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(Circulation. 1998;98:1297-1301.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Effect of High- and Low-Molecular-Weight Heparins on Thrombin-Thrombomodulin Interaction and Protein C Activation

Raimondo De Cristofaro, MD; Erica De Candia, MD; ; Raffaele Landolfi, MD

From the Hemostasis Research Center, Department of Internal Medicine, Catholic University, Rome, Italy.

Correspondence to Dr Raimondo De Cristofaro, Centro Ricerche Fisiopatologia dell'Emostasi, Istituto di Semeiotica Medica, Università Cattolica S. Cuore, Largo F. Vito 1, 00168 Roma, Italy.

	Abstract

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Background—Thrombin-thrombomodulin (TM) interaction, which is critical for accelerating the protein C anticoagulant pathway, involves the heparin-like domain of TM. This study was aimed at investigating the possible effect of heparin on thrombin-TM binding and protein C activation.

Methods and Results—The affinity of thrombin-TM interaction was studied by a functional method, based on the ability of thrombin-TM adduct to activate protein C, and by evaluation of the binding of thrombin to immobilized TM. Both experimental approaches showed that the affinity of thrombin-TM interaction was decreased by micromolar heparin concentrations. Heparin had no significant effect when a recombinant TM form, lacking the chondroitin sulfate moiety, was used. Furthermore, it was also shown that the inhibitory effect of heparin was directly proportional to the heparin molecular mass (molecular weight range, 3 to 16 kDa), which suggests that the effect was mediated by formation of electrostatic bonds between heparin and thrombin.

Conclusions—These results indicate that heparin at therapeutic concentrations reduces the affinity of thrombin for TM and the rate of protein C activation. The magnitude of this effect is proportionally linked to the molecular mass of heparin.

Key Words: heparin • glycoproteins • enzymes • proteins

	Introduction

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Heparin is widely used in the prevention and treatment of arterial and venous thrombosis. It affects the hemostatic system by accelerating antithrombin III–mediated inhibition of some coagulative serine proteases such as thrombin, factor Xa, factor IXa, factor XIa, and factor XIIa.¹ Heparin, however, can also interact with a number of proteins and cell surfaces,² and some of these interactions may be relevant to its pharmacological effect.³ In recent studies, for instance, we showed that heparin inhibits thrombin interaction with the platelet glycoprotein Ib^{4 5} and that such inhibition results in a delay of thrombin-induced platelet activation (unpublished data, 1998).

In the present study, we explored the effect of heparin on thrombin-thrombomodulin (TM) interaction.

TM is an endothelial membrane glycoprotein whose interaction with -thrombin causes an allosteric transition that enables the enzyme to activate the zymogen protein C.^{6 7} Activated protein C is a potent anticoagulant that inactivates factor Va and factor VIIIa and inhibits the further production of thrombin.^{8 9}

The interaction of thrombin with TM involves both a protein region of TM and its chondroitin sulfate moiety. The protein region, comprising the 5 to 6 epidermal growth factor (EGF)-like domains, interacts with the so-called fibrinogen recognition site (FRS) of thrombin, which is involved in the binding of some other macromolecular ligands such as fibrinogen and the 7-transmembrane thrombin receptor.^{10 11}

The chondroitin sulfate component of TM, on the other hand, may interact with the thrombin exosite referred to as the heparin binding site (HBS).^{10 12 13 14} In fact, it was demonstrated that thrombin molecules, mutated at some charged residues located at the HBS, bind TM with lower affinity than wild-type enzyme.¹⁵ The same effect (a roughly 20-fold affinity reduction) was also observed with the enzymatically deglycosylated TM molecule or mutant TM variants lacking the chondroitin sulfate moiety.¹⁶ In addition, TM per se can function like a glycosaminoglycan. In fact, in the absence of heparin, it can accelerate thrombin inhibition by antithrombin III, and this effect is canceled by TM digestion with chondroitinase.¹⁷

On this basis, we hypothesized that thrombin HBS occupancy by heparin might affect the thrombin-TM interaction. The present study was designed to investigate the in vitro effect of heparin on thrombin-TM interaction and on subsequent protein C hydrolysis. Furthermore, the effect of heparins with different molecular weights (range, 3 to 16 kDa) was also investigated.

