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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. The purpose of this study was to investigate the effects of β2-glycoprotein I (β2GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation.

Methods

The effect of β2GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of β2GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-β2GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured.

Results

Beta2-glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The β2GPI region responsible for stimulating tPA activity was shown to be at least partly contained within β2GPI domain V. In addition, β2GPI bound tPA with high affinity (Kd ∼20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (Kcat/Km) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of β2GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following β2GPI repletion. Stimulation of tPA-mediated plasminogen activity by β2GPI was inhibited by monoclonal anti-β2GPI antibodies as well as by anti-β2GPI antibodies from patients with APS.

Conclusion

These findings suggest that β2GPI may be an endogenous regulator of fibrinolysis. Impairment of β2GPI-stimulated fibrinolysis by anti-β2GPI antibodies may contribute to the development of thrombosis in patients with APS.

Beta2-glycoprotein I (β2GPI) belongs to the complement control protein superfamily and circulates in plasma at a concentration of ∼4 μM (1). Like other members of the complement control protein superfamily that contain one or more characteristic short consensus repeats (SCRs) (2), β2GPI contains 5 SCRs. However, domain V of β2GPI (β2GPI-V) consists of an atypical SCR containing a lysine-rich sequence (CKNKEKKC) that imparts a positive charge to this domain and mediates the binding of β2GPI to phospholipid (3). Plasmin cleavage between Lys317 and Thr318 of the β2GPI domain V abolishes phospholipid binding (4).

Beta2-glycoprotein I is an important antigen in the antiphospholipid syndrome (APS), and anti-β2GPI antibodies are an independent risk factor for thrombosis and recurrent loss of pregnancy (5–7). Both procoagulant and anticoagulant activities may be regulated by β2GPI (7–9). Thrombin generation has been shown to be impaired in the plasma of β2GPI-deficient mice (10).

Impaired fibrinolysis may contribute to the development of thrombosis (11, 12). Plasmin plays a central role in the lysis of fibrin thrombi, and the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is precisely regulated (13). Tissue plasminogen activator binds fibrin with high affinity and may be of particular importance for the lysis of fibrin thrombi in the vasculature (13). Several stimulators of fibrinolysis, including insoluble proteins, protein aggregates, microfilaments, fibrin fragments, and type IV collagen, may stimulate fibrinolysis, primarily by promoting the formation of a ternary tPA–fibrin–plasminogen complex (14, 15). Annexin A2 is an endothelial cell coreceptor for plasminogen and tPA that accelerates tPA-dependent plasminogen activation (16, 17), and the annexin A2/S100A10 heterotetramer may be even more potent in this regard (18–20). However, to date, no soluble plasma cofactors that promote tPA-dependent plasminogen activation have been identified.

Several studies have examined the interactions of β2GPI with the fibrinolytic system. Lopez-Lira et al (21) and Yasuda et al (22) reported low-affinity binding of Glu-plasminogen to intact or plasmin-cleaved β2GPI, respectively. However, interactions of β2GPI with tPA have not been described. Here, we report our findings that β2GPI binds tPA with high affinity and enhances tPA activity and tPA-dependent plasminogen activation. Depletion of β2GPI from plasma impairs the lysis of plasma clots, and anti-β2GPI antibodies inhibit the ability of β2GPI to stimulate fibrinolysis. Given the abundance of β2GPI in plasma, these findings suggest that β2GPI may be an endogenous regulator of fibrinolysis and that impairment of fibrinolysis by anti-β2GPI antibodies may contribute to APS-associated thrombosis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Purification of β2GPI from human plasma.

We used a modification of previously described methods (23) to purify β2GPI from human plasma. Briefly, polyethylene glycol (final concentration 15%) was added to plasma, and the precipitated proteins were collected by centrifugation at 10,000g for 30 minutes. The precipitate was resuspended, and β2GPI was isolated by sequential chromatography using heparin–Sepharose CL-6B (Sterogene Bioseparations, Carlsbad, CA) and Source 15S (Amersham Pharmacia Biotech, Uppsala, Sweden). The purity of the isolated β2GPI was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gels.

Cloning, expression, and purification of β2GPI in mammalian cells.

