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. Author manuscript; available in PMC: 2007 Aug 21.
Published in final edited form as: Arthritis Rheum. 2005 Dec;52(12):4018–4027. doi: 10.1002/art.21485

Identification of polyclonal and monoclonal antibodies against tissue-type plasminogen activator in the antiphospholipid syndrome

Cai-Sheng Lu 1, Arash A Horizon 2, Kwan-Ki Hwang 3, John FitzGerald 4, Wei-Shiang Lin 5, Bevra H Hahn 6, Daniel J Wallace 7, Allan L Metzger 8, Michael H Weisman 9, Pojen P Chen 10
PMCID: PMC1950581  NIHMSID: NIHMS25116  PMID: 16320350

Abstract

Objective

To test the hypotheses that some plasmin-reactive anti-cardiolipin antibodies (aCL) may bind to tissue-type plasminogen activator (tPA), and that some of the tPA-reactive aCL may inhibit tPA activity.

Methods

We studied the reactivity of 8 patient-derived monoclonal aCL with tPA and presence of IgG anti-tPA antibodies in patients with the antiphospholipid syndrome (APS). Of the reactive monoclonal aCL, we examined their effects on tPA activity.

Results

Six patient-derived plasmin-reactive monoclonal aCL bind to tPA. Analysis of plasma samples revealed that 10/80 (12.5%) APS patients and 1/81 (1.2%) SLE patients have antibodies against fibrin-associated tPA, using the mean plus 2 standard deviations of 28 normals as the cut off. Of the 6 monoclonal tPA-reactive aCL, two (CL1 and CL15) inhibited tPA activity.

Conclusion

Some of the plasmin-reactive aCL in APS patients may bind to tPA. Of the tPA-reactive aCL, some (like CL1 and CL15) may inhibit tPA activity and thus may be prothrombotic in the host.

INTRODUCTION

Accumulated studies show that antiphospholipid antibodies (aPL) represent a heterogeneous group of immunologically distinct antibodies (Ab) that recognize various phospholipids (PL), PL-binding plasma proteins and/or PL-protein complexes (1-7). The involved plasma proteins include β2 glycoprotein-1 (β2GPI), annexin-V, prothrombin (PT), thrombin, protein C (PC), activated PC (APC), protein S, plasminogen and plasmin (8-12). Importantly, thrombin, APC and plasmin belong to the trypsin superfamily; all three enzymes contain a trypsin/thrombin-like serine protease domain (13-15). At the amino acid levels, human thrombin and human APC share a 50.5% similarity, while human thrombin and human plasmin share a 48% similarity (10, 11).

Recently, we found that 5/7 IgG monoclonal anti-cardiolipin Ab (aCL), derived from two patients with the Antiphospholipid Syndrome (APS), react with human thrombin, APC and plasmin; and that one patient derived IgG monoclonal anti-PT Ab (aPT) that binds to cardiolipin also binds to APC and plasmin (10, 11). Interestingly, some display higher binding affinity to plasmin than that to thrombin and APC. Specifically, CL1 and CL15 bind to plasmin with relative Kd values of 5.6 × 10-8 M and 1 × 10-7 M, respectively. Importantly, one of these monoclonal Ab (mAb, CL15) could inhibit the anticoagulation function of APC and reduce the plasmin-mediated lysis of fibrin clots (11, 12).

Of note, hemostasis consists of platelet activation and aggregation, and initiation of blood coagulation system and fibrin formation. Briefly, the key coagulation cascade begins with induced expression of tissue factor (TF), and sequential activation of factor VII and factor X. The activated factor X (designated Xa) works with activated factor V (designated Va) to convert PT to thrombin, while factor IXa works with factor VIIIa to generate more factor Xa. Once thrombin is generated, it converts fibrinogen to form fibrin clots. At the same time, thrombin binds to thrombomodulin and activates PC; APC works with protein S to inactivate factors Va and VIIIa, thus reduce and terminate thrombin generation (16, 17). On the other hand, the fibrin clots activate the fibrinolytic system that comprises plasminogen, which is converted to plasmin mainly by the tissue-type plasminogen activator (PA). Plasmin dissolves fibrin in hemostatic plugs and in thrombi. As such, fibrin formation and fibrinolysis are in dynamic balance, and abnormalities in either hemostasis or fibrinolysis may promote thrombosis (17, 18).

