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Keywords:

  • antibodies;
  • Crohn's disease;
  • fibrinolysis;
  • thrombosis;
  • tissue-type plasminogen activator;
  • ulcerative colitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Summary. Background: Patients with inflammatory bowel disease (IBD) have an increased prevalence of thromboembolic events. The pathogenetic mechanisms of these events include reduced fibrinolysis, which may be caused by antibodies to tissue-type plasminogen activator (t-PA). Objectives: To evaluate anti-t-PA antibodies in patients with IBD, considering clinical, biochemical and functional characteristics. Patients and methods: We immunoenzymatically measured anti-t-PA antibodies in plasma from 97 consecutive IBD patients and 97 age- and sex-matched healthy controls. We also assessed the antibody interactions with different epitopes of t-PA, the antibody inhibition on t-PA activity and the correlations with clinical features and other serum antibodies. Results: IBD patients had higher median anti-t-PA antibody levels (5.4 U mL−1 vs. 4.0 U mL−1; P < 0.0001): 18 patients were above the 95th percentile of the controls (OR 5.3; 95% CI 1.7–16.3; P < 0.003), and the six with a history of thrombosis tended to have high levels (6.9 U mL−1). Anti-t-PA antibody levels did not correlate with IBD type, activity, location or treatment, or with age, sex, acute-phase reactants or other antibodies. The anti-t-PA antibodies were frequently IgG1 and bound t-PA in fluid phase; they recognized the catalytic domain in 10 patients and the kringle-2 domain in six. The IgG fraction from the three patients with the highest anti-t-PA levels slightly reduced t-PA activity in vitro. Conclusions: The prevalence of anti-t-PA antibodies is high in IBD patients. By binding the catalytic or kringle-2 domains of t-PA, these antibodies could lead to hypofibrinolysis and contribute to the prothrombotic state of IBD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

It is a well-established fact that patients with inflammatory bowel disease (IBD) are at increased risk of thrombotic events that may involve both the venous and the arterial district, but the exact pathogenetic mechanisms are not yet clear [1].

Recent data show that IBD patients have a threefold greater risk of developing deep vein thrombosis (DVT) and pulmonary embolism (PE) than the general population [2,3] and the annual incidence rate is approximately 0.5% [2]. Moreover, some authors suggest a possible association between venous thromboembolism and clinical features of IBD (i.e. extent and activity of the disease) [4].

A number of qualitative and quantitative abnormalities regarding platelets [5] and the coagulation cascade [6,7] have been suggested as possible causes of the thromboembolic events occurring in IBD patients, as well as an increased prevalence of known risk factors such as hyperhomocysteinemia [8,9] and reduced levels of vitamin B6 [10].

The fibrinolytic system has also been widely investigated in IBD. Fibrinolysis plays an important role in dissolving clots formed in the circulatory system. The fibrinolytic system is finely regulated by means of plasminogen activators such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (uPA), and natural inhibitors such as α2-plasmin inhibitor (α2-PI), plasminogen activator inhibitor 1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI). Among the plasminogen activators, t-PA is thought to play a relevant role in initiating fibrinolysis and thrombolysis. Literature data in IBD are still controversial, even though hypofibrinolysis seems to prevail [11–15].

A decrease in fibrinolytic capacity may lead to a prothrombotic condition; reduced t-PA and/or increased PAI-1 and TAFI levels seem to be associated with DVT [16,17] and myocardial infarction [18,19]. A hypofibrinolytic state may also be caused by the presence of anti-t-PA antibodies, which reduce t-PA activity by preventing t-PA from converting plasminogen to the active plasmin enzyme. It has been previously demonstrated that anti-t-PA antibodies interact with the catalytic domain of t-PA [20], thus suggesting that they may contribute to the prothrombotic state described in patients with antiphospholipid syndrome [20,21]. Furthermore, high anti-t-PA antibody levels have been described in other diseases characterized by alterations in immune and fibrinolytic systems, such as systemic lupus erythematosus [22] and systemic sclerosis [23]. Finally, IBD is characterized by the production of a wide range of antibodies [24–28], some of which are potentially involved in the increased thrombotic risk, including antiphospholipid antibodies [29] and anti-protein S antibodies [30].

