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Keywords:

  • antiphospholipid antibodies;
  • monocyte adherence;
  • p38 MAPK;
  • thrombosis;
  • tissue factor;
  • vascular cell adhesion molecule-1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

Summary. Background: The purpose of this study was to examine whether SB 203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, is effective in reversing the pathogenic effects of antiphospholipid antibodies. Methods: The adhesion of THP-1 monocytes to cultured endothelial cells (EC) treated with immunoglobulin G (IgG) from a patient with antiphospholipid syndrome (IgG-APS) or control IgG (IgG-NHS) in the presence and absence of SB 203580 was examined. The size of an induced thrombus in the femoral vein, the adhesion of leukocytes to EC of cremaster muscle, tissue factor (TF) activity in carotid artery and in peritoneal macrophages, the ex vivo expression of vascular cell adhesion molecule-1 (VCAM-1) in aorta preparations and platelet aggregation were studied in mice injected with IgG-APS or control IgG-NHS and with or without SB 203580. Results: SB 203580 significantly reduced the increased adhesion of THP-1 to EC in vitro, the number of leukocytes adhering to EC, the thrombus size, the TF activity in carotid arteries and in peritoneal mononuclear cells, and the expression of VCAM-1 in aorta of mice, and completely abrogated platelet aggregation induced by IgG-APS. Conclusion: These data suggest that targeting the p38 MAPK pathway may be valuable in designing new therapy modalities for treating thrombosis in patients with APS.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

Antiphospholipid syndrome (APS) is an autoimmune disorder of recurrent thrombosis and pregnancy loss associated with the presence of antiphospholipid antibodies (aPL), and persistently positive anticardiolipin (aCL) and/or anti-β2 glycoprotein I (anti-β2GPI) and/or lupus anticoagulant (LA) tests [1,2].

There is convincing evidence that aPL are pathogenic in vivo from studies that utilized animal models [3–5]. However, the mechanisms by which aPL mediate disease are only partially understood. It is known that more than one mechanism may be involved in causing thrombosis [i.e. inhibition of the activation of protein C, impairment of fibrinolysis, activation of endothelial cells (EC) or enhancing platelet activation [6]]. aPL-mediated thrombosis and EC activation may also involve complement activation [7].

The data strongly suggest that in vitro aPL induce a proinflammatory and procoagulant effect on EC and monocytes, as measured by expression tissue factor (TF) and adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and E-selectin (E-sel) and platelets. aPL-induced effects in vitro appear to be mediated, in part, by the activation of p38 mitogen-activated protein kinase (p38 MAPK) in EC, monocytes and platelets [8–19]. However, no studies have demonstrated whether those effects are seen in vivo or whether p38 MAPK inhibitors are effective in reversing the proinflammatory and prothrombotic effects of aPL in vivo. Therefore, we hypothesized that a specific inhibitor of p38 MAPK (4 methylsulfinylphenyl-4 pyridyl 1 imidazole, named SB 203580) should attenuate the proinflammatory and procoagulant effects produced by aPL in vivo. In this study, we examined whether aPL induce expression of TF and VCAM-1 in vivo. Furthermore, we studied whether specific inhibition of p38 MAPK reverses aPL-induced monocyte adherence to EC in vitro, white blood cell (WBC) adherence in vivo, thrombosis, EC and platelet activation, and TF activity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

Preparation of immunoglobulin G (IgG)

Total IgGs containing aPL (IgG-APS) from one patient with primary APS [without systemic lupus erythematosus (SLE)] were affinity-purified as previously described [4]. The patient was a 53-year-old white male with a history of one transient ischemic attack, two myocardial infarctions, three deep vein thromboses and one pulmonary embolism. The titers of aCL and anti-β2GPI antibodies (Abs) in his serum were 456 IgG phospholipid units (GPL)/mL and 256 standard IgG anti-β2GPI units (SGU)/mL, respectively. His LA test was positive.