Considering the high binding affinity of some aCL to plasmin, we searched for human proteins that are homologous to plasmin and are associated with hemostasis. The result showed that the human tissue-type PA (tPA) is the most homologous one, sharing a 42% similarity with human plasmin (19). Notably, both proteins share homologous protease domains and kringle domains (19). This similarity of tPA to plasmin led us to hypothesize that some of the plasmin-reactive aCL may bind to tPA and, of which, some may interfere with tPA-mediated activation of plasminogen, resulting in a hypofibrinolytic state. Of note, anti-tPA Ab had been reported in 3 of 39 patients with primary APS (20). We now report that 6/6 plasmin-reactive monoclonal IgG aCL/aPT bind to tPA, and that 10/80 (12.5%) APS patients have IgG Ab against fibrin-associated tPA. Moreover, of the 6 tPA-reactive mAb, CL1 and CL15 could inhibit tPA activity on fibrin surface.

MATERIALS AND METHODS

Patient-derived monoclonal aCL and aPT

Seven IgG monoclonal aCL and one IgG monoclonal aPT were analyzed in the present study. The aCL included CL1, CL15, CL24, IS1, IS2, IS3 and IS4 (21), and the single aPT was IS6 (22). Their generation and characterization had been reported previously (21, 22).

Patients and healthy controls

Eighty consecutive APS patients who met the Sapporo criteria at the Cedars Sinai Medical Center were recruited (23). For patient controls, 81 consecutive systemic lupus erythematosus (SLE) patients without APS were also recruited at the Cedars-Sinai Medical Center (24, 25). Medical charts and laboratory test reports for each patient entered in this study were reviewed by AAH to confirm patients’ diagnosis. Patients were then classified as primary APS if they had no associated autoimmune disease, or secondary APS if they also fulfilled criteria for another autoimmune disease.

Of the 80 APS patients, 44 are primary APS (55%) and 36 secondary APS (all have SLE). The gender breakdown was 72 females and 8 males. Fifty-seven of the 80 APS patients (71%) were positive for aCL and 38 patients had LAC. The average age was 44.1 years (range 21-82 years). The gender breakdown of 81 SLE patients was 76 females and 5 males, and the average age was 44.9 years (range 20-74 years).

Twenty-eight healthy donors at University of California Medical Center (Los Angeles, CA, USA) were included as normal controls. The gender breakdown was 18 females and 10 males. Their average age at the time of sample donation was 30.8 years (range 20-72).

ELISA for Ab against tPA and fibrin-associated tPA

The ELISA for anti-tPA Ab was done as follows. Briefly, high binding plates (Costar, Cambridge, MA) were coated with 10 μg/ml of human tPA (single-chain native t-PA isolated from human melanoma cells; Calbiochem, La Jolla, CA) or recombinant tPA (rtPA, single-chain rtPA isolated from Chinese hamster ovary cells; Genentech, San Francisco, CA) in phosphate buffered saline (PBS), pH 7.4. After incubating overnight at 4 °C, plates were blocked with PBS containing 0.25% gelatin. Purified IgG (1 μg/ml, which is determined to be in the linear range of the titration curves for mAb) in PBS/0.1% gelatin were distributed to wells in duplicate and incubated for 2 hours at room temperature (RT); a pooled normal human IgG (Jackson ImmunoResearch, West Grove, PA) and monoclonal IgG3 (Sigma, St. Louis, MO) were used as negative controls. After washing with PBS, bound human IgG was detected with HRP-conjugated goat anti-human IgG (γ-chain specific; Biosource International, Camarillo, CA), and peroxidase substrate tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD).

