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Circulation. 1997;95:118-124

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(Circulation. 1997;95:118-124.)
© 1997 American Heart Association, Inc.

Articles

Effect of Nonspecific Binding to Plasma Proteins on the Antithrombin Activities of Unfractionated Heparin, Low-Molecular-Weight Heparin, and Dermatan Sulfate

Benilde Cosmi, MD; James C. Fredenburgh, PhD; Janice Rischke, RT; Jack Hirsh, MD; Edward Young, PhD; Jeffrey I. Weitz, MD

McMaster University and Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada.

Correspondence to Dr Jeffrey Weitz, Hamilton Civic Hospitals Research Centre, 711 Concession St, Hamilton, Ontario L8V 1C3, Canada.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background Nonspecific binding to plasma proteins decreases the anti–factor Xa (anti-Xa) activity of unfractionated heparin (UFH) but not that of low-molecular-weight heparin (LMWH). However, plasma proteins could influence the anti-thrombin (anti-IIa) activity of LMWH. To explore this possibility, we compared the effects of plasma proteins on the anti-IIa activities of UFH and LMWH. We also examined their effects on the anti-IIa activity of dermatan sulfate (DS) because, like UFH, DS binds to plasma proteins.

Methods and Results There was almost complete recovery of anti-IIa activity when UFH, LMWH, or DS was added to plasma from each of 20 healthy volunteers. The addition of a chemically modified heparin with low affinity for antithrombin III to plasma containing UFH increased the anti-IIa activity in a concentration-dependent fashion by displacing UFH from plasma proteins. In contrast, addition of low-affinity heparin had no effect on the anti-IIa activity of LMWH. LMWH does not bind to plasma proteins because the bulk of the LMWH chains are <6000 D, and only heparin fractions >6000 D bind nonspecifically to plasma proteins. As further evidence that plasma proteins do not influence the anti-IIa activity of LMWH, the rate of thrombin inhibition in plasma in the presence of LMWH is virtually identical to that in buffer containing physiological amounts of the major antithrombins. In contrast, with UFH or DS, the rate of thrombin inhibition is twofold slower in plasma than in buffer.

Conclusions Nonspecific binding of UFH to plasma proteins most likely contributes to the variable anti-IIa response to UFH in patients with thromboembolic disease. Although DS also binds to plasma proteins, the clinical significance of this finding is unclear. In contrast, because LMWH does not bind to plasma proteins, the anti-IIa activity of LMWH should be just as predictable as its anti-Xa activity.


Key Words: anticoagulants • heparin • plasma • proteins


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Although UFH is an effective antithrombotic agent, it produces a highly variable anticoagulant response in patients with venous thromboembolic disease.1 As a result, careful laboratory monitoring is necessary to ensure an adequate anticoagulant response.2 3

UFH exerts its anticoagulant effect by binding to antithrombin III,2 4 5 an interaction mediated by a pentasaccharide sequence found on one third of the heparin chains.6 7 UFH also binds nonspecifically to a variety of other plasma proteins, including platelet factor 4, histidine-rich glycoprotein, vitronectin, fibronectin, and lipoproteins.8 9 10 11 12 These nonspecific interactions have the potential to limit the amount of heparin available to bind to antithrombin III, thereby decreasing its anticoagulant effect.

The plasma levels of heparin-binding proteins vary widely in patients with thrombosis. Large amounts of platelet factor 4, a potent inhibitor of UFH,8 9 are released during the clotting process. Other heparin-binding proteins are acute-phase reactants, the levels of which will increase in ill patients. The variability in the concentrations of heparin-binding proteins provides a plausible explanation for both the unpredictable anticoagulant response to heparin in patients with thromboembolic disease1 and the phenomenon of heparin resistance.13

