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(Circulation. 1996;94:1746-1751.)
© 1996 American Heart Association, Inc.

Articles

Thrombogenic Properties of Murine Anti-Cardiolipin Antibodies Induced by ß₂ Glycoprotein 1 and Human Immunoglobulin G Antiphospholipid Antibodies

Silvia S. Pierangeli, PhD; Xiao Wei Liu, MD; G. Anderson, PhD; J.H. Barker, MD; E.N. Harris, MPhil, MD, DM

the Antiphospholipid Standardization Laboratory (S.S.P.) and Department of Internal Medicine (E.N.H.), Morehouse School of Medicine, Atlanta, Ga, and the Department of Medicine, Division of Rheumatology (X.W.L.), Department of Physiology (G.A.), and Department of Surgery (J.H.B.), University of Louisville (Ky).

Correspondence to Silvia S. Pierangeli, PhD, Lab Director, Antiphospholipid Standardization Laboratory, Department of Microbiology and Immunology, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA 30310.

	Abstract

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Background Recurrent arterial thrombosis and venous thrombosis are frequent complications of the antiphospholipid syndrome (APS). Patients produce anti-cardiolipin antibodies, but the role of these antibodies in thrombus formation is uncertain. This study used a unique CD-1 mouse model of thrombosis to determine whether anti-cardiolipin and anti-ß₂ glycoprotein 1 (ß₂GP1) antibodies induced immunologically in these animals are thrombogenic.

Methods and Results The CD-1 mouse model enables measurement of the kinetics of a thrombus induced in the femoral vein of the animal. Animals are first anesthetized, then one femoral vein is exposed and subjected to a standardized, nonpenetrating "pinch" injury that induces a thrombus. The vein is transilluminated, and the growing thrombus is visualized on a television screen. The rate of formation and disappearance of the thrombus as well as its area can be measured by a computer attached to the television. Three groups of CD-1 mice (each group comprising seven animals) were studied. Group 1 mice were actively immunized with ß₂GP1, resulting in production of anti-ß₂GP1 and anti-cardiolipin antibodies. Group 2 mice were actively immunized with human immunoglobulin G (IgG) anti-cardiolipin antibodies and produced anti-human IgG as well as anti-cardiolipin antibodies (the latter by an idiotype–anti-idiotype reaction). These animals did not produce anti-ß₂GP1 antibodies. Group 3 mice were immunized with human serum albumin (HSA) and produced anti-HSA but not anti-cardiolipin antibodies. The kinetics of thrombus formation induced in the femoral veins of the experimental mice were compared. Results showed that the mean thrombus area as well as mean time during which thrombi persisted were significantly greater in group 1 and group 2 mice compared with group 3. There was no statistical difference between group 1 or group 2.

Conclusions Demonstration of a thrombogenic effect of murine anti-cardiolipin antibodies suggests that these antibodies may be pathogenic in humans with APS.

Key Words: antibodies • anticoagulants • thrombosis • antigens • apolipoproteins

	Introduction

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Antiphospholipid syndrome is a disorder of recurrent thrombosis, pregnancy loss, and thrombocytopenia associated with production of aCL antibodies.^{1 2 3} Recurrent strokes^{4 5} and myocardial infarction^{6 7} are frequent consequences of arterial thrombosis, and many occur at a young age.⁸ The pathogenic role of aCL in thrombus formation and the specificity of the antibodies involved are presently subjects of considerable interest and debate.⁹

Two separate groups of in vivo experiments suggest that aCL antibodies may play a direct role in causing pregnancy loss and thrombosis. It has been shown that passive immunization of pregnant mice with human polyclonal^{10 11} and mouse monoclonal aCL antibodies^{10 12 13 14} resulted in increased fetal loss^{10 14} and fetal resorption,^{11 12 13} and also caused lower mean weights of embryos and placentas^{11 12 13} compared with mice passively immunized with immunoglobulins from normal controls. In addition, similar fetal effects were demonstrated in mice in which aCL antibodies were induced by active immunization with any of the following immunogens: human monoclonal anti-DNA antibodies carrying the 16/6 idiotype,^{15 16} murine monoclonal aCL antibodies,¹⁷ and ß₂GP1.¹⁸ A second group of studies, using a unique mouse model that enabled study of the kinetics of formation and disappearance of a thrombus induced in the femoral veins of the animals, also suggested a pathogenic role for aCL antibodies.^{19 20} In the latter model, passive infusion of CD-1 mice with affinity-purified IgG or IgM aCL antibodies derived from humans with APS resulted in significantly increased areas of thrombi as well as length of time that thrombi persisted.²⁰

Given the above data that support a role for human aCL antibodies in pregnancy loss and thrombus formation, it is now important to determine whether these effects are confined to antibodies derived from humans or whether aCLs from other animal sources behave similarly. If the latter is demonstrated, it would suggest that the specificity of aCL antibodies rather than their source is the important determinant in thrombus formation.