Full-length β2GPI complementary DNA was cloned into pDNR-LIB vector (Sanying Biotechnology, Beijing, China). For expression of recombinant β2GPI, the β2GPI coding sequence was amplified using the primers 5′-C-ACC-ATG-GGA-CGG-ACC-TGT-CCC-AAG-3′ and 5′-GCA-TGG-CTT-TAC-ATC-GGA-TGC-ATC-A-3′ (24) and cloned into pcDNA3.1 expression vector. Then, β2GPI-pcDNA3.1 was transfected into 293T cells, and cell extracts prepared 48 hours after transfection were analyzed by immunoblotting using rabbit anti-human β2GPI and anti-His (C-terminal) antibodies. The 6× His–tagged recombinant β2GPI was purified using nickel–nitrilotriacetic acid (Ni-NTA) Superflow columns (Amersham Pharmacia Biotech).

Preparation of recombinant β2GPI domain V.

The sequence encoding β2GPI-V was amplified from β2GPI-pDNR-LIB using the primers 5′-CAC-GGA-TCC-AAA-GCA-TCT-TGT-AAA-GTA-CC-3′ and 5′-CTG-AAG-CTT-TTA-GCA-TGG-CTT-TAC-ATC-3′, and the PCR product was cloned into vector PQE30 (obtained from Z. H. Xie, Tsinghua University, Shenzhen, China) using Bam HI and Hind III restriction sites (underlined primer sequences). The domain V construct was transfected into competent Escherichia coli M15 cells grown in 100 μg/ml of ampicillin and 25 μg/ml of kanamycin, and expression was induced by exposure of cells to 1 mM IPTG at 37°C for 4 hours. The 6× His–tagged β2GPI-V was purified using Ni-NTA Superflow columns, and the purity of the recombinant polypeptide was confirmed by SDS-PAGE using 15% gels.

A domain V peptide corresponding to amino acids Gly274–Cys288 (GQKVSFFCKNKEKKC) of β2GPI was synthesized by Eurogentec (San Diego, CA).

Monoclonal anti-β2GPI antibodies.

The monoclonal anti-β2GPI antibody BD4 was raised against intact β2GPI using standard methods (25). This antibody had an affinity constant for β2GPI of 2.04 × 107M–1. Murine IgG1 monoclonal antibody 1D2, also raised against intact β2GPI, was obtained from Abcam (Cambridge, UK). The epitopes for these antibodies have not been defined. Both antibodies are of the IgG1 subclass.

Measurement of the effect of β2GPI on tPA-dependent plasminogen activation.

The effect of β2GPI on tPA-dependent plasminogen activation was measured using a fluorogenic plasmin substrate (H-D-Val-Leu-Lys-AMC) (product no. I-1390; Bachem Bioscience, King of Prussia, PA). Briefly, 10 nM tPA was incubated for 15 minutes at room temperature with increasing concentrations of native or recombinant β2GPI, β2GPI-V, β2GPI domain V peptide, or 0.5% bovine serum albumin (BSA; control). Fifty microliters of each reaction mixture was then transferred to 96-well microplates, to which 100 nM Glu-plasminogen (Enzyme Research Laboratories, South Bend, IN) and 200 μM H-D-Val-Leu-Lys-AMC was added. After mixing, substrate hydrolysis was measured at regular intervals as the relative fluorescence intensity units (I360/465 nm). Initial rates of plasmin generation were determined by linear regression analysis of plots of I360/465 nm versus time squared, using Prism 4.0 software (GraphPad Software, San Diego, CA) (17).

Parallel studies were performed using an identical approach, but substituting uPA for tPA.

Measurement of the effect of β2GPI on tPA-dependent plasminogen activation in a fibrin gel.

Fibrin–agarose gels were prepared by mixing 4 ml of agarose (1%; 55°C), 4 ml of fibrinogen (3 mg/ml), 320 μl of Glu-plasminogen (500 nM), and 320 μl of thrombin (1 unit/ml) in 10-ml polypropylene tubes. The contents of each tube were added to 90-mm plates and allowed to gel. After incubation at 4°C for 1 hour, glass pipettes were used to load 10 μl of the reaction mixtures containing 10 nM tPA, either alone or in the presence of β2GPI, β2GPI-V, β2GPI domain V peptide, or 0.5% BSA, into individual wells prepared in the gels. After 28 hours at 37°C, the area of lysis caused by each sample was measured.