The limitations of UFH have stimulated the development of anticoagulants, such as LMWH and DS, which produce a more predictable dose response. LMWH is produced by depolymerizing UFH and has a mean molecular weight about one third that of the parent compound.14 Like UFH, LMWH exerts its anticoagulant effect by catalyzing antithrombin III. A smaller percentage (about 15% to 20%) of LMWH chains contain the pentasaccharide sequence with high affinity for antithrombin III.14

In previous studies, we showed that LMWH exhibits considerably less nonspecific binding to plasma proteins than UFH when their inhibitory activities against activated factor X (anti-Xa activities) are compared in vitro15 16 or ex vivo.16 These observations may explain why the anticoagulant response, as measured by anti-Xa levels, is more predictable for LMWH than for UFH.14 We also demonstrated that the extent of nonspecific binding of heparin to plasma proteins is dependent on the molecular weight of the heparin chains, with the longer chains exhibiting the most nonspecific binding.16 Since it is the higher-molecular-weight fraction that is responsible for the anti-IIa activity of LMWH,14 it is possible that the pharmacokinetic advantages of LMWH observed when its anti-Xa activity is measured may not apply if the anticoagulant activity is monitored by use of anti-IIa levels. An inhibitory effect of heparin-binding proteins on the anti-IIa activity of UFH or LMWH may be important, because studies in vitro suggest that these agents exert their anticoagulant effects predominantly through inhibition of thrombin-mediated feedback activation of factors V and VIII.17 18 Inhibition of factor Xa also appears to be important, because the pentasaccharide, which has no anti-IIa activity, is an effective antithrombotic.19

DS, an effective anticoagulant for the prophylaxis of deep vein thrombosis20 21 and for patients undergoing hemodialysis,22 selectively inhibits thrombin by potentiating the activity of heparin cofactor II.23 DS binds to a variety of plasma proteins,24 25 26 27 which could influence its anti-IIa activity.

The purpose of this study was to compare the extent to which the nonspecific binding to plasma protein influences the anti-IIa activity of UFH, LMWH, and DS. To displace UFH or LMWH nonspecifically bound to plasma proteins, we used a chemically modified heparin that has virtually no affinity for antithrombin III. Although chemical modification reduces the affinity of heparin for antithrombin III by disrupting a key saccharide unit within the pentasaccharide sequence,15 the chemically modified heparin retains its affinity for plasma proteins because this interaction is charge dependent and does not involve the pentasaccharide sequence. Even though chemically modified heparin has little anti-Xa activity, it retains significant anti-IIa activity because of its ability to catalyze heparin cofactor II. This problem was overcome by first immunodepleting the plasma of heparin cofactor II and then measuring the anti-IIa activity of UFH or LMWH before and after the addition of low-affinity heparin. To examine the effect of plasma proteins on the anti-IIa activity of DS, we compared the ability of DS to catalyze thrombin inhibition in plasma with its activity in a buffer system containing physiological concentrations of the naturally occurring thrombin inhibitor. This method was also used to examine the influence of plasma proteins on the anti-IIa activity of UFH and LMWH.


*    Methods
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 *Methods
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Reagents
Human {alpha}-thrombin (specific activity, 3930 U/mg), generously provided by Dr J. Fenton II, New York State Department of Health, Albany, was labeled with 125I to a specific activity of 1.8x105 to 2.6x105 cpm/U with lactoperoxidase.28 Human {alpha}2-macroglobulin was isolated by zinc chelation chromatography.29 Purified human antithrombin III was obtained from Enzyme Research Laboratories. Heparin cofactor II was isolated from human plasma by affinity chromatography using an affinity-purified IgG against human heparin cofactor II purchased from Affinity Biologicals. When subjected to SDS-PAGE, antithrombin III and heparin cofactor II migrated as single bands with apparent molecular weights of 58 000 and 70 000, respectively. Furthermore, when titrated against thrombin by the method described by Olson et al,30 both inhibitors completely inactivated the enzyme when the inhibitor and thrombin were added in equimolar amounts.