The ELISA for Ab against fibrin-associated tPA was done according to Gris et al. (26, 27). First, to conjugate human fibrin to wells, polyvinyl-chloride microtiter plates (Costar, Cambridge, MA) were treated with 100 μl per well of 2.5% glutaraldehyde (Sigma) in 0.1 M sodium bicarbonate buffer, pH 9.5, and incubated at RT for 2 hours. After three washes with deionized water, each well was added 100 μl of 0.3 μM (0.11 μg/ml) human fibrinogen (Haematologic Technologies, Essex Junction, VT) in 0.1 M sodium phosphate buffer, pH 7.4, containing 1 mM CaC12. After an 18-hour incubation at 4 °C and then three washes with Buffer A (0.05 M sodium phosphate, pH 7.4, and 0.08 M NaCl), fibrinogen was converted to fibrin by adding to each well 100 μl of human α-thrombin (Haematologic Technologies) at 1 NIH U/ml in buffer A containing 0.1% bovine serum albumin (BSA), 1 mM CaC12, and 0.01% Tween 20. After a 30-minute incubation at 37 °C and three washes with 5 mM sodium phosphate buffer (pH 6.8), containing 0.5 M NaC1 and 0.05% Tween 20, wells were blocked with 0.02 M L-lysine in buffer B (0.05 M sodium phosphate buffer, pH 6.8, 0.08 M NaCl) containing 0.1% BSA, and 0.01% NaN3. The treated plates were sealed and stored at 4 °C until use.

Second, on the day of assay, each well was added tPA at 1 μg/ml in Buffer B containing 0.4% BSA and 0.01% Tween, and plates were incubated overnight at 4 °C. After wash, mAb (1 μg/ml) or test plasma samples (1:25 dilution) in buffer A containing 4% BSA were distributed to wells in duplicate and incubated for 2 hours at RT. Plates were washed with buffer A containing 0.2% BSA, 0.01% Tween 20, and 0.01% Thymerosal; and bound IgG was measured. For each sample, the optical density (OD) in wells coated with fibrin only (without tPA) was subtracted from the OD in wells coated with tPA-fibrin. Of note, when plasma samples were analyzed, a tPA-reactive IgG monoclonal Ab, CL15 at 1 μg/ml was used in each ELISA plate to serve as the reference sample, and the OD of each test sample was divided by that of CL15 in the same plate, resulting in a reference unit (RU) of each sample. CL15 at 1 μg/ml was in a linear range of its titration curve. It should be noted that our preliminary studies of plasma samples at 1:50 and 1:100 dilutions showed higher background OD from binding to fibrin without tPA, resulting in a lower signal (binding to tPA-fibrin)-to-background (binding to fibrin only) ratio than those of the samples analyzed at 1:25 dilution. These results suggest that fibrinogen or other proteins in the plasma at the 1:25 dilution (but not in 1:50 or 1:100 dilutions) are in sufficient concentrations to block some binding of IgG to fibrin.

The level of IgG in each sample was also determined in a sandwich ELISA using Goat anti-human Ig (Biosource International) and HRP-conjugated goat anti-human IgG (γ-chain specific). Plasma samples were analyzed at 1:100,000 dilution, and a pooled normal human IgG (Jackson ImmunoResearch) at 0.1 μg/ml was used as the reference in each plate. A competitive inhibition assay was used to study the binding properties of selected mAb to rtPA. Briefly, each mAb (0.5-2 μg/ml, as determined to be in the linear range of the titration curve of each test mAb) was preincubated for 1.5 hours with various concentrations of rtPA or human α-thrombin (Haematologic Technologies) in PBS/0.1% gelatin. Then, the mixture was distributed to the rtPA or α-thrombin-coated wells in duplicate. After incubation, bound IgG was measured. The amount of inhibition for a mAb at a given concentration of soluble rtPA (or α-thrombin) was calculated as follows: % inhibition of mAb binding to rtPA = [(OD from a test mAb alone) −(OD from the same mAb plus rtPA at the given concentration)] /(OD from the same mAb alone) × 100. The inhibition data of each mAb were used to calculate its relative Kd toward rtPA (28).