Effect of plasma β2GPI depletion on clot lysis.

The fibrinolytic activity of control and β2GPI-depleted plasma from 3 normal donors was compared. First, rabbit preimmune or anti-human β2GPI IgG was conjugated to Affi-Gel 10 (Bio-Rad, Richmond, CA) at a concentration of 5 mg of IgG per ml of beads. The beads were placed in separate columns, and 2 ml of plasma was passed through each. Immunoblotting of plasma passed through the anti-β2GPI column revealed no detectable β2GPI, and the first 1 ml of flow-through fraction from this column was used for subsequent studies. The corresponding flow-through fraction from the column containing preimmune IgG was used as a control.

The fibrinolytic activity in control and β2GPI-depleted plasma was determined using a 96-well microplate clot lysis assay described by Nagashima et al (26). Briefly, 15 μl of β2GPI-depleted or control plasma was mixed with 3 μl of 2 μM thrombomodulin and 60 μl of 0.04M HEPES, pH 7.0, containing 0.15M NaCl and 0.01% Tween 80. This mixture was added to another well containing 4 μl of a 75 units/ml concentration of thrombin, 2 μl of a 1M concentration of CaCl2, and 4 μl of a 0.5 μg/ml concentration of tPA. The total volume was adjusted to 120 μl with water. The absorbance of the clotted sample at 405 nm (A405) was monitored every 15 minutes for 180 minutes. Maximal absorbance occurred at 30 minutes, and the extent of lysis was defined as the difference between A405 at 30 minutes and 180 minutes.

To confirm that any difference in clot lysis between control and β2GPI-depleted plasmas was actually due to β2GPI depletion, a β2GPI-depleted plasma sample was replenished with 100 nM or 200 nM (final concentrations) purified human β2GPI. Clot lysis rates were then determined using the same protocol.

Effect of β2GPI on tPA amidolytic activity.

The activity of tPA was determined in the presence or absence of β2GPI using a fluorogenic tPA substrate (glutaryl-Gly-Arg-AMC) (product no. I-1195; Bachem). Assays were performed in 96-well plates containing 0.5% BSA or mixtures of tPA (10 nM) and increasing concentrations of β2GPI (0–5 μM). Reaction mixtures were incubated at room temperature for 15 minutes, at which point 200 μM I-1195 was added, and substrate hydrolysis was measured at regular intervals (I360/465 nm). Initial rates of hydrolysis (K) were calculated by linear regression of plots of I360/465 nm versus time squared, using Prism 4.0 software (17).

Binding of β2GPI to tPA.

Binding of β2GPI to tPA was initially analyzed using a microplate binding assay. High-binding enzyme-linked immunosorbent assay plates (Costar, Cambridge, MA) were coated with 10 μg/ml of single-chain tPA by overnight incubation at 4°C. After washing, wells were incubated for 4 hours at 25°C with 100 μl of increasing concentrations of β2GPI. Bound β2GPI was detected using rabbit anti-human β2GPI (0.33 μg/ml) followed by peroxidase-conjugated goat anti-rabbit IgG and tetramethylbenzidine. Binding specificity was determined by assessing the ability of fluid-phase tPA to inhibit the binding of 40 nM β2GPI.

We also analyzed the binding of β2GPI to tPA in real time using an IAsys Plus Biosensor system (Affinity Sensors, Paramus, NJ). The β2GPI was immobilized on a carboxymethyl dextran biosensor using amine coupling, and the amount of ligand bound to the β2GPI-coated surface was assessed following injection of tPA (0–14 nM). IAsys FASTfit software was used to determine the association and dissociation rate constants (Ka and Kd, respectively). The binding of plasminogen to β2GPI was assessed using an identical approach.

Anti-β2GPI antibody inhibition of plasminogen activation.

IgG fractions were purified from 83 plasma samples (supplied by the Guangdong Family Planning Science and Technology Institute, Guangzhou, Guangdong, China) using protein G–agarose. These samples were from 40 healthy control subjects and from 43 patients who met the Sapporo criteria for definite APS (27). The 43 patients with APS were positive for either IgG or IgM anticardiolipin antibody (32 patients), IgG or IgM anti-β2GPI antibody (24 patients), and/or or lupus anticoagulant (11 patients). Twenty-two APS samples contained anti-β2GPI antibodies but not anti-tPA antibodies, whereas the other 21 APS samples contained neither anti-β2GPI nor anti-tPA antibodies.

