 1-6. Odegard OR & Abildgaard U (1977) Antifactor Xa activity in thrombophilia studies in a family

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