Ancrod (Arvin) was purchased from Knoll Pharmaceuticals, whereas standard UFH from porcine intestinal mucosa (grade II; specific activity, 166.9 U/mg) and protamine sulfate were obtained from Sigma Chemical Co. Enoxaparine (Rhone-Poulenc) had a specific activity of 155 anti-Xa U/mg and 86 anti-IIa U/mg compared with the fourth International LMWH Standard. Dermatan sulfate was a kind gift from Dr F. Gianese, Mediolanum Farmaceutici, Milan, Italy. Heparin fractions of defined molecular weight (5000, 6000, 9000, 16 000, and 18 000) were purchased from Enzyme Research Laboratories, whereas polybrene was obtained from Aldrich Chemical Co. CNBr-activated Sepharose 4B was purchased from Pharmacia Fine Chemicals; the chromogenic substrate for thrombin, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, was obtained from Sigma Chemical Co; and PPACK was purchased from Calbiochem.

Low-affinity heparin with <1 U/mg of anti-Xa activity was prepared from UFH by controlled periodate oxidation and borohydride reduction31 32 and subjected to affinity chromatography using an antithrombin III–Sepharose column to remove residual high-affinity material.32 UFH and LMWH with high affinity for antithrombin III were prepared by affinity chromatography on the same antithrombin III–Sepharose column.

Preparation of Platelet-Poor Plasma
Blood was drawn from the antecubital veins of each of 20 healthy volunteers into plastic syringes prefilled with 1/10 vol of 0.13 mol/L trisodium citrate. After centrifugation at 1700g for 15 minutes at 4°C, the platelet-poor plasma from each volunteer was harvested and stored in aliquots at -70°C.

Preparation of Heparin Cofactor II–Depleted Plasma
A monospecific, affinity-purified goat IgG against human heparin cofactor II was coupled to CNBr-activated Sepharose 4B (2 mg/g dried gel) according to the manufacturer's instructions. A column (4x1.5 cm) containing 3.5 mL of antibody-linked gel was equilibrated with 0.15 mol/L NaCl buffered to pH 7.4 with 0.02 mol/L Tris-HCl (TBS). Plasma was depleted of heparin cofactor II by passage over the column at a flow rate of 20 mL/h at 23°C. Plasma-containing fractions were pooled and concentrated by ultrafiltration with an Amicon concentrator (Amicon Grace Co) with a molecular-weight cutoff of 10 000 until the protein concentration, as measured with the Bio-Rad protein assay (Bio-Rad) using human albumin as a standard,33 was the same as that in the starting plasma. After use, the column was regenerated with 3 column volumes of Immunopure gentle elution buffer (Pierce) followed by an equivalent volume of 0.1 mol/L Tris-HCl, pH 8.0, and was then reequilibrated with TBS.

The levels of antithrombin III and {alpha}2-macroglobulin in both the starting plasma and heparin cofactor II–depleted plasma were measured by radial immunodiffusion.34 Antithrombin III and heparin cofactor II activity also were measured with commercially available kits (Diagnostica Stago).

Preparation of Defibrinated Plasma
Aliquots of control and heparin cofactor II–depleted plasma were incubated with ancrod (0.34 IU/mL) for 20 minutes at 37°C, and the resultant clots were squeezed and then removed with a wooden stick. After incubation on ice for 15 minutes, any residual fibrin was removed by centrifugation at 10 000g for 5 minutes.