We also assessed the effect of affinity-purified anti-β2GPI antibodies isolated from patient plasma, as we have previously described (28), on the ability of β2GPI to enhance tPA-mediated plasminogen activation.

The effect of anti-β2GPI antibodies on β2GPI-dependent enhancement of plasminogen activation was determined by measuring plasmin amidolytic activity following incubation of plasminogen with tPA and β2GPI in the presence or absence of monoclonal anti-β2GPI antibodies, patient-derived anti-β2GPI+/anti-tPA– or anti-β2GPI–/anti-tPA– IgG fractions, or affinity-purified anti-β2GPI IgG. Initial rates of plasmin generation were calculated by linear regression analysis of plots of I360/465 nm versus time squared, using Prism 4.0 software.

Statistical analysis.

Statistical analyses were performed using Student's 2-tailed t-test or the Mann-Whitney U test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Stimulation of tPA-mediated plasminogen activation by β2GPI.

Native β2GPI, recombinant β2GPI, and β2GPI-V accelerated the initial rate of plasminogen activation by tPA in a concentration-dependent manner, as measured by cleavage of the plasmin substrate H-D-Val-Leu-Lys-AMC (Figures 1A and B). In contrast, the β2GPI domain V peptide GQKVSFFCKNKEKKC did not significantly enhance plasminogen activation (Figure 1A).

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Figure 1. Enhancement of tissue plasminogen activator (tPA)–dependent Glu-plasminogen activation by β2-glycoprotein I (β2GPI). A, Measurement of tPA-dependent plasminogen activation in the presence of bovine serum albumin (BSA), 1 μM human native β2GPI (β2GPI-n), recombinant β2GPI (β2GPI-r), β2GPI domain V (β2GPI-V), or β2GPI domain V peptide (β2GPI-p). Substrate hydrolysis was measured as relative fluorescence intensity units (I360/465 nm) as a function of time squared (minutes2) (37). B, Initial rates of plasmin generation (determined as in A) in the presence of increasing concentrations of native β2GPI, recombinant β2GPI, β2GPI-V, or BSA. Values are the mean of triplicate determinations (n = 3 experiments).

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β2GPI was similarly effective when a fixed concentration of tPA (10 nM) was used to activate increasing concentrations of plasminogen (0–200 nM). Analysis of these data using Lineweaver-Burke plots revealed that β2GPI and β2GPI-V, respectively, conferred a 19.7-fold increase and a 5.9-fold increase in the catalytic efficiency (Kcat/Km) of tPA-mediated Glu-plasminogen activation (Table 1). The observed increase in catalytic efficiency resulted from a decrease in the Km (9.8-fold) and enhancement of the maximum enzyme velocity (Vmax) (2-fold). By comparison, Cesarman et al (17) reported that annexin A2 caused a 63-fold increase in the catalytic efficiency of tPA-mediated Glu-plasminogen activation, reflecting a 9-fold decrease in the Km and a 5-fold increase in Vmax.

Table 1. Effects of native β2GPI and β2GPI-V on the kinetics of tPA-dependent activation of Glu-plasminogen*
 Km, μMKcat, second−1Kcat/Km, μM−1 second−1
  • *

    The maximum enzyme velocity (Vmax) and the Michaelis-Mentin constant (Km) were calculated using nonlinear regression. The catalytic constant (Kcat) was calculated as the Vmax/E0, where E0 represents the enzyme (i.e., tissue plasminogen activator [tPA]) concentration (10 nM). Vmax was derived from a standard curve of plasmin amidolytic activity. The catalytic efficiency (Kcat/Km) was then calculated. BSA = bovine serum albumin; β2GPI = β2-glycoprotein I.

Plasminogen   
 Plus BSA0.45380.11490.253
 Plus β2GPI0.04640.23104.983
 Plus β2GPI domain V0.31480.47181.499

Neither human albumin nor control human IgG affected tPA-dependent plasminogen activation. Parallel studies demonstrated that β2GPI did not affect the ability of single-chain urokinase to activate plasminogen.