Human IgG from a healthy individual (IgG-NHS) was purified by an identical method. The sterile-filtered IgG fractions were passed through a Detoxi-GelTM endotoxin-removing gel (Pierce, Rockford, IL, USA) to remove/neutralize any biological activity of endotoxin. Subsequently, the preparations were determined to be free of endotoxin contamination by the limulus amoebocyte lysate assay (E-Toxate, Sigma-Aldrich, St Louis, MO, USA; assay sensitivity: <0.05 IU mL−1). Protein concentration was determined by the Bradford method [20]. Levels of aCL and anti-β2GPI Abs were measured by ELISA as previously described [21,22]. IgG-APS had high titers of human aCL and anti-β2GPI Abs. This study was approved by the Morehouse School of Medicine Institutional Review Board committee and both patients provided informed consent.

In vitro experiments

Adhesion of monocytes to EC  In order to determine the effect of SB 203580 on IgG-APS-induced monocyte adherence, human THP-1 monocyte adherence was assessed by using a previously described fluorescence-based microplate adhesion assay [23]. Briefly, confluent human umbilical vein EC (HUVEC; ATCC, Manassas, VA, USA) monolayers were exposed to 20 μm SB 203580 (EMB Biosciences, La Jolla, CA, USA) in complete MCDB131 for a 2 h pretreatment before HUVEC were treated with either complete MCDB131, IgG-NHS, IgG-APS (200 μg mL−1) or 3 μg mL−1 lipopolysaccharide (LPS; Sigma-Aldrich) for 4 h in the absence or presence of 20 μm SB 203580 in a humidified 5% CO2 incubator. BCECF-loaded THP-1 monocytes (105 cells well–1) were incubated on the HUVEC monolayers for 30 min at 37 °C in a humidified 5% CO2 incubator. HUVEC monolayers were washed five times before the remaining fluorescence (i.e. adherent THP-1 cells) was read on a Cytofluor II fluorescence plate reader (Millipore Corp., Bedford, MA, USA) using fluorescein optics. The percentage of THP-1 monocyte adherence was calculated as:

  • image

In vivo experiments

Analysis of thrombus dynamics and adhesion of leukocytes to endothelium  CD1 male mice (9–10 animals/group) weighing approximately 20 g (Charles River Laboratories, Wilmington, MA, USA) were injected i.p. with 500 μg of IgG-APS or IgG-NHS twice (at time 0 and 48 h later). In some experiments, mice were treated i.p. with 25 mg kg−1 of SB 203580 in 50% dimethyl sulfoxide in phosphate-buffered saline (DMSO:PBS) or with 25 mg kg−1 SB 202474 (the inactive analog of SB 203580; EMB Biosciences) in 50% DMSO:PBS 30 min before the IgG injections. Surgical procedures were performed to study thrombus dynamics and adhesion of leukocytes (WBC) – as an indication of EC activation – to the postcapillary venular endothelium in the exposed cremaster muscle 72 h after the first IgG-APS or IgG-NHS injection, as previously described [4,7,10–13,24]. Thrombus size was measured in μm2 and the number of WBC adhering (sticking) to postcapillary venules in the cremaster muscle was determined [4,7,10–13,24]. The procedures described below were carried out in the mice after the analysis of thrombus dynamics and the adhesion of WBC to endothelium of cremaster muscle were completed.

Platelet aggregation

Blood was obtained from the mice in acid citrate dextrose anticoagulant (9/1 vol/vol) by cardiac puncture at the end of the surgical procedures described above. Platelet-rich plasma (PRP) was obtained and aggregation of platelets was measured using a Minigator II aggregometer (Payton Scientific, Buffalo, NY, USA), after the addition of 0.005 U mL−1 thrombin [25,26].

Determination of TF activity in mouse peritoneal cells

Peritoneal macrophages were obtained by flushing the peritoneal cavity of the mice with PBS for 5 min. This procedure was carried out in the animals immediately after the surgical procedures and after they were killed. Then, 2 × 107 peritoneal cells were washed twice with PBS and resuspended in 1 mL Tris-buffered saline (TBS)–0.1% Triton X-100, pH 7.4, and centrifuged at 20 800 g, 4 °C, for 30 min. The cells were washed twice and then resuspended in 50 μL TBS–0.1% Triton X-100, pH 7.4, and sonicated [27]. The TF activity of peritoneal cells lysates was determined using a commercial chromogenic assay (Actichrome TF, American Diagnostica, Stamford, CT, USA) that measures factor (F) Xa after activation by the TF–FVII complex. TF activity was expressed in pm mg–1 ml–1 protein.