Relevant to the issue of aCL specificity is the role of anti-ß₂GP1 antibodies. Several studies have shown that the plasma protein ß₂GP1 markedly influences aCL activity in ELISA tests.^{21 22 23 24 25 26} The effect of ß₂GP1 is variously explained by aCL antibodies binding ß₂GP1 directly (in effect, acting like anti-ß₂GP1 antibodies),^{21 22 25 26} binding determinants on both cardiolipin and ß₂GP1,²⁷ or binding "new" determinants on cardiolipin in the presence of ß₂GP1.^{23 24} It has also been demonstrated^{23 24 28 29} that active immunization of mice with ß₂GP1 induces both anti-ß₂GP1 and aCL antibodies. Subsequent work showed that aCL antibodies, induced by immunization with ß₂GP1, bound both cardiolipin and ß₂GP1.^{30 31 32 33} ß₂GP1 is believed to act as a natural anticoagulant in human plasma, and neutralization of its effect by aCL antibodies may predispose to thrombosis.^{21 22 23 24 25 26} aCL antibodies have also been induced by active immunization of mice with purified human aCL antibodies.^{15 17 31} The latter effect has been attributed to an idiotype–anti-idiotype mechanism^{15 17} or to formation of an aCL-phospholipid complex that is immunogenic.³¹

In light of the above results, it is important to determine the roles of murine aCL and anti-ß₂GP1 antibodies in thrombus formation. To examine this question, we used the previously described mouse model of thrombosis.^{19 20} Three groups of mice comprising seven to nine animals per group were immunized separately with ß₂GP1 (group 1), purified human IgG aCL (group 2), or HSA (group 3). As demonstrated previously, group 1 mice produced aCL and anti-ß₂GP1 antibodies,^{30 31 32 33} group 2 mice produced murine aCL and anti-human IgG antibodies³¹ but not anti-ß₂GP1 antibodies, and group 3 mice produced anti-HSA antibodies but were aCL-antibody negative.³¹ By comparing the kinetics of thrombus formation in these three groups of mice, we were able to assess the relative roles of murine aCL and anti-ß₂GP1 antibodies.

	Methods

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Animals
Normal male CD-1 (outbred) mice weighing 30 to 40 g each were purchased from Charles River Laboratories. The animals were housed in the American Association for the Accreditation of Laboratory Animal Care–approved animal care facilities of the University of Louisville, Ky. Animals were handled by trained personnel according to Institutional Animal Care and Use Committee guidelines.

Immunogens
Isolation of ß₂GP1
Human ß₂GP1 for immunization of mice was isolated from human serum (healthy control donors) by heparin sepharose chromatography, as described elsewhere.³⁴ Protein concentration was determined by use of the Lowry method. After the protein concentration was adjusted with sterile saline solution, the preparations were filter sterilized before injections.

HSA
HSA (99% purity) was purchased from Sigma Chemical Co.

Isolation of IgG From an APS Patient
IgG was isolated from the serum of a patient with APS by use of protein G sepharose chromatography. Purity was determined by SDS-PAGE (single band at 150 kD) and absence of contamination with ß₂GP1 was determined by immunoblot with the use of a rabbit anti-human ß₂GP1 antiserum. Protein concentration was determined by use of the Lowry method. After the protein concentration was adjusted with sterile saline solution, the preparations were filter sterilized before injections.

Immunization Schedule
Mice were immunized on days 1, 7, 14, and 21, as described previously.³¹ In brief, animals were injected subcutaneously with 150 µg of ß₂GP1, 150 µg IgG-APS, or 150 µg HSA in adjuvant (Adju-Prime, Pierce Chemical Co) at the times indicated. A specimen of blood was drawn from each animal at weekly intervals to test for the presence of various antibodies.