The aim of this study was to compare the presence and levels of anti-t-PA antibodies in IBD patients and healthy controls, and to evaluate the possible correlations between anti-t-PA antibodies and the clinical and serological features of the patients. Additionally, in patients with high anti-t-PA antibody titers, we evaluated the subclasses of anti-t-PA immunoglobulin G (IgG), their inhibitory effect on t-PA activity, and the epitopes involved in antigen–antibody interactions.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Patients

The study prospectively and consecutively enrolled 45 patients with ulcerative colitis (UC) (29 men and 16 women; mean age ± SD 42.8 ± 14.9 years) and 52 patients with Crohn's disease (CD; 21 men and 31 women; mean age 39.2 ± 12.3 years), who were referred to our out-patient gastrointestinal clinic. The diagnosis of IBD was based on clinical, endoscopic, radiological and histological findings, and the medical history of the patients was obtained from their clinical records. The demographic and clinical features of the IBD patients are summarized in Tables 1 and 2.

Table 1.   Demographic and clinical features of patients with inflammatory bowel disease
 Crohn's disease, (n = 52)Ulcerative colitis, (n = 45)
  1. 5-ASA, 5-aminosalicylic acid; ASCA, anti-Saccharomyces cerevisiae antibodies; p-ANCA, anti-neutrophil cytoplasmic antibodies; APA, anti-phospholipid antibodies.

Men/women21/3129/16
Age, mean ± SD (years)39.2 ± 12.342.8 ± 14.9
Duration of disease, mean ± SD (months)87.5 ± 69.986.6 ± 69.7
Disease activity (at sampling), n (%)
 Active13 (25.0)11 (24.4)
 Quiescent39 (75.0)34 (75.6)
Location/extension, n (%)
 Ileum9 (17.3)
 Ileum + colon24 (46.2)
 Colon19 (36.5)
 Pancolitis18 (40.0)
 Left-sided colitis24 (53.3)
 Procto-sigmoiditis3 (6.7)
Crohn's disease type, n (%)
 Non-penetrating, non-stricturing31 (59.6)
 Stricturing11 (21.2)
 Penetrating10 (19.2)
Previous thrombosis, n (%)2 (3.8)4 (8.9)
Extraintestinal manifestations, n (%)19 (36.5)8 (17.8)
p-ANCA+, n (%)3 (5.7)21 (46.7)
ASCA IgG+, n (%)17 (32.7)1 (2.3)
ASCA IgA+, n (%)27 (51.9)9 (20)
APA+, n (%)6 (11.5)2 (4.5)
Treatment, n (%)
 No therapy11 (21.1)3 (6.7)
 Only 5-ASA26 (50.0)30 (66.7)
 Steroids ± 5-ASA8 (15.4)3 (6.7)
 Immunosuppressors ± steroids ± 5-ASA7 (13.5)9 (20.0)
Table 2.   Extraintestinal manifestations in patients with inflammatory bowel disease
 Crohn's disease* (n = 52)Ulcerative colitis (n = 45)
  1. *Some patients had more than one extraintestinal manifestation.

Arthropathy, n (%)
 Peripheral16 (30.7)1 (2.3)
 Axial2 (3.8)0 (0.0)
Skin, n (%)2 (3.8)3 (6.7)
Eye, n (%)5 (9.6)0 (0.0)
Hepato-biliary tract, n (%)2 (3.8)2 (4.4)
Previous thrombosis, n (%)2 (3.8)4 (8.9)

Disease activity at the time of blood sampling was assessed by means of Crohn's Disease Activity Index [31], and by Truelove and Witts’ criteria for UC [32]. The clinical behavior of the CD patients was classified according to the Vienna classification [33].

Six patients (two with CD and four with UC) had a history of thrombosis; three had had previous DVT, one DVT and PE, one arterial thrombosis and one thrombosis of the portal and suprahepatic veins.