Stimulation of fibrinolysis in a fibrin gel by β2GPI.

At β2GPI concentrations of 1 μM and 5 μM, there was a 2.6-fold and a 3.1-fold increase, respectively, in lysis of a fibrin gel (Figure 2). A 5 μM concentration of β2GPI-V caused a 2.6-fold stimulation of fibrinolysis, although the Gly274–Cys288 β2GPI-V peptide had no effect. Control proteins BSA and IgG also had no activity. While the relative amounts of fibrin digestion in this assay seem proportionally small compared with the extent of plasminogen activation in the amidolytic assays, the kinetic parameters governing these assays are not comparable.

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Figure 2. Enhancement of fibrinolysis in fibrin gels by native β2-glycoprotein I (β2GPI) and β2GPI domain V (β2GPI-V). Fibrin gel lysis was assessed as described in Materials and Methods. A, Representative results of fibrin gel lysis in the presence of 10 nM tissue plasminogen activator (tPA) plus bovine serum albumin (BSA) (1), 10 nM tPA plus 5 μM β2GPI-V (2), and 10 nM tPA plus 5 μM native β2GPI (3). B, Activity of tPA (as determined from the size of lytic areas) in the presence or absence of increasing amounts of native β2GPI (β2GPI-n), β2GPI-V, or β2GPI peptide (β2GPI-p), calculated from a standard curve. Values are the mean ± SEM.

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Effect of plasma β2GPI depletion on clot lysis.

Plasma that had been perfused over an anti-β2GPI column contained no immunologically detectable β2GPI, as determined by immunoblotting (data not shown). To control for dilution, plasma from the same donors was perfused over a column containing preimmune (control) rabbit IgG. Both plasma samples were clotted, and their clot lysis rates were determined as described in Materials and Methods.

Table 2 depicts the extent of clot lysis using control and β2GPI-depleted plasma from 3 donors. Though the maximal A405 and extent of lysis varied, the decrease in absorbance between 30 and 180 minutes, reflecting clot lysis (26), was significantly diminished in the absence of β2GPI (P = 0.0260 versus the means of triplicate points in controls).

Table 2. Effects of β2GPI depletion on clot lysis*
 A405ΔA405% lysis
At 30 minutesAt 180 minutes
  • *

    Clot lysis in clotted samples of blood from 3 normal donors was evaluated as described in Materials and Methods. Absorbance of the clotted sample at 405 nm (A405) was monitored every 15 minutes for 180 minutes. Maximal absorbance occurred at 30 minutes. The extent of lysis was defined as the difference in the A405 at 30 minutes compared with 180 minutes.

Patient 1    
 Anti-β2GPI0.5950.5090.08614.4
 Control IgG0.3680.2040.16444.5
Patient 2    
 Anti-β2GPI1.090.810.2825.6
 Control IgG0.920.470.4548.9
Patient 3    
 Anti-β2GPI1.291.30−0.010
 Control IgG1.190.700.4941.1

Figure 3A shows the results of a representative experiment in which endogenous clot lysis rates in control and β2GPI-depleted plasma were compared. As shown in Figure 3B, the reconstitution of β2GPI-depleted plasma with purified β2GPI (100 and 200 nM final concentrations) led to complete restoration of lytic activity in the β2GPI-depleted plasma samples.

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Figure 3. Clot formation and lysis in the presence and absence of β2-glycoprotein I (β2GPI). A, Plasma from a normal donor was divided into equal aliquots and subjected to chromatography on a column to which either anti-β2GPI antibodies or preimmune rabbit IgG had been conjugated. After passage through the column containing anti-β2GPI antibodies, plasma was completely immunodepleted of β2GPI, as determined by immunoblotting. Flow-through from both columns was placed in 96-well microplates and clotted as described in Materials and Methods. Clots were monitored by measurement of the absorbance at 405 nm (A405) over time. Clotting led to an increase in the A405 value, which was maximal at ∼30 minutes, followed by a progressive decrease as the clots lysed. Results are also shown for normal plasma not subjected to chromatography. B, The same experiment described in A was performed on β2GPI-depleted plasma that was analyzed either directly or after the addition of 100 nM or 200 nM β2GPI. The addition of β2GPI to the β2GPI-depleted plasma restored its fibrinolytic activity. Values are the mean ± SEM.