Determination of aCL Antibodies by ELISA
Mouse aCL antibodies (IgG and IgM) were determined by use of an ELISA assay, as described elsewhere.²⁰ Alkaline phosphatase anti-mouse IgG and anti-mouse IgM were used as secondary antibodies in the ELISA system. The color reaction was stopped when a positive control (of 100 G phospholipid units) reached 1.0 OD units (20 to 30 minutes).

Determination of Anti-ß₂GP1 Antibodies by ELISA
Mouse anti-ß₂GP1 antibodies were detected by an ELISA method, as described previously.^{30 31} Rabbit anti-human ß₂GP1 used as a positive control in this assay was produced in our laboratory by immunization of a New Zealand White rabbit with human ß₂GP1 in complete Freund's adjuvant, as described elsewhere.³¹ The color reaction was stopped when the positive control (diluted 1/500) reached an OD of 1.0 (20 to 30 minutes).

Determination of Anti-Human IgG and Anti-HSA Antibodies
Presence of mouse anti-human IgG antibodies was determined by immunoprecipitation in gel (Ouchterlony technique) with goat anti-human IgG serum (-chain specific) (The Binding Site) used as a positive control. The presence of mouse anti-HSA was determined by Ouchterlony with a goat anti-HSA serum (Sigma Chemical Co) used as a positive control.

Surgical Procedure and Measurement of the Dynamics of In Vivo Thrombus Formation
We used a modification of a surgical procedure described previously.^{19 20 35} This procedure enables continuous and quantitative measurements of a standardized, focally induced, nonocclusive mural thrombus in a surgically exposed mouse femoral vein.

Mice were immunized initially with ß₂GP1 or IgG-APS according to the protocol outlined above, and by 2 to 3 weeks after the initial injection, they were producing high levels of aCL antibodies (OD values, 0.8 to 1.2 units). After individual mice were documented by ELISA as having high aCL antibody levels (see above), surgical experiments were performed.

With sodium pentobarbital (60 mg/kg IP) used as anesthesia, a longitudinal incision was made in the right groin of the CD-1 mice, extending to the knee. Through this incision, the right femoral vein (0.3 to 0.5 mm in diameter) was dissected free between the inguinal ligament proximally and the superficial epigastric branch distally (Fig 1). This dissection left a 1-cm segment of vein free for manipulation (thrombogenic injury).

View larger version (67K): [in this window] [in a new window]   Figure 1. Exposed femoral vein and fiber-optic light positioned under the vein in a mouse.

A standardized thrombogenic injury was produced in the vein by use of a "pinch" technique. To produce the injury with a standardized force, the forceps were brought together until the flat circular (0.1-mm diameter) opposing surfaces met to produce the pinch injury. On release of the pinching forceps, two small thrombogenic injury sites (corresponding to the contact surfaces) were produced on the superior surface of the vein. To observe and measure the pattern of thrombus formation, a fiber-optic device was used to transilluminate the vein, while a trilocular stereoscopic operating microscope (ERNST, Leitz GMBH Wetzlar) equipped with a closed-circuit video system (NEC-NC-A/CCD Camera, NEC USA Inc), Panasonic 12" color monitor (Matsushita Electronic Corp of America) and U Matic V-5800 recorder (Sony Corp) were used to visualize and measure the thrombus (Fig 2). Five separate measurements were obtained for each animal, and mean values were computed.

View larger version (77K): [in this window] [in a new window]   Figure 2. Photograph taken from the video monitor demonstrating the method for analysis of thrombus size. Commercial software (Optimas) was used to measure thrombus area from the digitized video image at selected time intervals. See "Methods" for further explanation.

The transilluminator was designed especially for transmitting light through small blood vessels. It consisted of a row of 12 acrylic optical fibers embedded in a black epoxy block. The block was designed so that it cradled the vein in a small half circle that was approximately the same diameter as the vessel. The optical fibers embedded in the block were directed in such a way that they were in direct contact with the vessel lying in the trough and perpendicular to the vessel. This ensured that all the light emerging from the fibers passed into the vessel. The light source was a 250-W tungsten bulb equipped with a variable-intensity control.