	Methods

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Materials
Recombinant hirudin (rH[IV]_K47), ecarin, prothrombin, and human zymogen protein C were purchased from Sigma Chemical Co. BSA (fatty acid free) was from Boehringer Mannheim. S-2366 (pyr-Glu-Pro-Arg-pNA) and protein C activator from Agkistrodon contortrix were purchased from Chromogenix. Human recombinant TM fragment 1-490 was purchased from American Diagnostica. This recombinant carboxyl-terminal–truncated form of TM contains the chondroitin sulfate side chain. The recombinant human TM fragment E346-K466 (TM_EGF4-6), containing a leucine substitution for methionine 388, was purchased from Alexis Corp. This recombinant form of human TM lacks the chondroitin sulfate chain, which in the full-length TM molecule is covalently linked to serine 474. Fractionated porcine intestinal heparins with different molecular weights (16 000, 9000, and 3000 kDa) were purchased from Enzyme Research Laboratories Inc. The molecular weight of these fractions was confirmed by high-performance liquid chromatography analysis performed by a Bio-Rad Bio-Silect SEC 250-5 gel filtration column (300x7.8 mm) fitted to a liquid chromatograph (Perkin-Elmer, series 10). Purified heparin oligosaccharides with known molecular weights, from Enzyme Research, were used to construct the reference curve. Detection of the heparin fractions was accomplished at 205 nm. We calculated the average disaccharide units for the different heparin forms assuming an average molecular mass/hexose residue of 296.2 Da.¹⁸

Purification of Proteins
Human -thrombin was purified and characterized as previously reported.¹⁹ TM was free from other contaminating proteins as judged by 4% to 20% SDS-PAGE. TM concentration was measured spectrophotometrically with an extinction coefficient at 280 nm E^0.1% (1 cm)=1.19. SDS-PAGE analysis of zymogen protein C showed a closely spaced doublet having a molecular weight of 62 kDa. Its concentration was computed spectrophotometrically with an E^0.1% (280 nm)=1.37.²⁰

Characterization of Thrombin-TM Interaction
Functional Assay
The apparent affinity constant of thrombin for TM was calculated by a functional assay based on the ability of the thrombin-TM complex to cleave the zymogen protein C with much higher specificity than the free enzyme. Activation of protein C was performed in 96-well polystyrene trays purchased from Dynatech, with a total reaction volume of 200 µL. The buffer solution was 20 mmol/L Tris, 0.1 mol/L NaCl, 2.5 mmol/L CaCl₂, 1% BSA, pH 7.50 at 25°C. A fixed TM or TM_EGF4-6 concentration (1 nmol/L) was used, whereas thrombin was tested over a concentration spanning from 1.64 to 42 nmol/L for TM experiments and from 6 to 160 nmol/L for TM_EGF4-6 studies. Protein C was used at a concentration of 1 µmol/L. At appropriate time intervals, reaction was stopped by a 10-fold excess of hirudin. The produced activated protein C (aPC) concentration was determined by addition of the S2366 chromogenic substrate (200 µmol/L). A standard curve was prepared with protein C that had been totally activated with the Agkistrodon contortrix venom, as previously detailed.²¹ We evaluated the hydrolytic activity of thrombin for PC by omitting TM from the solution. The specific rate of protein C hydrolysis by thrombin-TM adduct was always obtained by subtraction of the contribution of free thrombin. The rate of aPC produced, analyzed as a function of thrombin concentration, was used to calculate the apparent equilibrium dissociation constant of the thrombin-TM interaction (K_d) and the maximum rate of aPC hydrolysis (V_max), according to the method of Ye and coworkers.¹⁵ The measured rates of aPC production were analyzed as a function of thrombin concentration according to equation 4 of the article by Ye et al.¹⁵ The obtained parameter values were also analyzed as a function of different heparin forms.