Effects of anti-tPA mAb on tPA activity

It is known that tPA by itself is not efficient in converting plasminogen to plasmin, but becomes 400-fold more efficient when tPA binds to fibrin (18). Accordingly, to study the effects of anti-tPA Ab on tPA activity, we examined the effects of anti-tPA mAb on the amidolytic activity of fibrin-associated rtPA using a tPA chromogenic substrate S2765 (N-α-Benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydrochloride; American Diagnostica, Greenwich, CT). Briefly, rtPA at (100 nM, final concentration, in PBS containing 0.1% of gelatin) was mixed with either a test mAb or a control human IgG (at 100 μg/ml). After a 15-minute incubation at RT, the mixtures were distributed on wells contained solid-phase fibrin and incubated at 37 °C for one hour. After intensive wash with Tris buffered saline (TBS, 50 mM Tris-HCl, 130 mM NaCl, pH 8.3), 50 μl of S2765 at 0.9 mM was added to each well, and generation of p-nitroaniline was monitored by measuring OD at 405 nm.

Statistical analysis

The mean RU plus 2 standard deviations (SD) of the 28 normal controls was used as the cutoff, and the plasma samples with RU values consistently higher than the cutoff in two separate experiments were considered positive. Differences among APS, SLE and normal control groups were analyzed using the Kruskal-Wallis test followed by the Dunn’s multiple comparison test. Differences in the test Ab-induced inhibition of plasmin activity were analyzed using the Wilcoxon matched pairs test (two-tailed). A p value of less than 0.05 was considered significant.

RESULTS

All 6 plasmin-reactive monoclonal IgG aCL bind to tPA and rtPA

To test our hypothesis that some of the plasmin-reactive aCL may bind to tPA (due to their similarity at the amino acid level), we examined 6 plasmin-reactive and 2 non-plasmin-reactive monoclonal aCL for their reactivity with human tPA and rtPA. The reasons for studying both tPA and rtPA are that the rtPA has been shown to be indistinguishable from the native tPA (29), and that the rtPA is much cheaper than tPA. However, rtPA may lack certain epitope(s) of tPA. The results showed that all 6 plasmin-reactive monoclonal aCL bind well to tPA (Figure 1 A). These 6 mAb include CL1, CL15, CL24, IS3, IS4 and IS6. The first 3 mAb were from a secondary APS patient with primary SLE, and the latter 3 mAb were from a primary APS patient (21, 22).

Figure 1.

Figure 1

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Identification of monoclonal anti-tPA Ab. A, some patient-derived monoclonal aCL bind to tPA. B, all monoclonal anti-tPA Ab bind to rtPA. Microtiter wells were coated with tPA or rtPA, and the test mAb, normal human IgG or the monoclonal isotype controls (IgG1 or IgG3) were analyzed at 1 μg/ml. Except for IS1 and IS2 (which are IgG1), all mAb are IgG3. Bound IgG were measured and expressed in OD, and the mean and standard error of the mean (SEM) are given (n = 2).

In contrast, of the 2 non-plasmin-reactive monoclonal aCL, IS1 binds weakly to tPA, while IS2 does not bind to tPA at all (Figure 1A). Similar results were obtained with rtPA (Figure 1 B). Consequently, rtPA was used in subsequent studies.

The binding properties of mAb to rtPA

As a first step to assess the clinical significance of the above patient-derived mAb against tPA, we performed competitive inhibition to study the binding affinity of 6 mAb that bind strongly to both tPA and rtPA. The results showed that rtPA at 1 μM inhibited more than 50% of binding to rtPA by CL1, CL15, IS3 and IS6. In contrast, CL24 and IS4 were not inhibited > 50% by rtPA at the concentration up to 1 μM, suggesting a low affinity interaction with rtPA by these two mAb. Based on these inhibition data, the relative Kd values of the tPA-reactive mAb to rtPA were 3 × 10-7 M for CL15 and IS3, and were 4 × 10-7 M and 5 × 10-7 M for CL1 and IS6, respectively (Figure 2A).

Figure 2.