To view the vessel and thrombus, the operating microscope described above was used. The thrombus in the transilluminated vein appeared bright yellow-white through the microscope. This image was produced because the thrombus displaced the darker column of flowing red blood cells within the vessel lumen. Therefore, the degree of brightness of the thrombus indicated its size indirectly, because the larger the thrombus became, the more red blood cells were displaced. The growing thrombus was measured continuously over a time period of 50 minutes, and its area was analyzed by use of a computer-assisted gray-level analysis. The video image was digitized by a personal computer vision PC+ image analysis system (Bioscan Optimas, Bioscan Inc). The digitized image was composed of pixels of various gray levels depending on the light intensity (gray levels ranging from 0 to 255, with 0 representing a black image and 255 a white image). The number of pixel gray levels above background in the area of interest (section of vessel containing the thrombus) was measured at 30-second intervals over a period of 50 minutes. A typical time course of thrombus growth was characterized by rapid growth in the first 1 to 5 minutes and then a decrease in area over the ensuing 30 minutes (Fig 3).

View larger version (18K): [in this window] [in a new window]   Figure 3. aCL antibody levels in immunized mice. aCL antibody levels were determined by ELISA at different times in mice immunized with ß2GP1 (), IgG-APS (), or HSA () as indicated in "Methods." Values are expressed as mean±SD net OD units.

Measurements (in minutes) were made of the times of thrombus formation (time to maximum size), disappearance (time from maximum size to disappearance), and total time thrombus was present (formation plus disappearance). Three to five thrombi were successively induced in each animal, and mean values were computed. After experiments were complete, mean thrombus area and mean formation, disappearance, and total times were computed for each of the three groups of immunized animals. The person performing the experiments (X.W.L.) was blinded to the materials with which individual mice were injected.

Statistical Analysis
One-way ANOVA was used to compare the means of thrombus size and times (formation, disappearance, and total times) of the three groups. Tukey's honestly significant difference was used for post hoc analysis of the means. Data were checked for normality by use of the Kolmogorov-Smirnov test. The null hypothesis of normality was not rejected.

	Results

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Production of Antibodies by Mice
aCL antibodies were produced at high levels in animals immunized with ß₂GP1 (group 1) or human IgG-aCL (group 2) but not with HSA (group 3), as shown in Fig 4. Mice immunized with human IgG-aCL did not have detectable titers of human aCL antibodies at the time of the surgical procedure. Anti-ß₂GP1 antibodies were produced in animals immunized with ß₂GP1 but not with human IgG-aCL or HSA, as shown in Fig 4. Animals immunized with HSA produced anti-HSA antibodies (as detected by Ouchterlony, data not shown), and animals immunized with IgG-APS produced anti-human IgG antibodies (as detected by Ouchterlony, data not shown). Animals immunized with human IgG-aCL antibodies did not produce anti-ß₂GP1 antibodies.

View larger version (18K): [in this window] [in a new window]   Figure 4. Anti-ß2GP1 antibody levels were determined by ELISA at different times in mice immunized with ß2GP1 (), IgG-APS (), or HSA () as indicated in "Methods." Values are expressed as mean±SD net OD units.

Dynamics of Thrombus Formation
The dynamics of thrombus formation and disappearance were examined in individual mice in each of the three groups.

Mean thrombus area was significantly larger in mice producing aCL antibodies (groups immunized with ß₂GP1 [group 1] or with IgG-APS [group 2]) than in controls (HSA-immunized mice [group 3]), as shown in the Table. The overall hypothesis of no difference in mean thrombus size was rejected (P=.0031). Groups 1 and 2 were found to be different from group 3 but were not separated from each other by use of Tukey's honestly significant difference post hoc test, with P.05.

View this table: [in this window] [in a new window]   Table 1. Dynamics of Thrombus Formation in Immunized Mice

Mean thrombus disappearance times and mean total times were significantly longer in animals producing aCL antibodies (groups 1 and 2) than in controls (group 3). The overall hypothesis of no differences in means of disappearance times and total times was rejected (P=.0055 and P=.0033, respectively). Groups 1 and 2 were different from group 3 but were not separated from each other by use of Tukey's honestly significant difference post hoc test, with P.05.