All of the patients were screened for anti-t-PA antibodies, anti-Saccharomyces cerevisiae antibodies (ASCA), anti-neutrophil cytoplasmic antibodies (p-ANCA), and anti-phospholipid antibodies (APA) (patients were considered APA positive in the presence of anti-cardiolipin antibodies and/or lupus anti-coagulant). They were also screened for acute-phase reactants (erythrocyte sedimentation rate, ESR; α1-acid-glycoprotein, AAG; and C-reactive protein, CRP).

The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the local Ethical Committee. All the subjects gave their informed consent before enrollment.

Controls

The 97 age- and sex-matched controls (50 men and 47 women; mean age 41.7 ± 13.9 years) were randomly chosen from a larger cohort of approximately 400 subjects with no personal or family history of thrombosis, who had been previously enrolled as normal controls for a study of other hemostatic parameters.

Samples

The blood (collected in sodium citrate or without anticoagulant) was centrifuged at 2500 × g for 20 min, and the plasma and serum samples were frozen in small aliquots and stored at −80°C until tested.

Anti-t-PA antibodies

Anti-t-PA antibodies were detected using an ELISA method, as previously described [20]. Briefly, microtitration plates were coated with recombinant protein (rt-PA) (Actilyse; Boehringer Ingelheim, Ingelheim, Germany) and overcoated with bovine serum albumin. After the addition of the diluted plasma samples, the rt-PA-bound immunoglobulins were identified by class-specific mouse monoclonal antibodies, which were in turn detected by peroxidase-conjugated anti-mouse immunoglobulin antibodies. High anti-t-PA antibody titers were defined as values exceeding the 95th percentile of distribution in the matched healthy controls (11.0 U mL−1).

Characterization of anti-t-PA antibodies

The immunoassay was the same as that described above except for the fact that the first antibody was an anti-human immunoglobulin with subclass specificity for IgG1, IgG2, IgG3 or IgG4 [20].

IgG fraction isolation

The plasma IgG fractions of the patients and controls were isolated using a protein G column as previously described [20].

Production of the t-PA catalytic domain

The recombinant molecule consisting of the catalytic domain of the enzyme was obtained by inserting the cDNA coding for the catalytic domain and first three N-terminal residuals of human t-PA [t-PA del (Val 14-Cys 261)] into the pET11c vector, which was then transfected into Escherichia coli cells [34].

Evaluation of purified IgG binding to different forms of t-PA immobilized on microplates

The microplates were coated with rt-PA, t-PA obtained from human melanoma cells (mt-PA, single chain t-PA, Biopool, Umeå, Sweden), the recombinant molecule consisting of the t-PA catalytic domain, or the recombinant molecule consisting of the t-PA catalytic and kringle-2 domains (Reteplase; Boehringer Mannheim GmbH, Mannheim, Germany). The immunoglobulins specifically bound to the t-PA forms were detected by ELISA, as previously described [20].

The anti-t-PA antibodies bound to the kringle domain were defined as those that bound the recombinant molecule consisting of the t-PA catalytic and kringle-2 domains, but not the recombinant molecule consisting of the catalytic domain alone.

Interactions between anti-t-PA antibodies and t-PA in fluid phase

After incubating the patients’ plasma for 1 h at 37°C with increasing rt-PA concentrations (0.015, 0.031, 0.063, 0.125, 0.250 and 0.5 mg mL−1), we evaluated the free anti-t-PA antibodies still capable of binding to the microplate-immobilized rt-PA (ELISA).

Measurements of in vitro inhibition of t-PA activity

The isolated immunoglobulins from the three patients with the highest anti-t-PA antibody titers were tested for in vitro inhibition of t-PA activity by adding increasing amounts to plasma from a healthy control and measuring t-PA activity after 1 h incubation at 37°C. The activity of t-PA was measured using a chromogenic method [Spectrolyse (fibrin) t-PA assay kit; American Diagnostica, NY, USA]. Immunoglobulins purified from a healthy subject were used as the negative control, and goat anti-t-PA antibodies (American Diagnostica, Greenwich, CT, USA) as the positive control.

Anti-Saccharomyces cerevisiae antibodies

The determination of ASCA was performed by means of a commercially available ELISA kit (Medimar, Milan, Italy) according to the manufacturer's instructions, as previously described [35].