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Stimulation of tPA amidolytic activity by β2GPI.

The presence of β2GPI enhanced the tPA-dependent cleavage of I-1105, a tPA-specific substrate, in a concentration-dependent manner. A concentration of 0.3 μM β2GPI caused a 4-fold increase in the rate of substrate hydrolysis (Figure 4A). A stimulatory effect of β2GPI (1 μM) was also observed in the presence of increasing concentrations of tPA (0–50 nM) (Figure 4B).

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Figure 4. Stimulation of tissue plasminogen activator (tPA) amidolytic activity by β2-glycoprotein I (β2GPI). A, Amidolytic activity of tPA in the presence of increasing amounts of native β2GPI (β2GPI-n), β2GPI domain V (β2GPI-V), and bovine serum albumin (BSA). B, Effect of 1 μM native β2GPI or BSA on tPA amidolytic activity in the presence of increasing concentrations of tPA. Results are expressed in relative fluorescence intensity units (I360/465 nm) as a function of time squared (minutes2). Values are the mean of triplicate determinations (n = 3 experiments).

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Binding of β2GPI to tPA.

Examination of the binding properties showed that β2GPI bound to tPA-coated microplates in a concentration-dependent manner (Figure 5A) and that this binding was competitively inhibited by fluid-phase tPA (50% inhibition concentration [IC50] ∼310 nM) (Figure 5B). Binding was not inhibited by the β2GPI-V–derived peptide (Gly274–Cys288) previously reported to mediate the binding of β2GPI to anionic phospholipid (data not shown), suggesting that different molecular interactions facilitate these binding events.

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Figure 5. Binding of β2-glycoprotein I (β2GPI) to tissue plasminogen activator (tPA). A, Binding of native β2GPI (β2GPI-n) or bovine serum albumin (BSA) to tPA, as determined using a microplate assay (see Materials and Methods for details). Optical density (OD) was measured at 450 nm. B, Inhibition of the binding of 40 nM β2GPI to immobilized tPA by fluid-phase tPA or BSA. Results are expressed as the percentage of β2GPI bound in the presence of fluid-phase tPA relative to that bound in its absence. Values in A and B are the mean ± SEM. C, Binding of the indicated nanomolar concentrations of tPA to β2GPI over time, as determined by optical biosensor analysis. D, Binding of 13.8 nM tPA to β2GPI over time, as determined in C. Increasing concentrations of NaCl (indicated to the right) inhibited the binding of β2GPI to tPA.

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Optical biosensor analysis confirmed a specific interaction between β2GPI and tPA (Figure 5C). Analysis of real-time binding curves yielded a mean ± SEM Ka of 1.13 ± 0.07 × 105M–1 second–1 and a Kd of 6.19 ± 0.10 × 103M–1 second–1. Accordingly, the K values were determined to be 2.0 × 10–8M and 5.0 × 10–7M for Ka and Kd, respectively.

We observed only weak binding between Glu-plasminogen and intact β2GPI (Kd <1 μM) using the same optical biosensor system (data not shown), consistent with the findings of Lopez-Lira et al (21).

High concentrations of NaCl inhibited the binding of β2GPI to tPA, suggesting a strong ionic component to the binding interaction (Figure 5D).

Anti-β2GPI antibody inhibition of the stimulatory effect of β2GPI on tPA-dependent plasminogen activation.

Two monoclonal anti-β2GPI antibodies (1D2 and BD4) inhibited tPA-dependent plasminogen activation to a significantly greater extent than did control murine IgG1 (P < 0.0001; n = 4 experiments) (Figure 6A). Purified IgG fractions from patients with APS also inhibited plasminogen activation in a concentration-dependent manner (Figures 6A and B).