Thrombus formation time did not differ statistically between the three groups (P=.3910).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Mechanisms of Thrombosis
Investigators interested in factors that cause thrombus formation should note that APS is unique in that the immune system appears to be involved in thrombogenesis. On the basis of in vitro studies, it appears that aCL antibodies may affect coagulation protein-phospholipid interactions,^{1 2} resulting in disorders of clotting. Both the arterial and venous circulations appear subject to these effects; thus, patients appear as likely to suffer myocardial infarction as they are to develop deep vein thrombosis. Antibody effects demonstrated in vitro by which thrombosis may be generated include inhibition of protein C activation,^{38 39 40} inhibition of the effects of activated protein C,^{39 40} or binding to ß₂GP1.^{21 22 41} Other postulated mechanisms include activation of platelets^{42 43 44} or endothelial cells.^{45 46}

Results
This study had two objectives. The first was to determine whether aCL antibodies induced in mice would have the same thrombogenic effect previously demonstrated in similar mice passively immunized with human aCL antibodies.^{19 20} The second was to determine whether anti-ß₂GP1 antibodies would also influence thrombus formation in this model. Results showed that induced venous thrombi were significantly larger and persisted significantly longer in mice producing aCL antibodies than in aCL-negative controls. No significant difference was found between mice producing both aCL and anti-ß₂GP1 antibodies compared with mice producing aCL antibodies alone.

Animal Model of APS
The above findings provide further documentation in vivo for a role of aCL antibodies in the pathogenesis of thrombosis and pregnancy loss, major complications of APS. Several published studies have shown that polyclonal^{10 11 12 13} and monoclonal^{11 14} aCL antibodies derived from both human^{11 12 13} and murine sources^{11 15} can cause fetal loss in mice. These effects occurred when the antibodies were administered passively^{10 11 12 13 14} or induced actively by immunization with aCL¹⁷ or anti-16/6 idiotype anti-DNA antibodies.^{15 16} The influence of anti-ß₂GP1 on fetal loss in these murine models has not been determined conclusively. Two studies have been conducted in which aCL and anti-ß₂GP1 antibodies were induced in mice by immunization with ß₂GP1, and pregnancy outcomes in these mice were studied. One study reported an increase in fetal loss,¹⁸ but the other⁴⁷ found no effect on pregnancy outcome.

Animal Models of APS-Induced Thrombosis
Although several groups have demonstrated that aCL antibodies may cause fetal loss in mice, demonstration of an antibody-mediated effect on thrombosis has proved elusive. Smith and colleagues⁴⁸ first reported that three of nine lupus-prone MRL/lpr mice producing aCL antibodies had cerebral infarction at autopsy. However, when CD-1 mice were passively infused with human aCL antibodies in another study,¹⁹ no thrombi could be detected at autopsy. Demonstration that aCL antibodies might influence thrombus formation became possible with the use of the CD-1 mouse model described in that study.¹⁹ That model enables measurement of the size of a thrombus as well as the times of formation and disappearance of a thrombus induced in the femoral veins of experimental mice. A previous study²⁰ demonstrated that induced thrombi were larger and had longer disappearance times when mice were passively immunized with IgG, IgM, or IgA immunoglobulins from patients with APS than when mice were immunized with immunoglobulins of the same isotype from healthy individuals. In addition, passive immunization of these mice with affinity-purified human IgG or IgM aCL antibodies also enhanced thrombus size and persistence.²⁰ The present study demonstrates that thrombogenic effects are not confined to human aCL antibodies but can also be shown for the same antibodies induced in mice. Hence, induction of thrombus formation appears related to aCL specificity and does not appear to be a function of the source of these antibodies. Murine aCL antibodies cross-react with negatively charged phospholipids such as those found in humans³⁰ and also demonstrate ß₂GP1-dependent cardiolipin binding activity.^{32 33} In the present study, the presence of additional anti-ß₂GP1 antibodies did not appear to influence the thrombogenic effects of aCL antibodies. This does not exclude the possibility that anti-ß₂GP1 antibodies may have an effect independent of aCL antibodies. Demonstration of an independent anti-ß₂GP1 effect may prove elusive in this model, because no investigative group has induced polyclonal anti-ß₂GP1 antibodies without aCL antibodies by immunization with ß₂GP1. Having demonstrated that both human^{19 20} and murine antibodies influence thrombus formation in the mouse circulation, we are currently conducting experiments to determine the mechanisms by which these effects occur.

	Selected Abbreviations and Acronyms

 

aCL = anti-cardiolipin APS = antiphospholipid syndrome ß2GP1 = ß2 glycoprotein 1 HSA = human serum albumin OD = optical density

	Acknowledgments

 
This study was supported by a grant from the Jewish Hospital Heart and Lung Institute, Louisville, Ky. We are grateful to Dr Jane Goldsmith for helping us with statistical calculations.

Received December 11, 1995; revision received March 28, 1996; accepted April 11, 1996.

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