Determination of TF activity in carotid artery homogenates

Pieces of approximately 5 mm of uninjured carotid arteries were dissected from both sides in each animal and collected in a TBS–0.1% Triton X-100 buffer containing heparin as anticoagulant and then homogenized. TF activity was determined in homogenates of pooled carotid artery from four animals.

Determination of endothelial VCAM-1 expression in flat aorta preparations from mice using quantum dot bioconjugates and two-photon excitation laser scanning microscopy

CD1 mice (= 2 per treatment group) were injected i.p. with IgG-APS twice plus either SB 203580 or its control (DMSO:PBS), as described above. Some mice in groups of two were challenged with LPS (1 μg g−1 body weight, i.p.), or with LPS (1 μg g−1 body weight, i.p.) and SB 203580, or with saline alone, 4 h before they were killed. A comprehensive description of this methodology can be found elsewhere [28]. In short, animals were killed and the aortas were pressure-perfused, followed by fixation. Then, the thoracic descending aortas were incubated with a rat antimouse monoclonal antibody against VCAM-1 (BD Biosciences, San Jose, CA, USA), followed by washing and incubation with quantum dot bioconjugates [Qdot 655 goat F(ab)′2 antirat IgG conjugate; Invitrogen, Eugene, OR, USA]. Image collection was accomplished using a Zeiss LSM 510 Meta two-photon microscope (Zeiss, Boston, MA, USA) equipped with a near-infrared titanium-sapphire femtosecond laser (Mira 900 Ti:S Coherent) tuned and mode-locked at 750 nm. The fluorescence quantitation of the confocal images was performed using the frequency histogram function of the LSM-510 examiner software (Zeiss).

Data analysis

The results were expressed as means ± SEM or means ± SD, as indicated in each experiment. An independent t-test was used to compare the antibody levels in different groups of mice. Student’s unpaired t-test was used to compare the mean values of thrombus sizes and numbers of adhering WBC between treated and control groups. Quantitative fluorescence microscopy of VCAM-1 expression ex vivo was analyzed using one-way anova. P-values ≤ 0.05 denote a statistical difference between groups.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

aPL-induced monocyte adherence to HUVEC was attenuated by SB 203580

While human THP-1 monocyte adherence to unstimulated, naïve HUVEC monolayers was minimal, a 4-h exposure of HUVEC to IgG-APS significantly enhanced monocyte adherence by 5.3-fold (Fig. 1). Similarly, activation of HUVEC monolayers by a 4-h treatment with LPS augmented THP-1 monocyte adherence from 6% to 48% adherence, a 7.8-fold increase. IgG-NHS did not alter monocyte adherence. aPL-induced THP-1 monocyte adherence was inhibited by approximately 34% when HUVEC monolayers were exposed to SB 203580 in the 2-h pretreatment period and during the 4-hr exposure to IgG-APS (Fig. 1). Exposure of HUVEC monolayers for 6 h to DMSO (0.008%) did not affect cell viability or monocyte adherence (data not shown). LPS-induced adherence was attenuated by 43% in the presence of SB 203580 under identical experimental conditions. Interestingly, SB 203580 did not affect aPL-induced adherence when the inhibitor was not present during the 4-h activation phase of the experiment (data not shown). These data suggest that a component of the aPL-induced monocyte adherence results from the activity of p38 MAPK.

image

Figure 1.  The effect of p38 mitogen-activated protein kinase inhibition on antiphospholipid antibody-induced monocyte adherence. Representative of three separate experiments with each bar being the mean ± SEM (= 5) of each experimental condition. *Statistical difference ( 0.05) between control immunoglobulin G (IgG-NHS) and IgG-antiphospholipid syndrome (IgG-APS) or naïve and lipopolysaccharide (LPS). **Statistical difference between IgG-APS and IgG-APS + SB 203580 or LPS and LPS + SB 203580.

