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The Journal of Immunology, 2001, 166: 6118-6125.
Copyright © 2001 by The American Association of Immunologists

Lupus Antibody Bivalency Is Required to Enhance Prothrombin Binding to Phospholipid1

Susan L. Field*, Colin N. Chesterman*,{dagger}, Yan-Ping Dai* and Philip J. Hogg2,*

* Centre for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sidney, Australia; and {dagger} Department of Haematology, South Eastern Laboratory Services, Sydney, and Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 µM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lupus anticoagulants (LA)3 and anticardiolipin Abs, also known as antiphospholipid Abs, are associated with a variety of autoimmune diseases. Antiphospholipid Abs are associated with an increased risk for thrombosis, fetal loss, and neurological disorders and thrombocytopenia. This association is the antiphospholipid syndrome (1).

The LA Abs are directed at the phospholipid-binding proteins {beta}2-glycoprotein 1 ({beta}2-GP1) in the case of anticardiolipin (2, 3) and prothrombin and/or {beta}2-GP1 in the case of LA (4, 5, 6). For some reason, LA is associated with in vitro anticoagulant activity on the one hand and a powerful predisposition to in vivo thrombosis on the other. We have hypothesized that LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall (7).

Rao et al. (8) observed increased binding of prothrombin to plastic-adsorbed phosphatidylserine and umbilical vein endothelial cell monolayers in the presence of LA IgGs. A murine monoclonal anti-prothrombin Ab and seven LA IgGs also were shown to enhance binding of prothrombin to 75:25 phosphatidylcholine:phosphatidylserine vesicles in a concentration-dependent manner (7). To investigate whether enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production in flow, Field et al. (7) measured the effect of LA IgGs on thrombin production by purified prothrombinase components in a phospholipid-coated flow reactor. A murine monoclonal anti-prothrombin Ab and four of six LA IgGs from patients with a history of thrombosis increased thrombin production up to 100% over control in the first 15 min (7).

These in vitro observations are supported by Ferro et al. (9) who showed that thrombin production is increased in patients with LA. In particular, Musial et al. (10) showed that thrombin generation was enhanced in blood flowing ex vivo during the 3 min after a skin-bleeding time incision with antiphospholipid Ab-positive patients.

The purpose of the studies herein was to investigate the mechanism by which the LA IgGs enhanced interaction of prothrombin with phospholipid vesicles. LA IgG bivalency was found to be required for enhanced prothrombin binding to phospholipid vesicles. To better understand the mechanism of this effect of the LA IgGs, an equilibrium binding model for the interactions of prothrombin, LA IgG, and phospholipid vesicles was developed. The augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Control and patient IgGs

The purified anti-prothrombin-fragment 1 and anti-prothrombin murine mAbs were obtained from Enzyme Research (South Bend, IN). IgG fractions were prepared from three patients with LA diagnosed on the basis of the accepted criteria of the International Society on Thrombosis and Haemostasis Subcommittee on Lupus Anticoagulants/Phospholipid-Dependent Antibodies (11) and displaying prolongation of the Kaolin clotting time and the dilute Russell’s viper venom test. Three control subjects were laboratory staff with no evident illness. Patient characteristics have been described by Field et al. (7). Total IgG was prepared from control and patient sera with protein G Sepharose (Pharmacia, Uppsala, Sweden). After application of serum, the protein G-Sepharose was washed with 20 mM sodium phosphate, pH 7 buffer, containing 1 M NaCl. After reequilibration of the column with 20 mM sodium phosphate, pH 7, the IgG was eluted with 0.1 M glycine, pH 2.7, and the pH neutralized immediately with 1 M Tris, pH 9 buffer. The IgGs were aliquoted and stored at -20°C until use.

F(ab')2 of the LA IgGs were prepared with the ImmunoPure F(ab')2 preparation kit from Pierce (Rockford, IL) and were free of intact IgG or Fc fragments as judged by nonreducing SDS-PAGE. Fab were derived from the F(ab')2 preparations by reduction with 0.01 M cysteine for 2 h at 37°C and alkylation with 0.15 M iodoacetamide (12). The fragments were dialyzed against 20 mM HEPES, 0.14 M NaCl, pH 7.4 buffer, aliquoted and stored at -80°C until use.