Figure 2

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Competitive inhibition of mAb binding to tPA. The results are expressed in % inhibition, and the mean and SEM are given (n = 2). Crossreactivity of CL15 to rtPA and human α-thrombin (B). Binding of CL15 to either rtPA or thrombin in a solid phase was inhibited by soluble antigens (rtPA or thrombin). The experiment was performed in a similar manner to that in A except the cross-inhibition. The results are expressed in % inhibition, and the mean and SEM are given (n = 2-5).

To further characterize binding properties of our tPA-reactive mAb, we used CL15 to perform a cross-inhibition experiment using human α-thrombin. The α-thrombin was chosen because it is a coagulation protein that reacts with CL15 and belongs to the trypsin family, and α-thrombin contains only the catalytic domain without any kringle domains. Figure 2B shows that α-thrombin could inhibit CL15 from binding to rtPA, indicating that CL15 cross-reacts with both rtPA and α-thrombin, and that CL15 binds to an epitope in the catalytic domain of the enzymes. In addition, binding of CL15 to rtPA and α-thrombin in a solid phase was more effectively inhibited by rtPA than by α-thrombin, indicating that CL15 reacts with tPA with a higher affinity than that for α-thrombin.

Detection of anti-tPA Ab in some APS patients

Subsequently, we searched for IgG anti-tPA Ab in APS patients. Initially, we used the above ELISA format to analyze IgG anti-tPA Ab in the plasma samples from 80 APS patients and 28 healthy controls. Samples were analyzed at a 1:25 dilution in PBS containing 0.1% Tween 20, as reported by Cugno et al. (30). The results failed to show a significant difference between IgG anti-tPA Ab in APS patients and those in healthy controls (data not shown). At that time, Gris et al. reported a first early pregnancy loss (during the eighth and ninth weeks of pregnancy) was strongly associated with IgG Ab against the putative neo-epitope(s) of tPA upon its binding to fibrin (26). Of note, tPA has a weak affinity for plasminogen (KM = 65 μM) and thus is not efficient in converting plasminogen to plasmin in solution. However, when tPA binds to fibrin, tPA assumes a new configuration and reacts with plasminogen with an increased affinity (KM between 0.15 and 1.5 ◻M, about 40-400 folds), resulting in much more efficient in generating plasmin (31).

Accordingly, we re-analyzed our monoclonal aCL for binding to tPA and rtPA on fibrin surface (designated tPA-fibrin and rtPA-fibrin, respectively). Figure 3A shows that all 6 plasmin-reactive monoclonal aCL bind well to tPA-fibrin. However, CL24 and IS6 display only weak binding to tPA-fibrin. Similar results were obtained with rtPA-fibrin, except for that CL24 does not bind to rtPA-fibrin (Figure 3B), indicating that CL24 does not recognize an epitope on the rtPA associated with fibrin.

Figure 3.

Figure 3

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Reactivity of monoclonal anti-tPA Ab with tPA and rtPA on fibrin surface (designated tPA-fibrin and rtPA-fibrin, respectively). All 6 anti-tPA mAb bind to tPA-fibrin (panel A) and 5/6 anti-tPA mAb bind to rtPA-fibrin (panel B). Notably, all 3 mAb with higher affinity to tPA (i.e., CL1, CL15 and IS3) bind stronger to tPA-fibrin than the other mAb.

Thereafter, we searched for IgG Ab against tPA-fibrin in the plasma samples from 80 APS patients, 81 SLE patients without APS, and 28 healthy controls. Figure 4 shows that APS patients have higher titers of IgG anti-tPA-fibrin Ab than SLE controls (p < 0.05) and healthy controls (p < 0.01). Using the mean plus 2 SD, IgG anti-tPA-fibrin Ab were found in 10/80 (12.5%) APS patients. Since many of the patients with SLE may have hypergammaglobulinemia, the data were also normalized to the level of IgG in each plasma sample. Similar results were obtained after normalization. There remained statistically significant differences in the levels of such Ab between APS and SLE or normal controls, indicating the association of IgG anti-tPA-fibrin Ab to APS. Using the mean plus 2 SD of the normal controls as the cutoff, IgG anti-tPA-fibrin Ab were found in 4/80 (5%) APS patients and none in SLE patients.

Figure 4.