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aPL-induced thrombosis and EC activation in vivo were abrogated by SB 203580

The aCL and anti-β2GPI Abs titers in the sera of the mice are shown in Table 1. The titers of aCL and anti-β2GPI Abs were high and not significantly different from each other in mice injected with either IgG-APS + SB 202474 or IgG-APS + SB 203580 (= 0.91 and = 0.78, respectively).

Table 1.   Anticardiolipin (aCL) and anti-β2 glycoprotein I (GPI) titers in the serum of the mice
Mice/groupsaCL titer (GPL mL–1) Mean ± SDAnti-β2GPI (SGU mL–1) Mean ± SD
  1. Cut-off value for aCL test: 10 IgG phospholipid units (GPL)/mL; cut-off value for anti-β2GPI test: 20 standard IgG anti-β2 GPI units (SGU)/mL.

  2. IgG-APS, immunoglobulin G-antiphospholipid syndrome; IgG-NHS, control IgG.

IgG-APS + SB 202474169.4 ± 36.3202 ± 29
IgG-APS + SB 203580145.0 ± 36.0195 ± 38
IgG-NHS + SB 202474<10<20
IgG-NHS + SB 203580<10<20

IgG-APS + SB 202474 induced a significant increase in thrombus size in CD1 mice when compared with mice treated with IgG-APS + SB 202474 (Fig. 2). Importantly, the pretreatment of the mice with SB 203580 abrogated the aPL-induced thrombosis by 81%. Thrombus size in mice treated with IgG-NHS + SB 202474 was no different from mice treated with IgG-NHS + SB 203580 or with IgG-APS + SB 203580 (= 0.45 and = 0.32, respectively; Fig. 2).

image

Figure 2.  The effect of SB 203580 on antiphospholipid antibody-induced thrombosis. CD1 mice were injected with immunoglobulin G-antiphospholipid syndrome (IgG-APS) + SB 202474 or IgG-APS + SB 203580, or with control IgG (IgG-NHS) + 202474 or IgG-NHS + SB 203580, as described in Materials and methods. SB 203580 and SB 202474 were dissolved in 50% dimethyl sulfoxide in phosphate-buffered saline. The size of induced thrombi was measured in the mice and results are expressed in μm2 as means ± SD. *Statistically significantly different from the IgG-NHS + SB 202474 group; **statistically significantly different from the IgG-APS + SB 202474 group.

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The adhesion of leukocytes to EC (number of WBC), an indicator of endothelial activation, was increased in aPL-treated mice and was significantly decreased by pretreating the mice with SB 203580 (60% reduction). Adhesion of leukocytes to endothelium was no different in mice treated with IgG-APS + SB 203580 when compared with mice treated with IgG-NHS in DMSO:PBS (= 0.67; Fig. 3).

image

Figure 3.  The effect of SB 203580 on antiphospholipid antibody-induced adhesion of leukocytes to endothelium. CD1 mice were injected with immunoglobulin G-antiphospholipid syndrome (IgG-APS) in 50% dimethyl sulfoxide in phosphate-buffered saline (DMSO:PBS) or IgG-APS + SB 203580 in DMSO:PBS, or with control IgG (IgG-NHS) in DMSO:PBS, as described in Materials and methods. The number of adhering leukocytes (# white blood cells) was measured in the mice and results are expressed as means ± SD. *Statistically significantly different from the IgG-NHS group; **statistically significantly different from the IgG-APS + DMSO:PBS group.

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aPL-induced platelet aggregation was inhibited by SB 203580

Platelets from mice treated with IgG-APS produced a significant (55%) aggregation in the presence of low (sub-aggregating) doses of thrombin. This aggregation was completely abrogated by pretreatment of the mice with SB 203580 (Fig. 4). IgG-APS did not produce aggregation of platelets in the absence of thrombin (data not shown). IgG-NHS did not induce mouse platelet aggregation in the presence of low doses of thrombin (data not shown).

image

Figure 4.  The effect of SB 203580 on antiphospholipid antibody-mediated enhancement of platelet aggregation. Platelet-rich plasma from the mice treated with immunoglobulin G-antiphospholipid syndrome (IgG-APS) and 50% dimethyl sulfoxide in phosphate-buffered saline (DMSO:PBS) or with IgG-APS+SB 203580 (in DMSO:PBS) was incubated with 0.005 U mL−1 thrombin. Aggregation of platelets was recorded for 5 min in a platelet aggregometer, as described in Materials and methods.