Thrombin-Inhibitor Complex Formation as Detected by SDS-PAGE and Autoradiography
To compare thrombin-inhibitor complex generation in defibrinated heparin cofactor II–depleted plasma with that in defibrinated control plasma, radiolabeled human {alpha}-thrombin was added and thrombin-inhibitor complex formation was assessed by SDS-PAGE and autoradiography. 125I-labeled {alpha}-thrombin (1 µL) was added to 25 to 30 µL of plasma prewarmed to 37°C to obtain a final thrombin concentration of 6 U/mL and 10 000 to 15 000 cpm in the final sample to be loaded onto the gel. After 30 seconds of incubation, the reaction was stopped by the addition of PPACK (1 µmol/L final concentration), and the samples were then diluted with an equal volume of 0.5 mol/L Tris-HCl, pH 6.8, containing 2% SDS, 5% glycerol, and 0.05% wt/vol bromphenol blue and boiled at 95°C for 15 seconds. Electrophoresis was performed35 with 10% polyacrylamide slab gels and 4% stacking gels. The gels were fixed in a solution of 45% methanol/10% acetic acid, stained with Coomassie blue R-250, and then dried. For autoradiography, a Kodak XOmat AR film (Eastman Kodak Co) was placed between the dried slab gel and a Kodak XOmatic regular intensifier screen for 7 to 15 hours at -70°C36 and then developed in a Kodak RP XOmat processor.

Anti-IIa Activity of UFH, LMWH, or DS in Plasma
In 96-well microtiter plates, UFH (final concentration, 0.25 or 0.5 U/mL, equivalent to 0.1 and 0.2 µmol/L, respectively), LMWH (final concentration, 5 µg/mL, equivalent to 0.5 µmol/L), or DS (final concentration, 5 or 10 µg/mL, equivalent to 0.25 and 0.5 µmol/L, respectively) was added to wells containing 10-µL aliquots of defibrinated plasma from each of 20 healthy volunteers. Alternatively, UFH (final concentration, 0.1 U/mL) or LMWH (final concentration, 3 µg/mL) was added to wells containing 10-µL aliquots of pooled defibrinated heparin cofactor II–depleted plasma in the presence or absence of low-affinity heparin (in concentrations ranging from 1 to 120 µg/mL). The plasma samples were then diluted 1 to 20 with TBS, and 25 µL thrombin was added to each well to obtain a final thrombin concentration of 28 nmol/L. After 5 minutes' incubation at 37°C, 25 µL of a 2.2-mmol/L solution of chromogenic substrate (N-p-tosyl-Gly-Pro-Arg-p-nitroanilide) was added to achieve a final reaction volume of 250 µL, and the change in optical density was monitored at 405 nm for 60 seconds at 37°C with a microplate reader (Thermomax, Molecular Devices Corp). After the absorbance of a blank solution containing all the reagents except thrombin had been subtracted, the concentrations of glycosaminoglycans were calculated by comparison of the results with standard curves of the anti-IIa activity of UFH, LMWH, or DS in pooled normal plasma. The recovery was expressed as either U/mL, µg/mL, or percentage of the amount added. In the experiments using heparin cofactor II–depleted plasma, the factor of increase in anti-IIa activity after the addition of low-affinity heparin was calculated by division of the anti-IIa activity measured in the presence of various concentrations of low-affinity heparin by that found in its absence. When used in high concentrations, the low-affinity heparin had a small amount of anti-IIa activity in heparin cofactor II–depleted plasma, which was subtracted from the results obtained.

Comparison of Glycosaminoglycan-Induced Potentiation of the Rate of Thrombin Inhibition in Plasma With That in a Buffer System
The ability of the various glycosaminoglycans to catalyze thrombin inhibition in plasma was compared with their activity in a buffer system. In plasma, the rate of thrombin inhibition was measured in the absence or presence of 0.1 U/mL UFH, 3 µg/mL LMWH, or 5 µg/mL DS. Thus, the glycosaminoglycans (2 µL) were added to 94 µL of defibrinated plasma to obtain a final volume of 96 µL. Chromogenic substrate (2 µL of a 22.2-mmol/L solution) was added as a competitor to obtain a concentration of 0.44 mmol/L, and after the mixture was incubated for 5 minutes at 23°C, 2 µL {alpha}-thrombin was added so that the final volume was 100 µL and the final thrombin concentration was 70 nmol/L. To measure the rate of thrombin inhibition, 10-µL aliquots were removed every 10 seconds for a total of 60 seconds. Each aliquot was added to a well of a 96-well microtiter plate prefilled with a quenching solution consisting of 25 µL of 0.25-mg/mL protamine sulfate in the case of UFH or 1 mg/mL polybrene in the case of LMWH or DS. Chromogenic thrombin substrate (25 µL of a 2.2-mmol/L solution in 190 µL of TBS) was added to each well, and the residual thrombin activity was determined by monitoring of the change in absorbance at 405 nm in a microplate reader for 6 minutes at 23°C.