Anti-neutrophil cytoplasmic antibodies

p-ANCA were detected by means of indirect immunofluorescence on commercially available slides (Inova, San Diego, CA, USA), as previously described [35].

Anti-phospholipid antibodies

ACA were measured by ELISA ‘in-house’ techniques as described by Loizou et al. [36]. Lupus anti-coagulant was diagnosed according to the revised criteria proposed by the Subcommittee for the Standardization of Lupus Anticoagulants [37] using a battery of assays consisting of activated partial thromboplastin time (APTT, Thrombofax, Ortho, Raritan, NJ, USA, or automated APTT reagent, Organon Teknika, Durham, NC, USA), colloidal silica clotting time, diluted Russel viper venom time, mixture tests and confirmation tests.

Statistical analysis

The data were statistically analyzed by means of the Mann–Whitney U-test, Fisher's exact test, the Kruskal–Wallis test or Spearman's correlation as appropriate, using the Statistical Package for Social Sciences (SPSS Inc., v. 11.0 for Windows, Chicago, IL, USA). The significance level was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

The median anti-t-PA antibody levels were significantly higher in the IBD patients (5.4 U mL−1, interquartile range 4.0–9.5 vs. 4.0 U mL−1, interquartile range 2.7–5.7; P < 0.0001), with no difference between the CD and UC patients (5.3 U mL−1, interquartile range 4.0–8.5 vs. 5.4 U mL−1, interquartile range 4.0–10.9; P = NS).

The anti-t-PA antibody levels in the IBD patients did not show any significant association with sex, disease location/extension, or clinical activity; among the CD patients, there was no correlation with clinical behavior. There was no difference between the patients treated with immunosuppressors (azathioprine and/or steroids) and those treated with aminosalicylic acid only or untreated (Table 3).

Table 3.   Anti-tissue-type plasminogen activator (anti-t-PA) antibodies in patients with inflammatory bowel disease, by clinical features [results expressed in U mL−1: median values (interquartile range)]
 Crohn's disease, Anti-t-PA antibodiesPUlcerative colitis, Anti-t-PA antibodiesP
  1. 5-ASA, 5-aminosalicylic acid; NS, not significant.

Sex
 Men5.4 (4.2–14.5)NS6.0 (3.8–9.5)NS
 Women5.5 (4.0–8.5)5.3 (4.5–10.9)
Disease activity
 Quiescent5.4 (4.0–8.5)NS6.0 (4.8–10.9)NS
 Active5.0 (3.8–12.4)4.5 (3.5–9.0)
Location
 Ileum4.8 (4.2–7.0)NS 
 Ileum + colon5.5 (4.0–8.8)
 Colon5.4 (4.0–16.7)
Extension
 Pancolitis 5.7 (4.4–9.3)NS
 Left-sided colitis5.3 (3.8–11.4)
 Procto-sigmoiditis6.0 (4.5–10.9)
Crohn's disease behavior
 Non-penetrating, non-stricturing5.0 (4.0–10.0)NS 
 Stricturing7.0 (3.0–8.9) 
 Penetrating5.7 (4.6–7.7) 
Treatment
 No therapy7.2 (5.5–14.5)NS3.5 (2.0–15.0)NS
 Only 5-ASA4.9 (4.0–6.2)5.3 (4.0- 10.4)
 Steroids ± 5-ASA8.2 (4.6–15.5)8.9 (5.0–9.0)
 Immunosuppressors ± steroids ± 5-ASA4.5 (3.6–21.4)7.0 (4.0–19.7)
Extraintestinal manifestations
 Yes5.5 (4.8–13.5)NS8.9 (5.0–15.0)NS
 No5.0 (4.0–8.7)5.2 (3.6–9.5)

The six IBD patients with a history of thrombosis had higher median anti-t-PA antibody levels than those without (6.9 U mL−1, interquartile range 4.6–37.7 vs. 5.4 U mL−1, interquartile range 4.0–9.0), but this difference did not reach statistical significance.