Figure 4

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Presence of IgG anti-tPA-fibrin Ab in some APS patients. A, plasma samples from 80 APS patients, 81 SLE patients, and 28 normal controls were analyzed at the 1:25 dilution. During each assay, CL15 at 1 μg/ml was used in each ELISA plate to serve as the reference sample, and the OD of each test sample was divided by that of CL15 in the same plate, resulting in a reference unit (RU) for each sample. B, the same data are normalized against the IgG level of each sample and are expressed in RU/IgG. The horizontal bars indicate the mean RU or mean RU/IgG for each group; the dashed line represents the cutoff, which is mean RU (or mean RU/IgG) plus 2 SD of the 28 normal controls. A representative result from three experiments is shown. A significant difference between the groups was denoted as * (p < 0.05) or ** (p < 0.01).

Effects of anti-tPA mAb on tPA activity

As noted earlier, tPA by itself is not efficient in converting plasminogen to plasmin, but becomes very efficient after binding to fibrin. Accordingly, we examined the effects of anti-tPA mAb on the amidolytic activity of fibrin-associated rtPA using a small chromogenic substrate of tPA, S2765. The rtPA was used because rtPA is cheaper, and rtPA is comparable to tPA in their interactions with our mAb. Figure 5A shows that CL1 and CL15 could inhibit the fibrin-associated rtPA activity. Specifically, CL15 at 100 μg/ml could inhibit 34% of fibrin-associated rtPA activity (Figure 5B). This inhibitory effect of CL15 was concentration-dependent in the range of concentrations tested (Figure 3C). We did not use CL15 at any concentration higher than 100 μg/ml, which is unlikely to be physiologically relevant, as the positive APS patients have only about 53-70 μg/ml IgG anti-tPA Ab (based on 2.1 RU to 2.8 RU in Figure 4A; 1 RU is roughly equivalent to 1 μg/ml IgG anti-tPA Ab, and samples were analyzed at the 1:25 dilution).

Figure 5.

Figure 5

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CL1 and CL15 inhibit tPA activity on fibrin surface. Test mAb and control IgG (at 100 μg/ml) were mixed with 100 nM rtPA for 15 minutes at room temperature, and then the mixtures were distributed to fibrin-coated wells. After incubation for one hour and wash, a tPA substrate S2765 was added and OD450 was measured after two hours. The data are expressed in OD change (A) and % inhibition (B) from normal human IgG control. CL15 inhibits tPA activity on fibrin surface in a concentration-dependent manner (C). The mean and SEM are given (n = 8). * denotes a significant difference from normal human IgG control (p < 0.05).

DISCUSSION

To test our hypothesis that some APS patients may have certain plasmin-reactive aCL, which could bind to tPA, and interfere with tPA-mediated plasmin generation and the subsequent plasmin-mediated fibrinolysis, we first analyzed patient-derived monoclonal plasmin-reactive aCL and showed that all 6 mAb bound strongly to tPA. The relative Kd values of the tPA-reactive mAb to rtPA were 3 × 10-7 M for CL15 and IS3, and were 4 × 10-7 and 5 × 10-7 M for CL1 and IS6, respectively (Figure 2A). Of note, IS3, IS4, CL1 and CL24 bind to β2GPI (21). Of these four, we had found the relative Kd value for IS3 binding to β2GPI was in the same 10-6 M range (Hwang and Chen, unpublished data). Previously, Tincani et al. used β2GPI-column to purify IgG anti-β2GPI Ab from 5 APS patients and found these Ab bound to β2GPI with the relative Kd values ranging from 3.4 to 7.2 × 10-6 M (32). Viewed as a whole, these data suggest that presently observed reactivity of patient-derived IgG monoclonal aCL with tPA is significant.