For studies in a buffer system, a solution of 2 µmol/L antithrombin III, 1 µmol/L heparin cofactor II, and 3.5 µmol/L {alpha}2-macroglobulin was used in place of plasma. These concentrations of inhibitors were chosen because they represent the concentrations of antithrombins normally found in plasma. Since DS is a specific catalyst of heparin cofactor II, the antithrombin III was omitted when the catalytic activity of this glycosaminoglycan was investigated.

In both the plasma and the buffer system, the rate of thrombin inhibition was calculated as described by Olson et al.30 Thus, the residual thrombin activity at each time point was determined by measuring the initial rate of substrate hydrolysis, and k' was then calculated from the slope of a semilogarithmic plot of the residual enzyme activity as a function of time. Using the equation Et/E0=e-k't, where E0 is the initial thrombin activity, Et is thrombin activity at time t, and k' is the pseudo–first-order rate constant. Since the concentration of competing chromogenic substrate in the reactions was constant, its influence on k' was ignored. In the plasma system, the factor of increase in k' in the presence of various concentrations of low-affinity heparin was calculated by dividing the k' in the presence of low-affinity heparin by that in its absence.

Gel Filtration of LMWH and Heparin Fractions of Defined Molecular Weight
To determine the molecular weight distributions of the UFH, LMWH, and DS, the agents were subjected to HPLC analysis with a TSK G2000 SWXL gel filtration column (30 cmx7.8 mm, Supelco Canada) fitted to a liquid chromatograph (System Gold, Beckman Instruments Inc) equipped with a model 126 solvent delivery system and a manual injector. After the glycosaminoglycans had been suspended in sterile distilled water, each was applied to the column and eluted with 0.5 mol/L Na2SO4 at a flow rate of 0.5 mL/min. The fractions were monitored with both a Beckman model 167 variable-wavelength absorbance detector set at 235 nm and a Waters model 410 differential refractometer according to the method described by Nielsen.37 The column was calibrated with a series of oligosaccharides ranging in molecular weight from 1500 to {approx}17 800. The same system was also used (1) to separate UFH into well-defined molecular weight fractions of 4000, 7000, and 8000 and (2) to confirm that the molecular weights of the heparin fractions purchased from Enzyme Research Laboratories corresponded to those stated by the manufacturer.

Statistical Analysis
Mean values and their associated SEMs were calculated. The significance of differences in test results was determined by one-way ANOVA followed by pairwise comparisons of means (according to Bonferroni) when appropriate. The level of significance was taken as {alpha}=.05 (two-tailed).


*    Results
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 *Results
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Characterization of Heparin Cofactor II–Depleted Plasma
The immunodepleted plasma used in these experiments was devoid of heparin cofactor II because (1) there was no detectable heparin cofactor antigen or activity and (2) no thrombin–heparin cofactor II complexes were generated when 125I-labeled thrombin was incubated in plasma, even in the presence of UFH or DS (Fig 1Down, lanes 5 and 6, respectively). In contrast, the immunodepletion process had no effect on the levels of antithrombin III or {alpha}2-macroglobulin. Furthermore, the rate of thrombin inhibition in the plasma immunodepleted of heparin cofactor II was identical to that in the starting plasma when the immunodepleted plasma was reconstituted with 1 µmol/L heparin cofactor II (data not shown).