Eighteen (18.6%) of the IBD patients had anti-t-PA antibody levels that were higher than the 95th percentile of the normal controls (11.0 U mL−1; OR 5.3; 95% CI 1.7–16.3; P < 0.003; Fig. 1) with no significant difference between the patients with CD (nine of 52, 17.3%) and those with UC (nine of 45, 20.0%). Once again, there were no significant differences in the associations between abnormally high anti-t-PA antibody levels and gender, disease location/extension or clinical activity, or (in CD patients) clinical behavior.

image

Figure 1.  Plasma anti-tissue-type plasminogen activator (anti-t-PA) antibody levels in inflammatory bowel disease (IBD) patients and controls. Solid horizontal lines represent median values. The 97 IBD patients had higher levels than the 97 controls (P < 0.0001), and 18 IBD patients had anti-t-PA antibody levels above the 95th percentile of the controls (dashed line).

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The prevalence of high anti-t-PA antibody titers was twice as high among the IBD patients with a history of thrombosis (two of six, 33.3%) than among those without (16 of 91, 17.5%), but the difference was not statistically significant (P = 0.35; OR 2.3; 95% CI 0.4–13.9). Interestingly, the patient with previous portal and suprahepatic vein thrombosis had the highest anti-t-PA antibody titer (100 U mL−1).

Moreover, Spearman's correlations did not show any significant associations between anti-t-PA antibody levels and age (r = −0.05; 95% CI−0.26–0.15; P = 0.60), or acute-phase reactant values: ESR (r = −0.14; 95% CI−0.39–0.12; P = 0.28); CRP (r = −0.19; 95% CI−0.44–0.08; P = 0.16); AAG (r = −0.18; 95% C.I.−0.44–0.10; P = 0.20).

The median anti-t-PA antibody levels in the IBD patients who were p-ANCA, ASCA IgG, ASCA IgA or APA positive were not significantly different from those in the IBD patients not expressing these antibodies (Table 4).

Table 4.   Anti-tissue-type plasminogen activator (anti-t-PA) antibodies in patients with inflammatory bowel disease by anti-neutrophil cytoplasmic antibodies (p-ANCA), anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-phospholipid antibodies (APA) expression [results expressed in U mL−1: median values (interquartile range)]
 Anti-t-PA antibodiesP
  1. NS, not significant.

p-ANCA+5.7 (4.5–11.2)NS
p-ANCA−5.4 (4.0–8.3)
ASCA IgG+4.7 (4.0–7.4)NS
ASCA IgG−5.5 (4.3–10.1)
ASCA IgA+4.9 (4.0–6.5)NS
ASCA IgA−6.0 (4.1–9.8)
APA+5.2 (4.9–5.6)NS
APA−6.0 (4.9–10.7)

The IgG fractions isolated from the plasma of the 18 patients with high anti-t-PA antibody titers showed that the antibodies were IgG1 in 13, IgG2 in one, and IgG4 in four. Among the same 18 patients, all had IgG bound to human t-PA, 10 had IgG bound to the catalytic domain and six to the kringle-2 domain, and in two patients IgG was not bound to the catalytic or the kringle-2 domain.

The isolated immunoglobulins from the three patients with the highest anti-t-PA antibody titers revealed a slight reduction in in vitro t-PA activity (Fig. 2); the negative controls were immunoglobulins purified from a healthy subject, and the positive controls were goat anti-t-PA antibodies. Moreover, rt-PA added to the patients’ plasma at concentrations of up to 0.5 mg mL−1 inhibited the binding of anti-t-PA antibodies to microplate-immobilized rt-PA in a concentration-dependent manner (Fig. 3).

image

Figure 2.  Inhibition of plasma tissue-type plasminogen activator (t-PA) activity by immunoglobulins purified from the plasma of the three patients with the highest anti-t-PA antibody titers. Negative control: immunoglobulins purified from a healthy subject; positive control: goat anti-t-PA antibodies.

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image

Figure 3.  Inhibition of immunoglobulin G binding to microplate-immobilized recombinant tissue-type plasminogen activator (rt-PA) by soluble rt-PA. When added to the immunoglobulins of the three patients with the highest anti-t-PA antibody titers, rt-PA (at concentrations of 0.015, 0.031, 0.063, 0.125, 0.250 and 0.5 mg mL−1) inhibited the binding of anti-t-PA antibodies to the rt-PA immobilized on microplates in a dose-dependent manner. The results are expressed as the percentage binding recorded in the absence of added soluble proteins (buffer).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Patients with IBD are at greater risk of developing thrombosis. The coagulation cascade and fibrinolytic system have been widely investigated; however, the published data are still controversial and the exact pathogenetic mechanisms underlying this risk are still unclear.