When the same ELISA was used to study the presence of IgG anti-tPA in APS patients, we were unable to detect significant difference of anti-tPA Ab between APS patients and healthy controls. Therefore, we switched to a recently published protocol for studying Ab against the putative neo-epitope(s) of tPA upon its binding to fibrin. With this new ELISA format, we found only 4/6 monoclonal plasmin-reactive aCL bound well to tPA-fibrin and rtPA-fibrin (Figure 3). Importantly, using this latter ELISA format, we could detect significantly higher titers of IgG Ab against tPA-fibrin in APS patients than those in SLE or healthy controls (Figure 4). Using the mean plus 2 SD of the normal controls, IgG anti-tPA-fibrin Ab were found in 10/80 (12.5%) APS patients. These findings are consistent with those of other investigators that IgG autoantibodies against certain conformational epitopes expressed on the tPA-fibrin complex are associated with APS (26).

The low frequency of IgG anti-tPA-fibrin Ab in our APS group may be due to the small sample size (n = 28) of our normal control group, or the low sensitivity of our assay due to high background binding to fibrin alone. Alternatively, low frequency of IgG anti-tPA-fibrin Ab in our APS group may indicate that only a small subset of APS patients have IgG antibodies against tPA, and thus not a major autoantibody in APS. Upon this finding, we studied the functional significance of IgG anti-tPA-fibrin Ab, and found that CL1 and CL15 could inhibit tPA activity on fibrin surface (Figure 5A). Although the degree of inhibition by CL15 was only moderate (up to 34%; Figure 5), it is possible that the effect of such Ab would be significant enough to shift the balance toward a hypofibrinolytic state since tPA is the rate-limiting enzyme in fibrinolysis (33). Since none of mAb showed any effect on the amidolytic activity of rtPA in a fluid phase (data not shown), these data suggest that CL1 and CL15 probably inhibit the fibrin-associated rtPA activity via interference of the tPA binding to fibrin surface. Taken together, these findings suggest that some APS patients may have certain Ab against tPA-fibrin that could inhibit tPA binding to fibrin surface, resulting in a reduction of plasmin-mediated fibirinolysis.

In 2000, Cugno et al. showed that, of 39 patients with primary APS, 3 had high titers of IgG Ab against tPA and 4 had high titers of Ab against fibrin-bound tPA, which is the physiologically active form of tPA (20). More recently, Cugno et al. reported the presence of anti-rtPA Ab in14/91 (15.4%) consecutive APS patients and an inverse correlation between anti-tPA titers and plasma tPA activity when tPA activity was determined in 53 APS patients (30). Characterization of anti-rtPA Ab from two patients revealed that the Ab consisted of IgG1 in one patient and IgG3 in the other one; and that purified IgG from both patients bound to the catalytic domain of the tPA (30). These data are similar to our present findings: 1) the presence of IgG anti-tPA-fibrin Ab in 10/80 (12.5%) APS patients; 2) our monoclonal anti-tPA Ab are of the IgG3 subclass; 3) our monoclonal anti-tPA Ab apparently recognize the protease domain of tPA; and 4) CL15 at 100 μg/ml could inhibit 34% of fibrin-associated tPA activity. Our contention of the tPA epitope in the protease domain of tPA is based on the facts that all monoclonal anti-tPA also bind to APC and that APC does not have any kringle domain; and that CL15 binding to tPA could be inhibited by α-thrombin, which contains only a protease domain (Figure 2B). However, we could only observe significant difference between APS patients and SLE or healthy controls when tPA/rtPA was coated onto fibrin surface, but not to ELISA plate directly, as reported by Cugno et al. (30). The underlying reason for this discrepancy is not clear at this point.

Studies have suggested that the fibrinolytic system is an important defense mechanism against thrombosis. The reduction of fibrinolysis may allow the growth and development of thrombi, resulting in a prothrombotic state. Decreased levels of tPA and/or increased levels of PAI-1 have been reported in patients with deep vein thrombosis (34). Along this line, mice lacking plasminogen were predisposed to severe thrombosis (35). On the other hand, venous thrombosis occurs frequently in transgenic mice overexpressing the native form of PAI-1 under the control of the metallothionein promoter (36), while age-dependent spontaneous coronary arterial thrombosis occurs in mice that express a stable form of human PAI-1 (37). In this context, it is conceivable that CL15 may promote thrombosis in the host patient by inhibiting tPA-mediated plasmin generation on fibrin surface, as well as the plasmin-mediated fibrinolysis (12). Indeed, CL15 had been found to be prothrombotic in an in vivo pinch-induced thrombosis model (38).

