Activated Protein C Resistance in Pediatric Inflammatory Bowel Disease

Background: There is evidence for a hypercoagulable state in inflammatory bowel disease(IBD), and small vessel thrombosis has been identified in the bowel of patients with Crohn's disease, suggesting thrombosis as a possible etiologic factor. Activated protein C (APC) resistance is the most common inherited disorder leading to thrombosis and accounts for 30% to 40% of episodes of idiopathic venous thrombosis.

Cited Here... The prevalence of APC resistance was studied in 23 patients with IBD (17 with Crohn's disease, 6 with ulcerative colitis) and in 11 control subjects with recurrent abdominal pain or celiac disease, using an APC resistance screening method.

Cited Here... One patient with Crohn's disease had a positive screen result, two patients(one with Crohn's, one with ulcerative colitis) had borderline results, and results in all of the control subjects were normal. One patient with Crohn's disease had a history of a thromboembolic event but had a normal screen result.

Conclusions: Activated protein C resistance does not seem to play a major role in the etiology of the hypercoagulable state in inflammatory bowel disease.

Inflammatory bowel disease, a term that refers to both Crohn's disease and ulcerative colitis, is a disorder of unknown origin. Current theories include defective immunoregulation, infections, and microvascular events in the gastrointestinal tract, coupled with a genetic predisposition. In recent years, several studies have presented evidence for a hypercoagulable state in inflammatory bowel disease (IBD) ⁽¹⁾. This state includes arterial and venous thromboembolic phenomena, detected clinically and in autopsies ^(1-3). There is a paucity of IBD among patients with Von Willebrand's disease and hemophilia ⁽⁴⁾, and Dhillon et al. demonstrated microthrombi in normal tissue of patients with Crohn's disease and ulcerative colitis ⁽⁵⁾. Levels of some coagulation factors are elevated, but this is thought to be a secondary phenomenon. Platelet aggregation and increased thromboxane B₂ andβ-thromboglobulin levels have been detected during active and inactive disease ⁽⁶⁾, and protein C levels are normal⁽⁷⁾.

In 1994, Svensson and Dahlback described activated protein C (APC) resistance as a cause of venous thrombosis. Up to 40% of patients with recurrent venous thrombosis had APC resistance, compared with an occurrence of 3% to 7% in a control population, making it the most common identified procoagulant state ^(8,9).

The purpose of this study was to look for APC resistance in pediatric patients with IBD, to determine its possible role in the hypercoagulable state associated with IBD.

PATIENTS AND METHODS

Thirty-four children were enrolled in the study; 17 had Crohn's disease, 6 had ulcerative colitis, and 11 with recurrent abdominal pain or celiac disease served as control subjects. There were 16 boys and 7 girls in the IBD group, and 2 boys and 9 girls in the control group. There were significantly more girls in the control group (p < 0.01). The mean age in both groups was similar (IBD group 16.0 ± 3.1 years, control subjects 15.3± 2.1 years). One girl with Crohn's disease had a history of pulmonary embolism, but none of the other patients had a history suggestive of thromboembolic disease.

Blood was drawn into a test tube containing citrate used for prothrombin and activated prothrombin time. Centrifuged platelet-free frozen plasma was collected and frozen at -70 °C. The APC resistance screen was performed using the commercial APC resistance kit developed by Chromogenix (Malmö, Sweden). Plasma, activated partial thromboplastin time reagent and calcium chloride were incubated until clot formation and a second analysis was performed with activated protein C. Clot detection was performed by ACL 2000 analyzer. In the normal population, the ratio of activated partial thromboplastin time, with and without a standardized amount of APC in the presence of calcium chloride, is above 2.5. In the affected population, the ratio is below 2 for homozygotes; 2 to 2.5 is considered borderline⁽¹⁰⁾. The test was done during routine follow-up visits, irrespective of the disease activity or therapy.

Statistical Analysis

Statistical analysis was performed using Fisher's exact test.

RESULTS

One patient of 23 (4.3%) with Crohn's disease had a positive APC resistance screen ratio of less than 2, consistent with homozygous APC resistance, whereas none of the control subjects had an abnormal result. This difference was not statistically significant. In addition, 1 patient with Crohn's disease and 1 with ulcerative colitis had borderline ratios between 2 and 2.5.

DISCUSSION

Possible causes for thromboembolic disease and microvascular thrombosis in IBD include increased procoagulant factors, decreased fibrinolysis, increased platelet activity, or physical inactivity associated with disease. Increased clotting factors ^(2,5,8), increased fibrinogen, and decreased fibrinolysis have all been documented, but tend to correlate with disease activity ^(11-15), whereas many episodes occur during quiescence or after colectomy in the case of ulcerative colitis ⁽¹⁶⁾. Webberly et al.⁽¹⁶⁾ found increased platelet aggregation, spontaneous and induced, in patients with IBD, and more relevantly, in seven of eight patients with a history of thromboembolism. Changes in platelet aggregation were independent of disease activity.

Protein C is a vitamin K-dependent zymogen to a serine protease with anticoagulant properties. When the coagulation system is activated, factor IX converts factor VIII to VIIIa, which leads to Va activation and conversion of prothrombin to thrombin. Formation of factors Va and VIIIa are the rate-limiting steps in thrombin formation. Thrombin binds to thrombomodulin, activating protein C. Activated Protein C cleaves and inactivates coagulant factors Va and VIIIa ⁽⁹⁾. Protein C deficiency leads to thromboembolism and necrotic ulcerations of tissues.

In 1994, Svensson and Dahlback described APC resistance as a cause of venous thrombosis ⁽¹⁷⁾. Resistance is caused by a mutation at nucleoside position 1641 in factor V, which is not degraded, causing continuous thrombin formation. Since that time, recurrent venous thrombosis was observed in up to 40% of patients who had APC resistance, as opposed to 3% of a control population, making it the most common type of inherited procoagulant state-at least 10 times more common than any other inherited defect of coagulation. This trait is autosomally dominant and may be silent. Combination with another procoagulant state may lead to thrombosis⁽⁸⁾.

Only one of our 17 patients with Crohn's disease and none of the 6 with ulcerative colitis had an abnormal screen result that was compatible with homozygous APC resistance, and 2 others had borderline values. None of the control subjects had evidence of APC resistance, and the difference was not statistically significant. The only patient with a history of thromboembolism had a normal APC resistance screen result. The groups were not matched in relation to gender, but this is unlikely to be of importance in an autosomally recessive disease.

These findings do not support a causal role for APC resistance in the pathogenesis of IBD or in the hypercoagulable state associated with these diseases, although larger numbers may be needed for a definitive answer.

Gaffney et al. ⁽¹⁸⁾ reported the efficacy of intravenous heparin in refractory cases of ulcerative colitis, suggesting that modulation of the coagulable state may play a role in the etiology and future therapy of the disease. Further studies are needed to identify the causes of the hypercoagulable state and its therapeutic modification.