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  		Figure 1. Comparison of thrombin-inhibitor complex formation in defibrinated control or heparin cofactor II–depleted plasma as assessed by SDS-PAGE and autoradiography. As standards, 125I-labeled thrombin was incubated in buffer alone (lane 1) or in the presence of 2 µmol/L antithrombin III (ATIII) (lane 2) or 1 µmol/L heparin cofactor II (HCII) (lane 3) for 60 seconds before addition of PPACK. Labeled thrombin also was incubated for 60 seconds in defibrinated HCII-depleted plasma in the absence (lane 4) or presence (lane 5) of 25 µg/mL DS, 1.7 U/mL UFH (lane 6), or 90 µg/mL low-affinity heparin (lane 7) and in defibrinated control plasma in the presence of the same concentrations of low-affinity heparin (lane 8) or UFH (lane 9) before the addition of PPACK. Free thrombin (IIa) and thrombin complexed with ATIII (IIa-ATIII), HCII (IIa-HCII), or {alpha}2-macroglobulin (IIa-{alpha}2M) are indicated.

Recovery of UFH, LMWH, and DS in Normal Plasma
Known amounts of UFH, LMWH, or DS were added to aliquots of defibrinated plasma obtained from 20 healthy volunteers, and the resultant anti-IIa activity was then determined. As illustrated in the TableDown, there was virtually complete recovery of all of the glycosaminoglycans.


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  		Table 1. Recovery of Glycosaminoglycans in Control Plasma as Determined by Their Anti-IIa Activity

Nonspecific Protein Binding of UFH, LMWH, and Heparin Fractions of Defined Molecular Weight
To determine the extent of nonspecific protein binding, the anti-IIa activities of UFH, LMWH, and heparin fractions of defined molecular weights were measured in a pool of defibrinated heparin cofactor II–depleted plasma both before and after the addition of low-affinity heparin in concentrations ranging from 1 to 120 µg/mL. As illustrated in Fig 2Down, low-affinity heparin produces a concentration-dependent increase in the anti-IIa activity of UFH, which reaches a plateau at 90 µg/mL. Thus, at low-affinity heparin concentrations of 90 or 120 µg/mL, there is a 1.75-fold increase in anti-IIa activity. In contrast, the low-affinity heparin has little effect on the anti-IIa activity of LMWH. Identical results were obtained when the low-affinity heparin was added to defibrinated heparin cofactor II–depleted plasma containing either UFH or LMWH with high affinity for antithrombin III.



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  		Figure 2. Effect of addition of low-affinity heparin on the anti-IIa activity of UFH and LMWH. The anti-IIa activity of 0.25 U/mL (1.5 µg/mL) UFH ({circ}) or 0.21 U/mL (2.1 µg/mL) LMWH ({bullet}) in pooled, defibrinated heparin cofactor II–depleted plasma was measured before and after the addition of low-affinity heparin in the concentrations indicated. After correction of the values for the small amount of anti-IIa activity produced by the low-affinity heparin alone, the factor of increase in anti-IIa activity produced by low-affinity heparin was calculated by division of the activity after low-affinity heparin addition by that found before its addition. Each point represents the mean of three assays, each done in duplicate, and the bars represent the 95% CIs.

To determine the extent to which the molecular weight of the heparin chains influences nonspecific protein binding, heparin fractions of defined molecular weight (4000, 5000, 6000, 7000, 8000, 9000, 16 000, and 18 000) were added to heparin cofactor II–depleted plasma, and the anti-IIa activity was measured before and after the addition of either 45 or 90 µg/mL low-affinity heparin. As illustrated in Fig 3Down, the extent of nonspecific protein binding is dependent on the molecular weight of the heparin chains. Thus, there was no increase in the anti-IIa activity when low-affinity heparin was added to samples containing heparin fractions of <=6000 D. In contrast, both concentrations of low-affinity heparin produced an {approx}150% to 200% increase in anti-IIa activity when added to samples containing heparin fractions of >=8000 D. An intermediate increase in anti-IIa activity was seen when low-affinity heparin was added to plasma samples containing a heparin fraction of 7000 D.