Previously, Ieko et al. reported that β2GPI exerted a dose-dependent enhancement of the tPA activity in the presence of PAI-1; and that addition of the EY1C8 patient-derived monoclonal aCL reduced tPA activity by about 45% in a mixture of tPA (3.6 U/ml), PAI-1 (7.1 ng/ml) and β2GPI (3.8 μM) (39, 40). Taken together with above findings, these data suggest that there are two kinds of Ab in APS that may inhibit tPA activity in plasma, one (like EY1C8) suppresses tPA activity via interaction with β2GPI, and one (like CL15) inhibits tPA activity via interference of its binding to fibrin surface, which is independent of β2GPI.

In addition to the two aforementioned Ab, Takeuchi et al. showed that the EY2C9 monoclonal IgM anti-β2GPI Ab (from an APS patient) suppressed intrinsic fibrinolysis in the presence of β2GPI, apparently by enhancing the β2GPI-mediated weak suppression of intrinsic fibrinolysis (41). Similar to intrinsic coagulation, intrinsic fibrinolysis is initiated by the contact activation of coagulation factor XII (FXII). Then, activated FXII (FXIIa) converts prekallikrein to kallikrein, which, in turn, converts pro-urokinase (single chain-urokinase type of plasminogen activator; scuPA) to urokinase plasminogen activator (uPA). In this context, Takeuchi et al. suggested that β2GPI and anti-β2GPI Ab may either inhibit FXIIa from activating kallikrein or suppress kallikrein from activating uPA, leading to reduced plasmin generation.

Our accumulated analyses of 7 mAb generated by screening against cardiolipin in the presence of bovine serum reveal that 5/7 bind to PT, thrombin, PC, APC, plasminogen, plasmin and tPA (Table 1). Of these 5 mAb, CL1, CL24, IS3, and IS4 also react with β2GPI (21), the major autoantigen or cofactor for autoantibodies detected by the conventional aCL ELISA. More importantly, we previously showed that CL15 could inhibit both the anticoagulation function of APC and the plasmin-mediated degradation of fibrin (11, 12). Taken together with the present observation of CL15 on fibrin-associated tPA activity, these findings suggest that a single aCL may act on three different targets, and thus promote thrombosis by three different mechanisms. This unusual complexity may explain the unusual slow progress in complete characterization of all prothrombotic aCL in APS, as well as the delineation of underlying mechanisms of all prothrombotic aCL.

Table 1.

Summary of the characteristics of 8 monoclonal IgG aCL/aPT from two APS patients

mAb: IS1 IS2 IS3 IS4 IS6 CL1 CL15 CL24
Antigensa
CL/bovine serum + + + + + + + +
CL/β2GPI - - + + ND + + +
β2GPI - - + + + + - +
PT - - + + + + + +
thrombin - - + + + + + +
PC - - + + + + + +
APC - - + + + + + +
plasminogen - - + + + + + +
plasmin - - + + + + + +
tPA - - + + + + + +
tPA-fibrin - - + + + + + +
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a

Binding to CL and human β2GPI is compiled from Zhu et al. (21). Binding to human PT, thrombin, PC, APC, plasminogen and plasmin are compiled from references (10-12), and are given in + or -. ND, not done. Binding to tPA and tPA-fibrin is from Figures 1 and 3.

Acknowledgments

We thank Kelly Yu for technical assistance.

This work was supported by a research award from the Alliance for Lupus Research, a grant from the Southern California Chapter of the Arthritis Foundation and a grant AR42506 from the National Institutes of Health.

Contributor Information

Cai-Sheng Lu, Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA 90095.

Arash A. Horizon, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA.

Kwan-Ki Hwang, Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA 90095.

John FitzGerald, Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA 90095.

Wei-Shiang Lin, Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA 90095.

Bevra H. Hahn, Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA 90095.

Daniel J. Wallace, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA.

Allan L. Metzger, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA.

Michael H. Weisman, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA.

Pojen P. Chen, Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA 90095.

References

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