In this study, we evaluated for the first time the prevalence and plasma levels of antibodies against t-PA in IBD patients. Our group has recently provided evidence that, at least when present at high titers, these antibodies specifically interact with the catalytic domain of t-PA and reduce its activity, thus leading to a prothrombotic state [20]. It has been suggested that anti-t-PA antibodies may provide the missing link between the thrombotic events described in patients with antiphospholid syndrome [21] and the reduced fibrinolytic activity typical in this clinical setting [38,39].

Our results demonstrate that IBD patients have significantly higher median plasma anti-t-PA antibody levels than healthy controls, and that the increased levels do not correlate with their demographic or clinical features. However, median plasma anti-t-PA antibody levels were slightly higher in the patients with than in those without a history of thrombosis; the lack of statistical significance may be due to the small number of thrombotic patients.

The association between IBD and increased plasma anti-t-PA antibody levels is further supported by the finding that 18.6% of the IBD patients had levels that were higher than the 95th percentile of the control values. Furthermore, the prevalence of abnormally high anti-t-PA antibody levels was twice as high among the thrombotic patients; again, the lack of statistical significance may be attributable to the small size of the thrombotic population. At this point, it is interesting to underline that our patient with the highest anti-t-PA antibody titer (100 U mL−1) had a history of portal and suprahepatic vein thrombosis of unknown cause. Thus, by inhibiting t-PA function, anti-t-PA antibodies may induce hypofibrinolysis, which is a prothrombotic condition already described in IBD [12–15]. These findings are in line with our previous observation of an inverse correlation between plasma anti-t-PA antibody levels and t-PA activity [20].

On the other hand, it is also possible that IBD patients, and specifically those with previous thrombosis, have an ongoing activation of coagulation and fibrinolysis [40] that induces relatively continuing high levels of t-PA and therefore a higher and longer presence of antigen to produce antibodies against. Furthermore, the presence of anti-t-PA antibodies did not appear to be associated with the expression of other antibodies frequently observed in IBD patients (such as ASCA, p-ANCA and APA). Thus, anti-t-PA antibodies seem to have an independent mechanism of production with a different phenotypic association.

Our data show that the anti-t-PA antibodies in IBD patients frequently belong to the IgG1 subclass, which is probably T-lymphocyte dependent and activates the classic complement pathway [41]. Several reports indicate that complement activation is involved in IBD, although it does not seem to play a direct pathogenetic role [42–44].

The anti-t-PA antibodies in our patients were frequently directed against the catalytic domain of the t-PA molecule, correlating with our previous findings [20]. Moreover, in six patients, they were directed against the kringle-2 domain through which t-PA binds fibrin and increases its affinity for plasminogen and, in a further two patients, against the whole rt-PA molecule but not the catalytic or kringle-2 domain (probably because they recognized a different epitope). Anti-t-PA antibodies may therefore reduce t-PA activity both directly and indirectly, and a slight reduction in t-PA activity in vitro was observed when increasing amounts of IgG from the three patients with the highest anti-t-PA antibody titers were added to plasma from a healthy control.

Finally, the addition of the rt-PA molecule to the patients’ plasma inhibited the binding of anti-t-PA antibodies to microplate-immobilized rt-PA in a dose-dependent manner, thus demonstrating that anti-t-PA antibodies can also bind to the t-PA molecule in fluid phase, a condition that is more similar to the in vivo situation.

In conclusion, high levels of anti-t-PA antibodies are frequently found in the plasma of IBD patients, and their titers are slightly higher in those with a history of thrombosis. Anti-t-PA antibodies interact with the catalytic and kringle-2 domains of the t-PA molecule, reducing its activity, and so they should be added to the list of possible mechanisms responsible for the prothrombotic state described in IBD patients.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References
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