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  		Figure 3. The effect of low-affinity heparin addition on the anti-IIa activity of heparin fractions of defined molecular weights. Each heparin fraction (0.25 anti-IIa U/mL) was added to heparin cofactor II–depleted plasma, the anti-IIa activity was measured in the presence of low-affinity heparin at a concentration of either 45 µg/mL ({circ}) or 90 µg/mL ({bullet}), and the factor of increase in anti-IIa activity was calculated by dividing the anti-IIa activity after low-affinity heparin addition by that found before its addition. Each point represents the mean of three experiments performed in duplicate, and the bars represent the 95% CIs.

Gel Filtration of UFH, LMWH, and DS
The gel filtration profiles of the UFH, LMWH, and DS used in our studies are illustrated in Fig 4Down.



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  		Figure 4. Molecular weight distributions of dermatan sulfate (A), standard heparin (B), and enoxaparine (C) as determined by HPLC analysis. Each glycosaminoglycan was applied to a TSK G200 SWXL gel filtration column and was eluted with 0.5 mol/L Na2SO4 at a flow rate of 0.5 mL/min. The fractions were monitored by measurement of both the absorbance at 205 nm (A205, solid lines) and the refractive index (RI, broken lines). The column was standardized against oligosaccharides of known molecular weights that eluted as shown by the lines.

Comparison of Glycosaminoglycan-Induced Potentiation of the Rate of Thrombin Inhibition in Plasma With That in a Buffer System
To examine the influence of plasma proteins on the anti-IIa activity of the various glycosaminoglycans, their ability to enhance the rate of thrombin inhibition in plasma was compared with their activity in buffer containing physiological concentrations of the various thrombin inhibitors. With LMWH, the rate of thrombin inhibition in plasma was similar to that in buffer (Fig 5Down), consistent with the concept that the activity of LMWH is not influenced by plasma proteins. In contrast with UFH and DS, the rates of thrombin inhibition in buffer were almost twofold greater than those in plasma, suggesting that the anti-IIa activity of both UFH and DS is modified by plasma proteins.



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  		Figure 5. Comparison of the observed rates (kobs) of thrombin inhibition in plasma (solid bars) and thrombin inhibitor–containing buffer (open bars) in the presence of UFH (0.1 U/mL), LMWH (3 µg/mL), or DS (5 µg/mL). Each bar represents the mean of six experiments, and the lines above the bars represent the 95% CIs.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We used a chemically modified heparin that has virtually no antithrombin III activity to displace heparin that is nonspecifically bound to plasma proteins. The addition of high concentrations of low-affinity heparin to plasma containing UFH results in an almost twofold increase in the anti-IIa activity (Fig 2Up). This is similar to the extent to which low-affinity heparin addition increases the anti-Xa activity of UFH.15 16 In contrast, the addition of even high concentrations of low-affinity heparin has virtually no effect on the anti-IIa activity of LMWH (Fig 2Up). These findings can be explained on the basis of the molecular-weight profile of enoxaparine, which has a median molecular weight of 4500. Although enoxaparine contains heparin chains ranging from 3000 to 9000 D, the bulk of the material is <6000 D (Fig 4Up). Since only heparin chains of >=6000 D bind nonspecifically to plasma proteins (Fig 3Up), enoxaparine would not be expected to exhibit this property.

LMWH contains a greater proportion of heparin chains with low affinity for antithrombin III than UFH.14 38 Two lines of evidence exclude the possibility that this low-affinity fraction displaces the LMWH from plasma proteins. First, the addition of high concentrations of low-affinity heparin had no effect on the anti-IIa activity of LMWH (Fig 2Up), even when added to plasma containing only LMWH with high affinity for antithrombin III. In contrast, low-affinity heparin addition to plasma containing standard heparin with high affinity for antithrombin III still produced an almost twofold increase in the anti-IIa activity. Second, a >20-fold molar excess of low-affinity heparin is needed to produce an increase in the anti-IIa activity of UFH (Fig 2Up). It is likely that such high concentrations of low-affinity heparin are needed because of the large capacity of plasma proteins to nonspecifically bind heparin. On the basis of these considerations, it is not surprising that there is insufficient low-affinity material in UFH and LMWH to affect their protein binding.