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aPL-mediated upregulation of TF was abrogated by SB 203580

We investigated TF function in homogenates of carotid tissue and in peritoneal macrophages of mice injected with IgG-APS or IgG-NHS in the presence of SB 203580 or SB 202474. IgG-APS + SB 202474 induced a significant increase in TF activity in peritoneal macrophage lysates and in carotid artery homogenates when compared with macrophage lysates and artery homogenates from mice treated with IgG NHS + SB 202474 (means = 3.3 vs. 0.8 pm mg−1 mL−1 and 15.3 vs. 0.7 pm mg−1 mL−1). This correlated with thrombogenic properties and EC activation in vivo, as shown above. These effects were significantly reduced by pretreating the mice with 25 mg kg−1 of SB 203580 (Fig. 5A,B).

image

Figure 5.  The effect of SB 203580 on antiphospholipid antibody-mediated tissue factor (TF) activity in mouse peritoneal macrophages and homogenates of carotid arteries. CD1 mice were injected with immunoglobulin G-antiphospholipid syndrome (IgG-APS) + SB 202474 or IgG-APS + SB 203580, or with control IgG (IgG-NHS) + SB 202474 or IgG-NHS + SB 203580, as described in Materials and methods. TF activity was determined in (A) peritoneal macrophages and (B) homogenates of carotid arteries obtained from the mice. Results are expressed in pm mg–1 mL–1 protein. *Statistically significantly different from the IgG-NHS + SB 202474 group; **statistically significantly different from the IgG-APS + SB 202474 group.

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aPL-induced expression of endothelial VCAM-1 in aorta of mice was abrogated by SB 230580

We examined whether IgG-APS have an effect on expression of VCAM-1 in flat aorta preparations of mice ex vivo by a two-step immunohistochemistry method that used quantum dot bioconjugates and two-photon excitation laser scanning microscopy. Staining of the aortic endothelium clearly illustrates increased staining of VCAM-1 on the surface of the well of the descending thoracic aorta following exposure to LPS or IgG-APS (Fig. 6B). While VCAM-1 expression was at extremely low levels in untreated (saline) aortic preparations, Hoechst nuclear staining confirmed the presence of cells (Fig. 6B). Treatment of mice with SB 203580 significantly reduced VCAM-1 fluorescence intensity in aortic preparations that were isolated from mice treated with LPS or IgG-APS plus SB 203580. Fig. 6A illustrates that mice treated with LPS or with IgG-APS similarly expressed significantly higher levels of VCAM-1 (a 2.4-fold increase) in the aorta preparations compared with mice treated with saline. Treatment with SB 203580 significantly reduced the expression of VCAM-1 in cells treated with LPS or IgG-APS (Fig. 6A,B). IgG-NHS did not induce significant expression of VCAM-1 compared with mice treated with DMSO:PBS (untreated; data not shown). These data suggest that activation of p38 MAPK contributes to aPL-induced expression of VCAM-1 in endothelium in vivo.

image

Figure 6.  The effect of SB 203580 on endothelial vascular cell adhesion molecule-1 (VCAM-1) expression in flat arterial wall preparations using quantum dot bioconjugates and two-photon excitation laser scanning microscopy. Endothelial VCAM-1 immunoreactivity in aortas of CD1 mice was determined after treatment with lipopolysaccharide (LPS) or immunoglobulin G-antiphospholipid syndrome (IgG-APS) +/– SB 203580. The untreated group was injected with 50% dimethyl sulfoxide in phosphate-buffered saline. (A) Quantitative VCAM-1 expression is depicted in fluorescence intensity arbitrary units (mean ± SEM of 10 images/mouse and 2 mice/group). (B) Representative images of endothelial VCAM-1 expression of the different treatment groups. *Statistically significantly different from the LPS + SB 203580 group; **statistically significantly different from the IgG-APS + SB203580 group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

Previous studies have shown that aPL induce upregulation of VCAM-1 in EC and TF in monocytes and EC in vitro, and activate endothelium and enhance thrombus formation in vivo [4,8–10,13,18–30]. This study shows for the first time that VCAM-1 and TF are upregulated in mice after treatment with aPL in vivo.