Solid-Phase Assay
Binding of thrombin to TM was also performed by a solid-phase assay, as previously described by Jandrot-Perrus et al.²² All solid-phase experiments were performed with the use of 96-well polystyrene trays. Briefly, TM or TM_EGF4-6 (0.25 µg/mL) was adsorbed to the wells of a solid-phase plate (100 µL/well) by incubation overnight at 4°C in 50 mmol/L carbonate buffer, pH 9.50. We obtained the blanks by using 1% BSA (fatty acid free) in the same buffer instead of the 2 TM forms. The remaining binding capacity of the sample wells was saturated by a 2-hour incubation with 1% BSA in HEPES-buffered saline, pH 7.50 (buffer A). After aspiration, 150 µL of a thrombin solution was applied to each well at different concentrations (from 0.016 to 16 nmol/L) in 10 mmol/L Tris, 0.1 mol/L NaCl, 2.5 mmol/L CaCl₂, 0.05% Tween 20, pH 7.50 at 25°C. Thrombin was incubated for 1 hour. Each sample and blank well was washed with 250 µL of 0.05% Tween 20 in buffer A for 10 seconds. The washing buffer was aspirated, and the detection of thrombin was accomplished by incubating each well with 150 µL of 100 µmol/L Phe-Pip-Arg-pNA in 50 mmol/L Tris, 0.2 mol/L NaCl, 0.1% PEG 6000, pH 8.00 at 25°C. Under these conditions, color development at 405 nm is directly proportional to the thrombin bound to immobilized TM. Absorbance was measured at 405 nm by a Sorin-Biomedica Eti-System plate reader. The signal at 405 nm was always found to be linear over 1 hour of substrate incubation. The blanks, generally showing a signal equal to 15% of the corresponding sample, were subtracted from the absorbance value of the sample. Each determination (sample and blank) was performed in duplicate. The effect of high-molecular-weight (16 000 Da) and low-molecular-weight (3000 Da) heparin was evaluated by the above-reported solid-phase method with fixed concentrations of heparin ranging from 0 to 10 µmol/L. The apparent thrombin-TM and thrombin-heparin equilibrium dissociation constants were calculated by simultaneous analysis of all 88 experimental points pertaining to each data set, according to a procedure previously detailed.^{23 24} The analysis, based on a modified Marquardt algorithm, was accomplished by Sigmaplot software (Jandel Scientific).

	Results

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The effect of heparin on thrombin-TM interaction was studied by a functional method, based on the activation of protein C, and by measuring thrombin binding to immobilized TM.

Functional Studies Based on Protein C Activation
The functional method used to measure the apparent equilibrium binding constant of thrombin-TM interaction was found to be accurate and reproducible, according to results previously reported by Ye and coworkers.¹⁵ The K_d value of the thrombin-TM interaction was 2.4±0.15 nmol/L, in good agreement with the K_d of 1.3 to 3.1 nmol/L determined for cell-associated full-length human TM.²⁵ The presence of heparin caused a rightward shift of the binding curve (see Figure 1), indicating a decrease of TM affinity. At increasing heparin concentrations, there was a progressive decrease of TM affinity, while the V_max of protein C cleavage was the same (ranging from 6.1 to 7 nmol · L^-1 · min^-1). The latter finding enabled us to rule out a noncompetitive inhibition by heparin. Figure 2 shows the best-fit value of the thrombin-TM dissociation constant as a function of the high-molecular-weight heparin (HMWH) concentration (molecular weight, 16 kDa). The linear dependence of K_d values from heparin concentration demonstrates that a competitive model, in which heparin binding to thrombin inhibits TM binding to the enzyme and vice versa, can provide a good analysis of the experimental data. The best-fit values of the thrombin-heparin equilibrium dissociation constant were found in the micromolar range, in agreement with previously published values.²⁶

View larger version (18K): [in this window] [in a new window]   Figure 1. Protein C activation (aPC) by thrombin-TM adduct in absence and presence of heparin. Initial rates of protein C cleavage were determined and fitted to the appropriate equation, as described in the text. Points obtained at 0 () and 10 µmol/L () HMWH are shown. Continuous lines were drawn according to the best-fit-parameter values as follows: Kd=2.4±0.15 nmol/L, Vmax=6.1±0.07 nmol · L-1 · min-1 for points obtained at 0 µmol/L heparin; Kd=20.6±2.15 nmol/L, Vmax=6.2±0.09 nmol · L-1 · min-1 for points obtained at 10 µmol/L heparin.

View larger version (17K): [in this window] [in a new window]   Figure 2. Analysis of apparent Kd values of thrombin-TM interaction as a function of HMWH concentration. Straight line is the best fit to the experimental points, drawn according to the equation Kdapp=Kd°[1+(H/Ki)], where Kdapp is apparent Kd value, Kd° is the dissociation equilibrium constant of thrombin-TM binding in the absence of heparin, H is heparin concentration, and Ki is equilibrium dissociation constant of thrombin-heparin interaction. Best-fit parameter values were Kd°=2.4±0.2 nmol/L and Ki=1.47±0.1 µmol/L. Dotted lines represent 95% CI.

The inhibitory effect of heparin on thrombin-TM interaction was also investigated with heparin fractions with molecular weight ranging from 3 to 16 kDa. The experimental data showed that the apparent affinity of heparin for thrombin, K_a, increased as a function of the molecular weight of the heparin chain. Figure 3 shows that a linear dependence exists between the apparent affinity of heparin for thrombin and the number of saccharide units present in heparin chains with a molecular weight ranging from 3 to 16 kDa. This result is in accord with a nonspecific electrostatic heparin binding to thrombin.²⁶ Application of such an electrostatic model²⁶ enabled us to show that the minimum number of saccharide units able to interact with thrombin was 6, as expected from structural requirements of the HBS of thrombin.^{13 14 26} This finding corroborates the hypothesis that the inhibitory effect of heparin on thrombin-TM interaction is due to its binding to thrombin and not to other effects.