To confirm our observation that nonspecific binding to plasma proteins influences the catalytic activity of UFH but has little effect on the activity of LMWH, we compared the ability of UFH and LMWH to increase the rate of thrombin inhibition in plasma with their activity in a buffer system containing physiological concentrations of the major thrombin inhibitors. As illustrated in Fig 5Up, in the presence of the same amount of UFH, the rate of thrombin inhibition in plasma is almost twofold lower than it is in buffer. In contrast, with LMWH, the rate of thrombin inhibition in plasma is similar to that in the buffer system. These findings support the concept that plasma proteins modify the anti-IIa activity of UFH but have little or no effect on the anti-IIa activity of LMWH.

With DS, the rate of thrombin inhibition is approximately twofold lower in plasma than in buffer (Fig 5Up), suggesting that like UFH, DS also binds nonspecifically to plasma proteins. These findings are consistent with those of other investigators who demonstrated DS binding to a variety of plasma proteins, including histidine-rich glycoprotein, vitronectin, complement factor H, and apolipoprotein B.24 25 26 A recent study by Zammit and Dawes27 suggests that fibrinogen is the major DS-binding protein in plasma. Since we used defibrinated plasma, we cannot assess the importance of fibrinogen in the modulation of DS activity. However, our studies suggest that even in the absence of fibrinogen, other plasma proteins have a modulating effect on DS activity.

Although similar in structure to UFH, DS is less negatively charged.39 Our observation that plasma proteins influence the anti-IIa activities of UFH and DS to a similar extent suggests that protein binding is not simply a charge-related phenomenon. Heparin chain length is an important determinant of the extent of plasma protein binding, because only heparin fractions >6000 D (ie, composed of >20 saccharide units) bind to plasma proteins (Fig 3Up). Since the median molecular weights of UFH and DS are {approx}14 000 and 20 000 D, respectively, it is not surprising that these agents bind to plasma proteins.

In summary, we have shown that unlike UFH and DS, the anti-IIa activity of LMWH is unaffected by proteins found in platelet-poor plasma. LMWH has been shown to produce a more predictable anti-Xa response than UFH in patients.14 If our results in platelet-poor plasma can be extrapolated to the situation in vivo, in which, in addition to plasma proteins, heparin binds to endothelium and to platelet factor 4 released from platelets, our findings suggest that the anti-IIa response to LMWH also will be more predictable than that of UFH. Given that the anticoagulant effect of LMWH reflects, at least in part, its ability to inhibit thrombin-mediated activation of factors V and VIII,17 18 our results may help to explain why LMWH can be used safely and effectively in patients with thromboembolic disease without the need for laboratory monitoring. Finally, our studies suggest that like UFH, DS also binds to plasma proteins. The clinical significance of this finding, however, is unknown.


*    Selected Abbreviations and Acronyms
 
anti-IIa = anti-thrombin
anti-Xa = anti–factor Xa
DS = dermatan sulfate
HPLC = high-performance liquid chromatography
LMWH = low-molecular-weight heparin
PPACK = D-Phe-Pro-Arg CH2Cl
TBS = Tris-buffered saline
UFH = unfractionated heparin


*    Acknowledgments
 
This work was funded by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario. Dr Weitz is a Career Investigator of the Heart and Stroke Foundation of Ontario. The authors thank Susan Crnic for her excellent secretarial support.

Received January 29, 1996; revision received August 10, 1996; accepted August 19, 1996.


*    References
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 up arrowDiscussion
 *References
 

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