Preparation of phospholipid vesicles

DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), DOPS (1,2-dioleoyl-sn-glycero-3-phosphatidylserine; Avanti Polar Lipids, Alabaster, AL), and biotin-DHPE (N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; Molecular Probes, Eugene, OR) were combined in the molar ratio 75:25 DOPC:DOPS or 74:25:1 DOPC:DOPS:biotin-DHPE and the solvent evaporated under argon. Small unilamellar vesicles were prepared by sonication and fractionation of the vesicles by gel filtration on Sepharose 4B (13). Large unilamellar vesicles were prepared by dissolving the dried lipid in n-octyl-{beta}-D-glucopyranoside, removing the glucopyranoside by dialysis against four changes of 50 mM HEPES, 0.125 M NaCl, 0.02% NaN3, pH 7.4 buffer over 4 days and fractionation of the vesicles by gel filtration on Sepharose CL-2B (14). Phospholipid concentration was determined by measuring inorganic phosphate (15). An average large-vesicle diameter of 280 nm was estimated by flow cytometric analysis with 2-µm standard microspheres (FACStarPlus; Becton Dickinson, San Jose, CA).

Binding of prothrombin to phospholipid vesicles measured by ultracentrifugation

Binding of iodinated prothrombin to phospholipid vesicles was measured by separating bound from free prothrombin in an air-fuge (16, 17). Prothrombin (Enzyme Research) was iodinated with immobilized iodogen according to the manufacturers instructions (Pierce) to a specific activity of 40,000 cpm/µg. 125I-Prothrombin (100,000 cpm), prothrombin (0.1 µM), 75:25 DOPC:DOPS vesicles (15 µM), and control or LA IgG (0–50 µM) was added to polyallomer centrifuge tubes containing 50 mM HEPES, 125 mM NaCl, 10 mM CaCl2, 5 mg/ml BSA, pH 7.4 buffer. The reactions were incubated for 20 min and the phospholipid vesicles sedimented in an air-fuge (Beckman, Palo Alto, CA) for 20 min. The centrifugation sedimented >=95% of the phospholipid vesicles. The supernatant was aspirated and bound and free 125I-prothrombin determined. Nonspecific binding of 125I-prothrombin to control tubes not containing phospholipid vesicles represented <=20% of the binding to phospholipid vesicles in the presence of 10 µM control IgG. The moles of prothrombin bound to phospholipid vesicles was calculated from the total prothrombin and the ratio of bound vs total 125I-prothrombin.

Binding of prothrombin to phospholipid vesicles measured by right-angle light scattering

Relative 90° light-scattering measurements were made in a SLM 48000 spectrofluorometer with quartz cuvettes. The excitation and analysis monochromators were set at 320 nm. The light scattering from the small phospholipid vesicles and prothrombin and/or LA IgG was compared with the light scattering from phospholipid and/or LA IgG alone. The volume of sample was 1.5 ml, and the quantity of phospholipid was 30 µg. The ratio of the light-scattering intensity for the prothrombin-phospholipid complex (IS2) to that of the phospholipid alone (IS1) was analyzed as described by Nelsestuen and Lim (18) to determine the concentration of prothrombin bound to phospholipid. In experiments with IgG, the data was expressed as the ratio IS2/IS1. It was not possible to reduce this data further without a knowledge of the actual concentration of anti-prothrombin IgG in the LA IgGs.

Binding of prothrombin to phospholipid vesicles measured by surface plasmon resonance

Avidin (Neutralite avidin; Molecular Probes) was immobilized on the amino-Silane surface of an IAsys microcuvette (Advanced Laboratory Solutions, Melbourne, Australia) with polymerized gluteraldehyde to cross-link amino groups on the cuvette and ligand. The procedure followed essentially the manufacturer’s instructions, using 10 mM Na phosphate, pH 7.7 as immobilization buffer, 4.2% (v/v) polymerized gluteraldehyde, and 0.1 mg/ml avidin. Phosphate buffer containing 1 mg/ml BSA was used as blocking agent and 10 mM HCl as regeneration buffer. DOPC:DOPS:biotin-DHPE phospholipid vesicles (74:25:1) were bound to the avidin-coated IAsys microcuvette by incubation with 1.1 mM vesicles in 50 mM HEPES, 125 mM NaCl, 3 mM CaCl2, and 0.5 mg/ml BSA, pH 7.4 buffer.

The phospholipid-coated cuvette was washed to remove unbound vesicles and then incubated with either prothrombin (1 µM), prothrombin (1 µM) followed by LA IgG (10 µM), prothrombin (1 µM) and LA IgG (10 µM), or LA IgG (10 µM) followed by prothrombin (1 µM). The sampling interval was 0.3 s, the stirrer was at 60 revolutions per second, and the reaction temperature was 20°C. The binding buffer was 50 mM HEPES, 125 mM NaCl, 3 mM CaCl2, 0.5 mg/ml BSA, pH 7.4. Interaction with prothrombin was for 6–12 min, with LA IgG for 7–14 min, and with prothrombin and LA IgG for 7–10 min. Prothrombin and LA IgG were displaced with binding buffer, followed by binding buffer containing no CaCl2. The on and off profiles were fit to a single exponential with IAsys FastFit (Advanced Laboratory Solutions, Melbourne, Australia).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of anti-prothrombin mAbs and LA IgGs on binding of prothrombin to phospholipid vesicles measured by ultracentrifugation

The effect of either an anti-prothrombin-fragment 1 or anti-prothrombin mAb on binding of 0.1 µM prothrombin to solution-phase phospholipid vesicles is shown in Fig. 1GoA. The anti-prothrombin Ab recognizes prothrombin-fragment 2 (residues 156–271) in solid-phase ELISA. Prothrombin was incubated with unilamellar 280-nm diameter 75:25 DOPC:DOPS phospholipid vesicles in the absence or presence of IgG, and bound prothrombin was determined by sedimenting the vesicles in an air-fuge. The anti-prothrombin-fragment 1 mAb did not influence prothrombin binding. In contrast, the anti-prothrombin mAb significantly enhanced prothrombin binding to phospholipid in a concentration-dependent manner.



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  		FIGURE 1. Effect of anti-prothrombin mAbs and LA IgGs on binding of prothrombin to phospholipid vesicles measured by ultracentrifugation. Binding of prothrombin to 75:25 DOPC:DOPS vesicles was measured by separating bound from free prothrombin in an air-fuge. The moles of prothrombin (II) bound to phospholipid vesicles was calculated from the total prothrombin and the ratio of bound vs total 125I-prothrombin. A, Effect of either an anti-prothrombin-fragment 1 or anti-prothrombin mAb on binding of 0.1 µM prothrombin to phospholipid vesicles. B, Effects of control, LA3, LA5, and LA6 IgG on binding of 0.1 µM prothrombin to phospholipid vesicles. The data is expressed as the fraction of total prothrombin bound. The data points and errors for the LA IgGs represent the mean and range of duplicate determinations. The data points and errors for control are the mean and SE of results from separate experiments with the three different control IgGs.

 
The total IgG from the plasmas of three control and three patients with LA were purified on protein G Sepharose. The ages of the three patients with LA were 17 (LA3), 14 (LA5), and 62 (LA6) years (7). The control subjects had no history of thrombosis, whereas LA3 and LA6 had histories of arterial and/or venous thrombotic events. LA5 had no thrombotic history but presented with a severe bleeding diathesis associated with hypoprothrombinaemia and had diagnostic lupus serology with positive anti-NF and Abs for dsDNA. IgG anticardiolipin Abs were present in the plasma of LA3 and LA5 but not LA6. None of the three LA IgGs react with plastic-adsorbed {beta}2-GP1 (6, 7). The LA IgGs were not contaminated by coagulation factors, as determined by SDS-PAGE and functional assays (7).

The effect of increasing control and LA IgG concentration on binding of prothrombin to solution-phase phospholipid vesicles is shown in Fig. 1GoB. All three LA IgGs enhanced prothrombin binding to phospholipid vesicles in a concentration-dependent manner. The sedimentation technique used for these studies can be compromised by nonspecific trapping of the ligand in the pellet or dissociation of ligand from the pelleted complex, particularly if the off-rate for the interaction is rapid. To confirm the effects of LA IgGs on prothrombin binding shown in Fig. 1Go, we used the technique of right-angle light scattering.

Effect of LA IgGs on binding of prothrombin to phospholipid vesicles measured by right-angle light scattering

Binding of prothrombin to 75:25 DOPC:DOPS vesicles was measured by right-angle light scattering. The isotherm for binding of prothrombin to the phospholipid vesicles is shown in Fig. 2GoA. The data was well fit to a single rectangular hyperbolae with a dissociation constant, and stoichiometry was 0.68 ± 0.08 µM and 0.12 ± 0.01 µM, respectively. These parameter values are similar to other estimates of this interaction (19).



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  		FIGURE 2. Effect of LA IgGs on binding of prothrombin to phospholipid vesicles measured by right-angle light scattering. Binding of prothrombin to 75:25 DOPC:DOPS vesicles was measured by right-angle light scattering. A, Isotherm for binding of prothrombin to the phospholipid vesicles. The solid line represents the best nonlinear least squares fit of the data to the rectangular hyperbolic binding equation. The calculated dissociation constant and stoichiometry was 0.68 ± 0.08 µM and 0.12 ± 0.01 µM, respectively. The data points are the average of duplicate determinations. B, Effect of control, LA3, LA5, and LA6 IgGs (50 µM) on binding of prothrombin to phospholipid vesicles. The data is expressed as the light-scattering intensity for incubation of IgG, prothrombin, and phospholipid (IS2) divided by the light-scattering intensity for IgG and phospholipid alone (IS1). The data points and errors for control are the mean and SE of results from separate experiments with the three different control IgGs. The data points for the LA IgGs are the average of duplicate determinations.

 
The effect of control and LA IgGs (50 µM) on binding of prothrombin to phospholipid vesicles is shown in Fig. 2GoB. The data is expressed as the light-scattering intensity for incubation of IgG, prothrombin, and phospholipid divided by the light-scattering intensity for IgG and phospholipid alone. Addition of the IgGs to the phospholipid vesicles alone resulted in a small increase in light scattering attributable to change in the refractive index of the solution, but there was no significant difference between control and LA IgGs (not shown). These results indicated that the LA IgGs did not interact directly with phospholipid (see below). However, the presence of LA IgG markedly changed the binding of prothrombin to the phospholipid vesicles. The control IgGs did not effect prothrombin binding. These findings and those of Fig. 1Go support the formation of complexes of LA IgG, prothrombin, and phospholipid.

Surface plasmon resonance was used to estimate the on and off rates for formation of LA IgG-prothrombin-phospholipid complexes.

Estimation of the kinetics of formation of LA IgG-prothrombin-phospholipid complexes

DOPC:DOPS:biotin-DHPE phospholipid vesicles (74:25:1) were immobilized on a avidin-coated IAsys microcuvette and incubated with either prothrombin (1 µM), prothrombin (1 µM) followed by LA IgG (10 µM), prothrombin (1 µM) and LA IgG (10 µM), or LA IgG (10 µM) followed by prothrombin (1 µM; Fig. 3Go). Addition of LA IgG after prothrombin binding enhanced the resonance unit amplitude. Very similar amplitude enhancements were observed if prothrombin and LA IgG were added together or if prothrombin was added after LA IgG. Two different control IgGs had no effect on prothrombin binding to phospholipid with either order of addition (not shown). These results support the formation of prothrombin-LA IgG-phospholipid complexes.



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  		FIGURE 3. Estimation of the kinetics of formation of LA IgG-prothrombin-phospholipid complexes. DOPC:DOPS:biotin-DHPE vesicles (74:25:1) were immobilized on an avidin-coated IAsys microcuvette, and the binding of prothrombin and/or LA IgGs was measured. The cuvette was incubated with either prothrombin (1 µM), prothrombin (1 µM) followed by LA IgG (10 µM), prothrombin (1 µM) and LA IgG (10 µM), or LA IgG (10 µM) followed by prothrombin (1 µM). The binding buffer was 50 mM HEPES, 125 mM NaCl, 3 mM CaCl2, 0.5 mg/ml BSA, pH 7.4. Interaction with prothrombin was for 6–12 min, with LA IgG for 7–14 min, and with prothrombin and LA IgG for 7–10 min. Prothrombin and LA IgG were displaced with binding buffer, followed by binding buffer containing no CaCl2.

 
The profiles were fit to a single exponential to obtain on and off rates and amplitude enhancements. In some cases, the probability of a second exponential being insignificant was zero; however, the rate of the second exponential was at least an order of magnitude slower than the first exponential and was ignored in this study. The assembly of prothrombin and LA IgG on phospholipid vesicles was relatively rapid and occurred with approximately the same rate as for binding of prothrombin alone (Table IGo). The half-life for association of 1 µM prothrombin was 20 s, compared with 19–46 s for assembly of prothrombin and LA IgG on the vesicles. In contrast, the off rates for prothrombin and LA IgG were 2- to 4-fold slower than the off rate for prothrombin. The half-life for dissociation of 1 µM prothrombin was 10 s, compared with 19–41 s for dissociation of prothrombin and LA IgG.


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  		Table I. On and off rates for prothrombin and LA IgG-prothrombin interaction with phospholipid vesicles measured by surface plasmon resonance

 
The amplitudes for prothrombin/LA IgG were calculated from the response where prothrombin was added after addition of LA IgG, thereby negating the response of the LA IgG alone, which was small to negligible. Amplitude enhancements over that for prothrombin binding alone were between 15% and 3.65-fold for the three LA IgGs (not shown).

Effect of LA F(ab')2 or Fab on binding of prothrombin to phospholipid vesicles

The requirement for LA IgG bivalency for enhanced prothrombin binding to phospholipid vesicles was tested by examining the effects of LA F(ab')2 or LA Fab on prothrombin binding in the centrifugation assay. F(ab')2 were derived from the intact IgGs with pepsin, and Fab were generated from F(ab')2 by reduction and alkylation (12). Reduced and alkylated IgG was used to control for possible perturbation of the antigenic binding site of the Fab. In contrast to intact LA IgG or LA F(ab')2, Fab did not promote prothrombin binding to phospholipid vesicles up to concentrations of ~40 µM (Fig. 4Go). Intact IgG and reduced and alkylated IgG promoted prothrombin binding to phospholipid vesicles equivalently. It is noteworthy that LA F(ab')2 were approximately twice as effective on a molar basis that intact LA IgG at promoting prothrombin binding. Fab of the LA IgGs also did not promote prothrombin binding to phospholipid vesicles in the light-scattering assay (not shown).



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  		FIGURE 4. Effect of LA F(ab')2 or Fab on binding of prothrombin to phospholipid vesicles. Binding of prothrombin to 75:25 DOPC:DOPS vesicles was measured by separating bound from free prothrombin in an air-fuge. The moles of prothrombin (II) bound to phospholipid vesicles was calculated from the total prothrombin and the ratio of bound vs total 125I-prothrombin. The effect of LA IgG, reduced and alkylated IgG, F(ab')2, or Fab on binding of 0.1 µM prothrombin to phospholipid vesicles is shown. Molar concentrations were calculated with molecular masses of 150, 100, and 50 kDa for IgG, F(ab')2 and Fab, respectively. The data is expressed as the fraction of total prothrombin bound. The data points are the average of duplicate determinations.

 
Model of the effect of LA IgG on prothrombin binding to phospholipid

The results shown in Figs. 1–4GoGoGoGo indicated that LA IgG and prothrombin form complexes on a phospholipid surface, and that IgG bivalency was required for these complexes to form. With these observations in mind, an equilibrium binding model for the interaction of LA IgG/prothrombin with phospholipid vesicles was developed (Fig. 5Go).



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  		FIGURE 5. Model of the effect of LA IgG on prothrombin binding to phospholipid. Prothrombin (II) interacts with LA IgG with dissociation constant, KA, and with phospholipid with dissociation constant, KL. Prothrombin-phospholipid complex also interacts with LA IgG with dissociation constant, KA. Note that prothrombin interacts with either Fab arm of the LA IgG. Interaction of a second molecule of prothrombin with LA IgG-prothrombin complex or LA IgG-prothrombin-phospholipid complex also occurs with dissociation constant, KA. Because of the linked equilibria of this model, interaction of prothrombin-LA IgG-prothrombin complex with phospholipid occurs with dissociation constant, KL. The factor, {alpha}, is defined as the ratio of the concentration of prothrombin in a 10-nm diameter shell around the phospholipid vesicles vs the prothrombin concentration in the solution phase. The model is described by Equation 2Go.

 
In the model, prothrombin interacts with LA IgG with dissociation constant, KA, and with phospholipid with dissociation constant, KL. Prothrombin-phospholipid complex also interacts with LA IgG with dissociation constant, KA. Note that prothrombin interacts with either Fab arm of the LA IgG. Interaction of a second molecule of prothrombin with LA IgG-prothrombin complex or LA IgG-prothrombin-phospholipid complex also occurs with dissociation constant, KA. Because of the linked equilibria of this model, interaction of prothrombin-LA IgG-prothrombin complex with phospholipid occurs with dissociation constant, KL. The factor, {alpha}, represents the high local concentration of prothrombin on the phospholipid surface relative to its concentration in the solution phase. The factor, {alpha}, was estimated from the diameter of the phospholipid vesicles and the dissociation constant and stoichiometry for binding of prothrombin to the vesicles.

The concentration of prothrombin bound to the phospholipid vesicles, [PL], is calculated by using the quadratic form of the binding equation (Equation 1Go; Ref. 20).

(1)
Prothrombin and phospholipid is defined by P and L, respectively. Subscript T denotes total concentration of reagent. Thrombin has a diameter of 4.5 nm (21); therefore, vesicle-bound prothrombin was anticipated to be within a 10-nm shell on the vesicle surface. The concentration of prothrombin in the 10-nm diameter shell around the 280-nm diameter phospholipid vesicles was calculated from PL and the stoichiometry of 10,000 molecules/vesicle. Lu and Nelsestuen (19) calculated that 13,000 molecules of prothrombin can bind to 328-nm 75:25 DOPC:DOPS vesicles. KL (Fig. 2GoA) and LT was fixed at 1 µM. The factor, {alpha}, was defined as the ratio of the concentration of prothrombin in the 10-nm diameter shell vs the prothrombin concentration in the solution phase. The theoretical relationship between {alpha} and the total concentration of prothrombin is shown in Fig. 6GoA, and {alpha} varied from ~10 to ~60, depending on the concentration of prothrombin. The lower the total prothrombin concentration, the higher the value of {alpha}. This result indicated that the concentration of prothrombin on the surface of the vesicles is at least an order of magnitude higher than the solution-phase concentration.



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  		FIGURE 6. Simulation of the effect of LA IgG on prothrombin binding to phospholipid. A, Predicted relationship between prothrombin concentration on the surface of phospholipid vesicles vs the concentration in the solution phase. The concentration of prothrombin in a 10-nm diameter shell around 280-nm diameter phospholipid vesicles is calculated from Equation 1Go assuming a LT of 1 µM, KL of 1 µM, and stoichiometry of 10,000 molecules/vesicle. The factor, {alpha}, is defined as the ratio of the concentration of prothrombin in the 10-nm diameter shell vs the prothrombin concentration in the solution phase. The model shown in Fig. 5Go was simulated by numerical integration of Equation 2Go. B, Prothrombin-binding isotherms were constructed by varying LA IgG (0–3 µM) at fixed prothrombin (0.1 µM) and phospholipid (1 µM) with KA and KL fixed at 1 µM and {alpha} of either 1, 10, or 60. C, Prothrombin-binding isotherms were constructed by varying LA IgG (0–3 µM) at fixed prothrombin (0.1 µM) and phospholipid (1 µM) with a KL of 1 µM and {alpha} of 30 and KA of either 1, 10, or 100 µM.

 
The equilibrium binding model in Fig. 6Go is described by Equation 2Go. LA IgG is defined by A.

(2)




where



and the concentration of P bound to L in the presence of A, [P]B, is

Prothrombin-binding isotherms were constructed by numerical integration of Equation 2Go with least-squares minimization (Scientist software; Micromath, Salt Lake City, Utah) and varying LA IgG (0–3 µM) at fixed prothrombin (0.1 µM), which is the same experimental design shown in Figs. 1Go and 4Go.

The IgG concentration in plasma is ~60 µM. A LA IgG concentration of 3 µM would correspond to ~5% of the total plasma IgG. It is anticipated that this fraction would be the upper limit in vivo. Prothrombin concentration is varied around the plasma concentration of 1.4 µM. KL and LT were fixed at 1 µM (see above), whereas KA was either 1, 10, or 100 µM and {alpha} was either 1, 10, or 60.

Prothrombin binding to phospholipid is not influenced by LA IgG when {alpha} is 1 (Fig. 6GoB). However, prothrombin binding is enhanced in the presence of LA IgG when {alpha} > 1. In other words, the concentration of prothrombin on the surface of the vesicles promotes the formation of LA IgG-prothrombin-phospholipid complexes. The augmentation of prothrombin binding by LA IgG is reduced when the affinity of LA IgG for prothrombin is weakened (Fig. 6GoC). For instance, when the dissociation constant for prothrombin binding to LA IgG is 100 µM, LA IgG had no significant effect on prothrombin binding. Therefore, in this model, the augmentation of prothrombin binding by LA IgG is influenced predominantly by the concentration of prothrombin on the surface of the vesicles and the affinity of LA IgG for prothrombin.

There was no effect of Fab of the LA IgGs on prothrombin binding to phospholipid vesicles (Fig. 4Go). The model shown in Fig. 5Go was revised to accommodate monovalent LA Fab in place of the bivalent LA IgG (not shown). When the bivalency of the Ab was eliminated, there was no predicted augmentation of prothrombin binding, which is in agreement with the experimental observations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of the studies herein was to investigate the mechanism by which the LA IgGs enhanced interaction of prothrombin with phospholipid vesicles. The effect of LA IgGs on binding of prothrombin to unilamellar 75:25 DOPC:DOPS phospholipid vesicles was measured. No attempt was made to affinity purify the anti-prothrombin Igs, as these Igs would not necessarily have reflected the net properties of the total Ig population on prothrombin binding. Also, binding to solution-phase phospholipid vesicles and not to plastic-adsorbed phospholipid was measured. This was an important distinction, as only one of the three LA IgGs (LA5) reacted with plastic-adsorbed DOPS in the presence of prothrombin, whereas all three LA IgGs formed complexes with prothrombin and solution-phase phospholipid vesicles (7). This probably reflects differences in presentation of prothrombin epitope(s) for LA IgG when prothrombin is bound to plastic-adsorbed DOPS vs bound to fluid bilayer phospholipid vesicles.

A murine monoclonal anti-prothrombin Ab and all three LA IgGs enhanced prothrombin binding to phospholipid vesicles in a concentration-dependent manner measured by either ultracentrifugation or right-angle light scattering. The results were consistent with the formation of LA IgG-prothrombin-phospholipid complexes. These results are supported by those of Rao et al. (8) who described increased binding of prothrombin to plastic-adsorbed DOPS and umbilical vein endothelial cell monolayers in the presence of four different LA IgGs.

The assembly of LA IgG and prothrombin on phospholipid vesicles was demonstrated by surface plasmon resonance. Addition of 10 µM LA IgG after binding of 1 µM prothrombin enhanced the resonance unit amplitude. Very similar amplitude enhancements were observed if prothrombin and LA IgG were added together or if prothrombin was added after LA IgG. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. The slower off rates may have been a consequence of the bivalency of the LA IgG-prothrombin-phospholipid vesicle interaction. Bivalent binding has been shown to strongly effect the kinetics of desorption (22, 23) and Willems et al. (25) observed a similar slow dissociation of bivalent anticardiolipin IgG-{beta}2-GP1 complexes from planar phospholipid bilayers. To test this hypothesis, F(ab')2 or Fab of the LA IgGs were made and examined for their ability to promote prothrombin binding to phospholipid vesicles.

The Fab of all three LA IgGs did not influence prothrombin binding to phospholipid vesicles at concentrations up to 40 µM. Therefore, LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles. In contrast, F(ab')2 of the LA IgGs were more effective than the intact IgGs at promoting prothrombin binding. This may reflect the enhanced mobility of the hinge region in the F(ab')2 and a more favorable binding and packing of the Ab fragment and prothrombin on the phospholipid surface. The same dependence on Ab bivalency for activity is observed for enhanced binding of {beta}2-GP1 to phospholipid by anticardiolipin Abs (24, 25, 26, 27). Willems et al. (25) reported that anticardiolipin Abs enhance binding of {beta}2-GP1 to planar phospholipid bilayers measured by ellipsometry. The Fab of the anticardiolipin Abs have no appreciable effect on {beta}2-GP1 binding. Takeya et al. (26) showed that anti-{beta}2-GP1 mAbs directed against the third and fourth domains of {beta}2-GP1 enhance binding of {beta}2-GP1 to plastic-adsorbed phospholipid and phospholipid vesicles. The Fab of the mAbs were without effect. Similarly, Arnout et al. (27) demonstrated that certain anti-{beta}2-GP1 mAbs, but not their Fab, enhance binding of {beta}2-GP1 to polar phospholipid monolayers measured by surface plasmon resonance.