View larger version (18K): [in this window] [in a new window]   Figure 3. Analysis of the equilibrium affinity constant of thrombin-heparin interaction as a function of the number of disaccharide units present in the heparin chain. Numbers reported in parentheses represent molecular weight of the heparin forms. Straight line was drawn according to the equation Ka=Ka°(N+1-l), where Ka° is the intrinsic thrombin-binding site affinity, N is the total number of disaccharide units of the heparin chain, and l is the number of disaccharide units per thrombin-binding site. The value of calculated Ka was found to be 47.2±4 µmol/L, with l having a value of 3.6±1.8. Dotted lines represent 95% CI.

To further validate the hypothesis that the heparin effect was mediated by competition with the heparin-like domain of TM, other experiments were performed with TM_EGF4-6, which lacks the chondroitin sulfate moiety. Thrombin interaction with TM_EGF4-6 was found to induce in thrombin a similar catalytic efficiency for protein C hydrolysis, but the affinity for the enzyme was reduced with respect to full-length TM, being 16 nmol/L. Notably, this affinity was not significantly reduced by either high- or low-molecular-weight heparin (see Figure 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Protein C activation (aPC) by thrombin-TMEGF4-6 adduct in absence and presence of heparin. Initial rates of protein C cleavage were determined and fitted to the appropriate equation, as described in the text. Points obtained at no heparin (), 20 µmol/L () LMWH (3 kDa), and 10 µmol/L HMWH (16 kDa) () are shown. Continuous lines were drawn according to the best-fit-parameter values as follows: Kd=15.9±2 nmol/L, Vmax=8.2±0.2 nmol · L-1 · min-1 for points obtained in absence of heparin; Kd=18.8±3 nmol/L, Vmax=8.6±0.3 nmol · L-1 · min-1 for points obtained at 20 µmol/L LMWH; and Kd=19.1±4 nmol/L, Vmax=8.3±0.5 nmol · L-1 · min-1 for points obtained at 10 µmol/L HMWH.

Finally, control experiments demonstrated that in the absence of TM, neither HMWH and low-molecular-weight heparin (LMWH) (3 and 16 kDa, respectively), up to a concentration of 10 µmol/L, affected protein C activation. This result excluded any potential inhibitory effect of heparin on the hydrolysis of protein C by free thrombin.

Solid-Phase Assay
Binding of thrombin to immobilized TM was characterized by an equilibrium dissociation constant equal to 2.3±0.2 nmol/L, in agreement with the value derived from the functional assays. This value increased as a function of heparin concentration, as shown by Figure 5A. By assuming a simple competitive scheme in which binding of heparin to thrombin can inhibit the enzyme binding to TM, it was possible for us to also compute the best-fit parameter value for heparin interaction with thrombin. The inhibition constant, K_i, which is the equilibrium dissociation constant of heparin binding to thrombin, was 1.5 µmol/L for HMWH (molecular weight of 16 kDa), whereas it was 5.1 µmol/L for LMWH (molecular weight, 3 kDa). These values are in agreement with those obtained in the functional studies described above (1.4 and 4.6 µmol/L for HMWH and LMWH, respectively). Furthermore, simultaneous fitting of all the experimental data enabled us to demonstrate that the maximal binding capacity of immobilized TM did not change as a function of heparin, thus excluding any effect of heparin on the concentration of TM bound to the microtitration plates.

View larger version (18K): [in this window] [in a new window]   Figure 5. Determination of apparent Kd of (A) thrombin-TM interaction and (B) thrombin-TMEGF4-6 interaction by solid-phase assay under the experimental conditions reported in the text. A, Continuous lines were drawn according to best-fit parameter values obtained for a single site binding isotherms at 0 (), 3.75 µmol/L (), 1.875 µmol/L (), and 0.935 µmol/L () HMWH concentration. A405 nm/min is proportional to thrombin bound to immobilized TM. B, Continuous lines were drawn according to best-fit parameter values obtained for a single site binding isotherms at 0 () and 10 µmol/L () HMWH concentration. The Kd of thrombin-TMEGF4-6 was 21±1.4 nmol/L in the absence of heparin and 27±3 nmol/L in its presence.