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Figure 6. Inhibition of β2-glycoprotein I (β2GPI)–mediated enhancement of plasminogen activation by anti-β2GPI antibodies. Plasminogen activation was determined as described in Materials and Methods. Percentage inhibition was calculated by comparing reaction rates in the presence versus absence of anti-β2GPI antibodies. A, Analysis of anti-β2GPI antibody–containing IgG fractions from 4 patients with antiphospholipid syndrome (APS) (IgG76, IgGC, IgG35, and IgG58), monoclonal anti-β2GPI antibodies (BD4 and 1D2), and normal human IgG (IgG1). B, Inhibition of tissue plasminogen activator (tPA)–dependent plasminogen activation by increasing concentrations of anti-β2GPI IgG from an APS patient (IgG76) or control IgG (IgG1). C, Ability of anti-β2GPI antibodies affinity-purified from the plasma of 3 patients with APS (abGPI-11, abGPI-17, and abGPI-27) and normal human IgG (IgG1) to inhibit β2GPI-dependent tPA-mediated plasminogen activation. Each of the purified anti-β2GPI antibody preparations inhibited plasminogen activation in a concentration-dependent manner. Values in A–C are the mean ± SEM. D, Effect of IgG fractions from 40 normal controls, 22 APS patients with anti-β2GPI, but not anti-tPA, antibodies (anti-β2GPI+/anti-tPA–), or 21 APS patients with neither anti-β2GPI nor anti-tPA antibodies (anti-β2GPI–/anti-tPA–) on tPA-dependent plasminogen activation in the presence of β2GPI. One outlying point in which an IgG fraction from a patient with anti-β2GPI caused marked inhibition of plasminogen activation (∼60% activity) was removed for this analysis. Horizontal lines show the mean. PAPS = primary APS.

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To determine whether this effect was associated with anti-β2GPI antibodies or anti-tPA antibodies (29), we used 2 approaches. First, we affinity-purified anti-β2GPI IgG from 3 patients, and tested the ability of the purified anti-β2GPI IgG to inhibit the ability of β2GPI to enhance tPA-dependent plasminogen activation. As depicted in Figure 6C, the 3 affinity-purified anti-β2GPI antibody preparations inhibited plasminogen activation in a concentration-dependent manner, whereas a control human IgG preparation (IgG1) had no effect.

Subsequently, we isolated whole IgG fractions from 22 patients with APS whose plasma contained IgG anti-β2GPI, but not anti-tPA, antibodies (β2GPI+/tPA–) and from 21 APS patients whose plasma contained neither anti-β2GPI nor anti-tPA antibodies (β2GPI–/tPA–). The ability of these plasma samples to inhibit β2GPI-mediated stimulation of tPA-dependent plasminogen activation was then analyzed. At a concentration of 80 μg/ml (533 nM), IgG fractions from β2GPI+/tPA– patients caused significantly more inhibition of plasminogen activation than did those from anti-β2GPI–/anti-tPA– patients or plasma samples from normal controls (Figure 6D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

The results of our studies demonstrate that β2GPI binds tPA with high affinity and stimulates tPA-dependent plasminogen activation. We found that β2GPI enhanced the catalytic efficiency (Kcat/Km) of tPA-dependent plasminogen activation by ∼20-fold, as a result of a decrease in the Km and an increase in the Vmax (Table 1). The enhancement of plasmin generation and the stimulation of fibrinolysis by β2GPI were also demonstrated in a fibrin gel (Figure 4). Moreover, the depletion of plasma β2GPI led to delayed clot lysis, which was restored upon repletion of purified β2GPI, suggesting that these findings are relevant to plasma.

The activity of full-length recombinant β2GPI expressed in 293T cells was similar to that of purified plasma β2GPI (Figure 1), suggesting that isolated plasma β2GPI was not denatured and, thus, that the activity of β2GPI was not attributable to nonspecific effects of denatured protein (30). The ability of recombinant β2GPI domain V to stimulate tPA-dependent plasminogen activation was ∼50% that of intact β2GPI, suggesting that β2GPI-V, as well as other regions of β2GPI, contribute to this activity. However, the β2GPI Gly274–Cys288 peptide neither enhanced tPA-dependent plasminogen activation nor significantly inhibited the binding of β2GPI to immobilized tPA. Thus, if this region is involved in the interaction of β2GPI with tPA, it likely requires conformational restraints imposed in the context of intact β2GPI. Additional studies using site-directed mutagenesis and β2GPI mutants lacking specific β2GPI domains may be of value in further defining the role of this region, if any, in the interactions of β2GPI with tPA.