The results shown herein indicate that LA IgG promote prothrombin binding to phospholipid vesicles and that LA IgG bivalency is required for this effect. To better understand the mechanism of this effect of the LA IgGs, an equilibrium binding model for the interactions of LA IgG, prothrombin, and phospholipid vesicles was developed. In the model, prothrombin interacts with either Fab arm of the LA IgG and/or phospholipid. Interaction of prothrombin with large phospholipid vesicles resembles binding of prothrombin to a planar surface. Subsequent interactions of prothrombin on the surface of the vesicle are favored because of the high surface concentration of prothrombin relative to its concentration in the solution phase. This property has been factored into the model. It is apparent from the simulation that surface concentrations of prothrombin on large diameter 75:25 DOPC:DOPS vesicles are 10–60 times higher than the concentration of prothrombin in solution phase. The lower the total prothrombin concentration, the larger the difference between surface and solution-phase concentration.

Prothrombin binding to phospholipid was predicted to be augmented by LA IgG when the surface prothrombin concentration was taken into account. The predicted promotion of prothrombin binding by LA IgG was reduced when the affinity of LA IgG for prothrombin was weakened. Moreover, monovalent Fab were not predicted to promote prothrombin binding, which agreed with the experimental observations. Therefore, the augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.

The simulations shown in Fig. 6Go generally underestimated the magnitude of the effects of the LA IgGs on prothrombin binding to phospholipid (compare Figs. 1Go, 2Go, and 6Go). The contribution of the loss in entropy to the change in free energy attributable to complex formation is less for interactions on surfaces than in solution (25, 28). This phenomenon is predicted to strengthen interactions of LA IgG and prothrombin on the phospholipid surface, which could have accounted for the smaller effects of the LA IgGs in the simulations. Moreover, LA IgG may have orientated prothrombin on the phospholipid surface such that the free energy of formation of quaternary prothrombin-LA IgG-prothrombin-phospholipid complexes was smaller than the product of their dissociation constants. In contrast, the model did not allow for steric restrictions in interactions between prothrombin and LA IgG on the phospholipid surface, which might have weakened overall affinities. It is important to note that the model is the simplest that can explain the data. It is likely that the LA IgGs will bind to multiple different epitopes on prothrombin, which could result in complexes of various sizes and stoichiometries. Also, the parts of prothrombin to which the Abs bind will most likely effect the efficiency with which the Ab promotes prothrombin binding to phospholipid.

All LA IgGs tested promoted prothrombin binding to phospholipid vesicles. This may be a general property of most, if not all, LA Abs. We have suggested have LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall (7). The fact that the LA IgGs can enhance thrombin production in flow (7) and that thrombin production is increased in patients with LA (9, 10) suggests that this effect of LA Abs on prothrombin binding may contribute to their pathogenesis.


   Acknowledgments
 
We thank Dr. Kerry Taylor (Mater Hospital, Brisbane, Queensland, Australia) for supplying a patient blood sample and clinical details (LA5), and Margaret Aboud (Royal North Shore Hospital, New South Wales, Australia) for supplying clinical details. We are grateful to Prof. Chris dos Remedios (Sydney University, New South Wales, Australia) for assistance with the light-scattering experiments, and Prof. Chris Parish and Dr. Kathy Brown (John Curtin School of Medical Research, Canberra, Australia) for assistance with developing the surface plasmon resonance experiments.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council of Australia and a Research Infrastructure Grant from the New South Wales Department of Health. Back

2 Address correspondence and reprint requests to Dr. Philip Hogg, Center for Thrombosis and Vascular Research, School of Pathology, University of New South Wales, Sydney, NSW 2052 Australia. Back

3 Abbreviations used in this paper: LA, lupus anticoagulant; {beta}2-GP1, {beta}2-glycoprotein 1; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DOPS, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine; biotin-DHPE, N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine. Back

Received for publication June 7, 2000. Accepted for publication March 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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