Similar experiments performed with TM_EGF4-6 demonstrated that HMWH did not significantly change the affinity of thrombin for such a form of TM, as shown by Figure 5B. This finding, in agreement with the results of the above-described functional experiments, further indicated that the inhibition by heparin of the thrombin-TM interaction is due to its competitive activity toward the chondroitin sulfate moiety of TM.

	Discussion

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The present study demonstrates that heparin inhibits thrombin-TM interaction and reduces the rate of protein C activation. Under our experimental conditions, heparin acted as a competitive inhibitor of TM binding to thrombin. This finding is in accord with the hypothesis that the interaction also involves the thrombin HBS and with the evidence that TM per se has structural and functional similarities to glycosaminoglycans.¹⁷ The experiments performed with TM_EGF4-6 also confirmed the above-mentioned hypothesis. Thus, TM_EGF4-6, which lacks the chondroitin sulfate chain, bound to thrombin with a reduced affinity, and this binding was not significantly affected by heparin.

It is known that the EGF 5-6 domains of TM bind to thrombin and that this binding involves an anion exosite different from HBS, ie, the FRS. An interaction between the HBS and FRS may exist, although the detailed mechanisms of the overall binding process as well as of its stoichiometry are not yet fully elucidated.^{17 27} These unresolved issues were not specifically addressed by the present study.

Another finding of the present study was the linear relationship between the molecular weight of heparin and its inhibitory effect on thrombin-TM interaction and protein C activation. This finding indicates that the strength of heparin binding is directly related to the number of disaccharide repeats and thus to the electrostatic charge of the molecule. This finding confirms the observation that although the antithrombin III–heparin interaction requires a specific pentasaccharide sequence, thrombin-heparin binding is mostly driven by nonspecific electrostatic bonds.²⁶

The functional consequence of heparin binding to thrombin was a dose-dependent reduction of TM-mediated protein C activation. The protein C system plays a key role in the hemostatic equilibrium, and thus the question arises as to whether heparins may attenuate the antithrombotic efficacy of this molecule in vivo. In our experiments, the heparin inhibition of protein C activation was significant for TM concentrations in the nanomolar range. Endothelial cells of different vascular compartments express roughly the same amount of TM molecules on their surface (50 000 to 100 000 copies).²⁸ However, by virtue of the great difference in vessel diameters, the TM concentration increases from 0.2 nmol/L at the endothelial interface of a small or medium artery to 500 nmol/L in the microcirculatory compartment. A theoretical prediction of the heparin effect in these 2 circulatory districts is shown in Figure 6. Such a prediction hinges on in vitro studies only and does not refer to data taken under flow conditions. However, it enables one to realize that the intensity of the heparin effect as well as of the influence of the molecular weight of heparin is more relevant at low TM concentrations, such as those found in the macrocirculatory compartment. Whether the reduced effect of LMWH on the TM-dependent anticoagulant pathway may result in significant therapeutic advantage remains to be established. This possibility, however, is not unlikely given the critical role played by the protein C pathway in the venous and possibly the arterial district.^{29 30 31}

View larger version (17K): [in this window] [in a new window]   Figure 6. Plot showing theoretical protein C (PC) activating capacity as a function of therapeutic heparin concentrations. PC activating capacity was considered proportional to the concentration of thrombin-TM adduct formed under both low (0.2 nmol/L) and high (500 nmol/L) TM concentrations. These TM concentrations are found in the macrocirculation and microcirculation, respectively. Lines were drawn according to a competitive model in which heparin binding to thrombin inhibits thrombin interaction with TM, as described in the text. The following binding parameters were used for the analysis: Kd value for thrombin-TM interaction equal to 2 nmol/L and Kds for thrombin interaction with heparin equal to 1.5 µmol/L for HMWH and 5 µmol/L for LMWH species. Continuous and dotted lines refer to HMWH (16 kDa) and LMWH (3 kDa), respectively, in the presence of low TM concentration, whereas the bold dashed line refers to HMWH in the presence of high TM concentration.

	Acknowledgments

 
This study was financially supported by grant No. 94.02636.CT04 from the National Research Council (CNR) of Italy.

Received January 27, 1998; revision received May 26, 1998; accepted May 27, 1998.

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				E. De Candia, R. De Cristofaro, and R. Landolfi Thrombin-Induced Platelet Activation Is Inhibited by High- and Low-Molecular-Weight Heparin Circulation, June 29, 1999; 99(25): 3308 - 3314. [Abstract] [Full Text] [PDF]

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