Although we recognize the importance of other pathogenic mechanisms induced by aPL, this study specifically addressed the effects of aPL on EC, platelets and mononuclear cells. Interestingly, in this study, we showed effects of aPL by the same IgG-APS on both sides of the vascular endothelium (arterial and venous). These findings are not surprising as the IgG was purified from a patient with both arterial and venous thromboses, and they would suggest a common mechanism mediated by these Abs. However, some patients with APS develop either arterial or venous thromboses or no thrombosis at all, and it is possible that those patients may have different mechanisms of aPL-induced endothelial activation and thrombosis.

Data from previous in vitro studies show that activation of p38 MAPK occurs in platelets, monocytes and EC after incubation with aPL [16–18]. We have shown that aPL induce significant upregulation of TF on EC, an increase in interleukin (IL)-6 and IL-8, and phosphorylation of p38 MAPK, and that these effects were inhibited by SB 203580 in vitro [16]. Recently, López-Pedrera also showed that aPL from patients with APS induce monocyte TF expression through the simultaneous activation of nuclear factor-κB/Rel proteins via the p38 MAPK pathway and the MEK-1/ERK pathway [19]. These in vitro p38 MAPK-dependent effects of aPL were recently confirmed in monocytes by Bohgaki et al. [18]. The present study illustrates for the first time that specific inhibition of p38 MAPK by SB 203580, attenuated IgG-APS-induced thrombosis in vivo and EC activation in vivo and in vitro. In addition, our data indicate that aPL induced TF activity in carotid artery tissue and in mononuclear cells (peritoneal macrophages), and VCAM-1 in aortic EC, all of which were significantly decreased by pretreatment with SB 203580. To test this last hypothesis, we used a highly innovative modified immunohistochemical en face method using quantum dot bioconjugates and two-photon excitation laser scanning microscopy [28]. This technique improves contrast resolution and allows detailed cellular structures to be imaged without the common problem of vascular auto-fluorescence, and has been shown to be very promising for mapping and quantifying the expression of endothelial markers ex vivo.

In a previous study, we demonstrated that aPL-treated platelets produce significantly higher levels of thromboxane B2 than controls, which is also completely abrogated by treatment with SB 203580 [17]. In the present study, we demonstrated that pretreatment of mice with SB 203580 completely inhibited platelet aggregation in aPL-treated mice, which correlated with a significant decrease in thrombus size. This suggests that platelet activation by aPL is an important thrombogenic mechanism in vivo that probably involves activation of p38 MAPK.

The pyridinylimidazole compounds, exemplified by SB 203580, inhibit p38 MAPK selectively by competitive binding in the ATP pocket [31–34]. p38 MAPK inhibitors have also been shown to be efficacious in animal models of inflammatory diseases, arthritis, other joint diseases, septic shock, and myocardial injury. Recently, structurally diverse p38 MAPK inhibitors were tested in preclinical studies [31–34]. In our study, we utilized SB 203580 because it has proven to be effective in reducing the pathogenic effects of aPL in vitro [16–19]. We do not exclude that other inhibitors of p38 MAPK activation may also have effects on aPL-mediated pathogenic effects. We recognize that, because of the nature of our mouse models, the treatment with the inhibitor would be administered for a relatively short period of time (72 h). Hence, this study does not address the long-term effects and possible adverse reactions of SB 203580 in other mouse models.

In summary, we show that a p38 MAPK inhibitor modulates the activation of platelets, monocytes and EC, three recognized target cells for aPL, in vivo and may be an innovative approach to treat and/or prevent thrombosis induced by aPL .

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

These studies were partially funded by a Research Centers in Minority Institutions–National Institutes of Health grant # G12-RR03034 and a Minority Biomedical Research Support Grant from the National Institutes of Health (GM58268-02).

Disclosure of Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflicts of Interest
  9. References
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