Annexin A2 and the annexin A2/S100A10 heterotetramer are endothelial cell receptors for plasminogen and tPA (16, 31). Binding of tPA and plasminogen to annexin A2 enhances plasmin generation by facilitating the coassembly of these reactants and lowering the Km for their interaction (16, 17). Cesarman-Maus et al (32) have described anti–annexin A2 antibodies in patients with APS that inhibit the fibrinolytic activity of this complex. While investigators in our laboratory have demonstrated a high-affinity interaction between β2GPI and annexin A2 (33), we have not yet directly assessed the effects of β2GPI on the ability of annexin A2 to enhance tPA-mediated plasminogen activation, either in isolation or on cell surfaces. However, Lopez-Lira et al (21) reported an increase in plasmin generation on the surface of HMEC-1 cells, a human microvascular endothelial cell line, following the addition of β2GPI, and we have observed that fluid-phase annexin A2 and β2GPI stimulate tPA-mediated plasminogen activation in an additive manner (data not shown).

Additional studies will clearly be required to better characterize the effects of β2GPI on cell surface plasminogen activator activity and to better define the roles of specific receptors in these effects. Such studies will require a full consideration of the complex nature of the conformation-dependent interactions of β2GPI with phospholipids and proteins, such as apolipoprotein E receptor 2 (34), factor XI (35), annexin A2 (33), and lipoprotein(a) (36).

Lopez-Lira et al (21) reported that β2GPI binds Glu-plasminogen and stimulates streptokinase-mediated plasminogen activation. Our preliminary studies also suggest that there is low-affinity binding (Kd >1 μM) between Glu-plasminogen and β2GPI, although the nature of the interactions between these 2 proteins requires further investigation. However, tPA was not present in the system described by Lopez-Lira et al, and thus, our findings extend their results by demonstrating a direct interaction of β2GPI with tPA. Taken together, however, these results suggest that in the presence of fibrin, β2GPI may potentially promote fibrinolysis through multiple pathways, including 1) direct stimulation of tPA amidolytic activity, 2) lowering of the Km for tPA-mediated plasminogen activation, and 3) binding to fibrin and providing additional binding sites for plasminogen and tPA at the fibrin surface (21).

Yasuda et al (22) reported that nicked β2GPI, which has been cleaved by plasmin between Lys317 and Thr318 in domain V, but not intact β2GPI, binds with low affinity to Glu-plasminogen (Kd 0.37 μM). Those investigators also observed that nicked β2GPI suppressed plasmin generation in the presence of tPA, plasminogen, and fibrin. Taken together with our findings, these results suggest that intact β2GPI may stimulate tPA-mediated plasminogen activation and subsequently undergo cleavage by newly formed plasmin. In turn, cleaved (“nicked”) β2GPI, if generated in sufficient concentrations in plasma, might limit additional plasmin generation (22).

The pathogenic effects of many “antiphospholipid” antibodies may be mediated through interactions with β2GPI. Indeed, Takeuchi et al (11) observed diminished fibrinolysis in euglobulin fractions from APS patients, attributing this effect to impaired factor XII activation and activity. It has also been reported that β2GPI protects tPA from inhibition by plasminogen activator inhibitor type 1, an activity blocked by monoclonal antiphospholipid antibodies (12).

In this study, we demonstrated that monoclonal anti-β2GPI antibodies and affinity-purified anti-β2GPI antibodies from APS patients inhibit in a concentration-dependent manner the ability of β2GPI to enhance tPA-mediated plasminogen activation. Moreover, IgG fractions from APS patients with anti-β2GPI antibodies caused significantly more inhibition of tPA-mediated plasminogen activation than did those from control subjects or APS patients without anti-β2GPI antibodies. This activity was not due to anti-tPA antibodies (29) and, thus, appears to be attributable to anti-β2GPI antibodies, suggesting another potential mechanism by which heterogeneous antiphospholipid antibodies may contribute to the development of thromboembolic disease (7).

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Drs. Cai and McCrae had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Bu, Cai, McCrae.

Acquisition of data. Bu, Gao, Xie, Zhang, He.

Analysis and interpretation of data. Bu, Zhang, McCrae.

Manuscript preparation. Bu, Cai, McCrae.

Statistical analysis. Bu, McCrae.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES