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The factor V Leiden mutation increases the risk of venous thrombosis in patients with inflammatory bowel disease
GASTROENTEROLOGY 1998;115:830–834
The Factor V Leiden Mutation Increases the Risk of Venous Thrombosis in Patients With Inflammatory Bowel Disease HOWARD A. LIEBMAN,* NADER KASHANI,* DOUGLAS SUTHERLAND,* WILLIAM MCGEHEE,* AND LORI KAM‡ Divisions of *Hematology and ‡Gastroenterology, Department of Medicine, University of Southern California School of Medicine, Los Angeles, California
Background & Aims: Thromboembolic disease is a significant cause of morbidity and mortality in patients with inflammatory bowel disease (IBD). The aim of this study was to determine the incidence and possible association of the factor V Leiden mutation with the development of thrombosis in patients with IBD. Methods: This retrospective study included 11 patients with IBD and arterial or venous thrombosis and 51 patients with IBD and no history of thrombosis who were matched for age, sex, ethnic/racial origin, and type of IBD (controls). The presence of the factor V Leiden mutation was determined by coagulation assay and confirmed by a polymerase chain reaction method. Results: Four of 11 IBD patients (36%) with thrombosis and 2 of 51 IBD controls (4%) were heterozygotes for the factor V Leiden mutation (relative risk, 14.00; 95% confidence interval, 1.55–169.25; P 5 0.009, Fisher exact test). All thrombotic events in the patients with activated protein C resistance were venous with a calculated prevalence of 50% (4 of 8 patients) and a relative risk of venous thrombosis in IBD patients with factor V Leiden of 23 (95% confidence interval, 2–294; P 5 0.005). Conclusions: In patients with IBD, inheritance of the factor V Leiden mutation results in a significant increased risk of venous thrombosis.
hromboembolism is a well-described complication of inflammatory bowel disease (IBD) with a reported incidence of 1%–6%.1–5 Although clinically significant thrombosis occurs in only a fraction of patients with IBD, it contributes significantly to patient morbidity and mortality. Autopsy studies report a much higher incidence of thrombosis, ranging from 7% to 39%, which is often responsible for patient mortality.1,6–8 Thromboembolic complications are more frequently associated with clinically active disease,5 but laboratory studies using sensitive markers of activation of the coagulation system have documented endothelial injury and increased thrombin generation in patients with both active and quiescent IBD.9–13 With this growing evidence that most patients with IBD have active thrombin generation, the question
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emerges as to what other factors contribute to the development of clinically overt thrombosis in patients with IBD. In 1993, Dahlback et al.14 were the first to recognize a new mechanism for familial thrombophilia. This disorder is characterized by in vitro resistance to the anticoagulant effects of activated protein C (APC). Subsequent population studies have found APC resistance (APCR) to be the most prevalent cause of inherited thrombophilia with a reported prevalence of 2%–5% in populations of European descent.15–18 The molecular defect responsible for APCR in a majority of patients has been identified and has been shown to result from a mutation in the factor V molecule (R506Q, factor V Leiden), resulting in the loss of an APC cleavage site necessary for rapid inactivation of factor Va.19–21 Recent studies show that inheritance of the factor V Leiden mutation potentiates the thrombotic risk in subjects with other acquired22–24 or congenital25,26 prothrombotic states. We hypothesized that the factor V Leiden defect would be one inherited prothrombotic risk factor that could significantly increase the risk of thrombosis in patients with IBD. Therefore, we screened 11 patients with a history of IBD and thrombosis for the factor V Leiden mutation and compared the prevalence of this mutation in them with that of 51 patients with IBD and no history of thrombosis. We show that APCR caused by factor V Leiden is a major contributory factor to the development of venous thrombosis in patients with IBD and the combined risk of venous thrombosis in patients with IBD, and that this mutation is a significantly greater risk factor than factor V Leiden mutation alone in causing thrombosis. Abbreviations used in this paper: APC, activated protein C; APCR, APC resistance; LAC-USC, Los Angeles County–University of Southern California (Medical Center). r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00
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Materials and Methods The study population included 11 patients with a history of IBD and thromboembolic complications and 51 IBD controls. A review of the records from Los Angeles County– University of Southern California (LAC-USC) Medical Center Anticoagulation Clinic, the IBD clinic, and hematology consultation records identified 14 patients with IBD and a history of either arterial or venous thrombosis. Only 11 of these patients were available to perform studies for APCR and factor V Leiden. A control population of 51 patients with IBD without a history of thrombosis were selected to be matched to the patients with thrombosis for age, sex, ethnic/racial origin, and type of IBD (ulcerative colitis or Crohn’s disease). The prevalence of the factor V Leiden mutation in the IBD patients with and without thrombosis was compared with that of a group of 81 patients with a history of idiopathic venous thrombosis and 142 racially matched control subjects without a history of venous or arterial thrombosis. Many of the patients with thrombosis, with and without a history of IBD, have been described previously.27 Patients were seen at LAC-USC Medical Center, the Kenneth Norris, Jr–University of Southern California Cancer Center, or at USC University Hospital. Patients with a history of IBD and deep venous thrombosis had documentation of venous thrombosis by ultrasonography. In patients with a diagnosis of pulmonary embolism, the diagnosis was based on appropriate clinical and laboratory findings combined with a high-probability ventilation-perfusion lung scan. Arterial thrombotic events, including stroke and myocardial infarction, were documented by generally acceptable diagnostic criteria including computerized tomography and angiography. Blood samples were collected in 3.2% sodium citrate, and platelet-poor plasma was prepared immediately by centrifugation at 10,000 rpm at 4°C for 10 minutes. Plasma samples were stored at 220°C until tested. All plasma samples were assayed within 1 week of collection. Plasma samples were assayed for the presence of factor V Leiden using the tissue factor– dependent factor V assay, as originally described by Le et al.28 and reported by our laboratory.27 This coagulation assay was demonstrated to have 100% sensitivity and specificity for the factor V Leiden defect in a blinded study compared with a polymerase chain reaction–based assay for the molecular defect of factor V Leiden.27 All patients in whom the factor V Leiden defect was detected were further studied using the polymerase chain reaction assay.27 Genomic DNA was isolated from 200 µL of the cellular pellet, which remained after removal of plasma for coagulation studies, using the QAI amp Blood Kit (Qiagen, Chatsworth, CA). In 8 IBD patients without thrombosis, only the molecular analysis for factor V Leiden was performed because fresh plasma was not available for coagulation studies. All patients with IBD and a history of thrombosis were also evaluated with functional assays for antithrombin III, protein C, protein S, plasminogen, the Exner test for the lupus anticoagulant, and anticardiolipin antibody assays using com-
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mercial kits (Protein C, Proclot Protein C; ACL Instrument Laboratories, Lexington, MA; and Staclot Protein S, Diagnostica Stago, Asieres-sur-Seine, France) and according to methods reported previously.29
Results The characteristics of the study populations are shown in Table 1. There were no significant differences in age or sex between the IBD patients with thrombosis and patients without thrombosis. The average age of the patients with IBD at the time of their thrombotic event was 39 years (range, 21–64 years), and the patients were studied an average of 2.5 months (range, 4 days to 36 months) after their thromboembolic event. Ulcerative colitis was the most frequent form of IBD in patients with thrombosis, a finding in agreement with most published studies of thrombosis and IBD.5 Despite our efforts to match the control IBD population with the patients with a history of thrombosis, there was a greater number of IBD patients of Latino origin in the control population (36% vs. 53%); however, this difference was not statistically different (P 5 0.48). This racial imbalance reflects the dominant ethnic background of the patients seen in the IBD clinic at LAC-USC Medical Center. Previous studies in our patient population have found a prevalence of factor V Leiden of 2.9% (2 of 69) in Latino subjects without a personal or family history of thrombosis (Table 1). We compared our IBD patients with and without thrombosis with 81 patients who had a history of idiopathic venous thrombosis and 142 control subjects without a history of IBD or thrombosis. The non-IBD thrombotic patients tended to be older than the IBD patients with thrombosis; this difference was statistically significant (P , 0.05). Table 1. Characteristics of the Study Population IBD Thrombosis (n 5 11)
Non-IBD Controls (n 5 51)
Age, yr (mean, range) 39 (21–64) 44 (12–73) Sex (M/F) 8/3 35/16 UC/CD 7/4 31/20 APCR1 (factor V Leiden) 4 2 Racial background (%) European, non-Latino 6 (55) 21 (41) Latinoa 4 (36) 27 (53) African American 1 (9) 2 (4) Asian 0 (0) 1 (2)
Thrombosis (n 5 81)
Controls (n 5 142)
54 (28–74) 51 (26–84) 47/34 82/60 — — 15
6
39 (48) 34 (42)
60 (42) 69 (49)
7 (9) 1 (1)
11 (8) 1 (1)
UC, ulcerative colitis; CD, Crohn’s disease. individuals with family origins from Central or South America.
aLatino:
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Four of the 11 patients (36%) with a history of thromboembolic disease had APCR (Tables 1 and 2). All 4 were subsequently determined to be heterozygotes for factor V Leiden. Only 2 of the 51 (4%) patients with IBD and no history of thromboembolic disease had APCR and were heterozygotes for factor V Leiden. This difference was highly significant (P 5 0.009, Fisher exact test), despite the small number of patients studied. Therefore, the risk of thrombosis in patients with IBD who are carriers of the factor V Leiden mutation compared with noncarriers is increased 14-fold (relative risk, 14.00; 95% Cl, 1.55–169.25). The presence of the factor V Leiden mutation was only associated with venous thromboembolism. Therefore, 4 of the 8 patients (50%) with a history of venous thrombosis had the factor V Leiden mutation, representing a relative risk of venous thrombosis in IBD patients with factor V Leiden of 23 (95% CI, 2–294; P 5 0.005). In comparison, 15 of our 81 (19%) non-IBD patients with a history of thrombosis had APCR due to factor V Leiden (2 homozygotes and 13 heterozygotes). In the ethnic and racially matched controls without a history of IBD or thrombosis, 6 of the 142 subjects were heterozygotes for factor V Leiden. Therefore, in the non-IBD patients, the risk of thrombosis in carriers of the factor V Leiden mutation is increased 5-fold (relative risk, 5.15; 95% confidence interval, 1.77–16.84). These findings strongly support the hypothesis that IBD significantly amplifies the thrombotic risk associated with the factor V Leiden mutation. We found no other clinical manifestations that distinguished patients with APCR and thrombosis from the patients with thrombosis who were APCR negative. However, it is interesting that the only patient with clinically quiescent IBD at the time of his venous thrombosis had the factor V Leiden (Table 2). Table 2. Characteristics of Patients With IBD and Thrombosis
Age, yr (mean, range) Sex (M/F) UC/CD Activity (1) a Thrombotic event DVT PE MI Stroke Other arterial
APCR1 (n 5 4)
APCR2 (n 5 7)
41 (27–64) 3/1 2/2 3/4
38 (21–56) 5/2 5/2 7/7
3 1 — — —
2 2 1 1 1
UC, ulcerative colitis; CD, Crohn’s disease; DVT, includes both lower and upper extremity venous throbmosis; PE, all patients in this study with pulmonary embolus had documented lower extremity venous thrombosis. aIBD active at time of thrombotic event.
Two of the patients with thrombosis had deficiencies in specific coagulation inhibitors. One patient had a functional protein S of 43% (normal, 65%–140%), with normal levels of protein C, antithrombin III, and plasminogen and a normal APCR. Another patient with a normal APCR was diagnosed with sepsis, venous thrombosis, and massive pulmonary embolism. She had clinical and laboratory evidence of severe liver disease with significant prolongations in her prothrombin time (23.6 seconds, ratio of 2.03) and activated partial thromboplastin time (67 seconds, ratio of 2.22). Coagulation studies also found low levels of antithrombin III (28%) and protein C (46%), probably resulting from decreased hepatic synthesis and consumption.
Discussion Thromboembolic complications are a rare but a significant complication of IBD. The reported incidence of thrombosis in three large retrospective studies was approximately 1.3%–6.4%.1,4,5 However, the contribution of thromboembolic complications to patient morbidity and mortality is significant. In the study by Talbot et al.,5 23 of 92 patients (25%) with documented thromboembolic complications died during their acute episodes. Therefore, a better understanding of the mechanisms contributing to the development of thrombosis in IBD could greatly reduce patient morbidity. There are growing experimental data confirming a relationship between IBD and increased intravascular thrombin generation.9–13 Activation of coagulation appears greatest during active IBD; this is consistent with the reported increased incidence of thrombosis occurring with exacerbations of active IBD.5 However, several studies have also shown the presence of increased plasma markers of activated coagulation during periods of clinical inactivity of IBD,9,13 suggesting an ongoing procoagulant state. These findings may be explained by the chronic production of the inflammatory cytokines reported to occur in both active and quiescent IBD.30,31 Cytokines such as tumor necrosis factor a and interleukin 1 have been shown to induce procoagulant changes in endothelial cells, platelets, and monocytes.32,33 Despite evidence of increased thrombin generation in a majority of patients with IBD, no study adequately explains why clinically significant thrombosis only occurs in a minority of patients. Several investigators have attempted to correlate the development of clinical thrombotic events with deficiencies of hemostatic inhibitory proteins such as antithrombin III, protein C, or protein S.13,34,35 Although some patients with IBD and an inherited deficiency of these
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proteins may present with a predisposition to recurrent clotting during acute exacerbations of their IBD,36 recent studies have shown that activation of coagulation in IBD patients seems not to be related to deficiencies in these proteins.13 Deficiencies of these anticoagulant proteins account for approximately 5%–9% of venous thrombosis cases in the general population37; therefore, it is not surprising that they are not a major contributory factor to the risk of clinical thrombosis in IBD. However, other newly characterized acquired and inherited prothrombotic defects such as hyperhomocysteinemia38,39 and the prothrombin G20210A mutation40,41 occur with greater frequency and could prove to be important cofactors in the development of thrombosis in IBD. APCR caused by factor V Leiden is now recognized as the most prevalent inherited cause of thrombophilia, accounting for 11%–20% of acute venous thrombotic events.17,18 The reported prevalence in populations of European descent is 2%–5%,15–18,42 and the mutation appears to be absent in native African, native American, and Asian populations.42 Inheritance of the factor V Leiden mutation seems to significantly increase the risk of thrombosis in patients with other procoagulant defects such as protein C deficiency,25 oral contraceptive use,22,23 or pregnancy.23 We therefore hypothesized that inheritance of factor V Leiden mutation maybe a major contributory factor in the development of thrombosis in IBD. In this retrospective study, we found the prevalence of factor V Leiden to be approximately 36% of patients with IBD and thromboembolic events. All thrombotic events reported in the IBD patients with factor V Leiden were of a venous origin, a finding consistent with previous studies that failed to show an association between factor V Leiden and arterial thrombotic events.18 Therefore, the prevalence of factor V Leiden in the patients with IBD and venous thrombosis is 50%. This represented a 23-fold increased risk of venous thrombosis in IBD patients who inherited this mutation, significantly greater than the 5-fold increased risk of venous thrombosis observed in our non-IBD subjects. All the patients with APCR reported in this study were heterozygotic for the factor V Leiden mutation. Therefore, the relative risk calculations made for this cohort are for the heterozygotic state only. It could be assumed that the risk of thrombosis for the rare IBD patients who are homozygotic for factor V Leiden is significantly greater, and we predict that the clinical manifestations of these patients would be dominated by thromboembolic events. Unanswered questions posed by this study relate to the treatment of IBD patients with and without a history of
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thromboembolic complications who have APCR. In patients who have experienced thrombotic events, evidence showing that even quiescent IBD may be associated with ongoing thrombin activation suggests that these patients should remain on long-term oral anticoagulation treatment. This recommendation is further supported by a recent study showing a significant increase in recurrent thrombotic events in patients with APCR.43 The more difficult question relates to the management of the patients with IBD who have no history of clinical thrombosis and who are found to have APCR. Despite an increased risk of thrombosis, there is insufficient evidence to suggest routine prophylatic anticoagulation. However, short-term anticoagulation during acute exacerbations would be justified, and recent studies on the use of minidose heparin in refractory ulcerative colitis suggest that this approach could be a safe method of management.44 In summary, we have found that inheritence of APCR due to the factor V Leiden mutation is a major contributory factor in the development of venous thrombosis in patients with IBD. Screening for this coagulation defect appears justified in all IBD patients of appropriate ethnic backgrounds who have a history of thrombosis or a family history of venous thromboembolic events.
References 1. Bargen JA, Barker NW. Extensive arterial and venous thrombosis complicating chronic ulcerative colitis. Arch Intern Med 1936;58: 17–31. 2. Ricketts WE, Palmer WL. Complications of chronic non-specific ulcerative colitis. Gastroenterology 1946;7:55–56. 3. Dennis C, Karlson KE. Surgical measures as supplements to the management of idiopathic ulcerative colitis: cancer, cirrhosis and arthritis as frequent complications. Surgery 1952;32:892–912. 4. Edwards FC, Truelove SC. The course and prognosis of ulcerative colitis. Part III. Complications. Gut 1964;5:1–15. 5. Talbot RW, Heppell J, Dozois RR, Beart RW. Vascular complications of inflammatory bowel disease. Mayo Clin Proc 1986;61: 140–145. 6. Warren S, Sommers SC. Pathogenesis of ulcerative colitis. Am J Pathol 1949;25:657–674. 7. Sloan WP Jr, Bargen JA, Gage RP. Life histories of patients with ulcerative colitis: a review of 2000 cases. Gastroenterology 1950;16:25–38. 8. Graef V, Baggenstoss AH, Sauer WG, Spittell JA Jr. Venous thrombosis occurring in non-specific ulcerative colitis. 1966;117: 377–382. 9. Edwards RL, Levine JB, Green R, Duffy M, Mathews E, Brande W, Rickles FR. Activation of blood coagulation in Crohn’s disease. Increased plasma fibrinopeptide A levels and enhanced generation of monocyte tissue factor activity. Gastroenterology 1987;92: 329–337. 10. Hudson M, Hutton RA, Wakefield AJ, Sawyer AM, Pounder RE. Evidence for activation of coagulation in Crohn’s disease. Blood Coag Fibrinol 1992;3:773–778. 12. Stevens TRJ, James JP, Simmonds NJ, McCarthy DA, Laurenson IF, Maddison PJ, Rampton DS. Circulating von Willebrand factor in inflammatory bowel disease. Gut 1992;33:502–506.
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13. Souto JC, Martinez E, Roca M, Mateo J, Pujol J, Gonzalez D, Fontcuberta J. Prothrombotic state and signs of endothelial lesion in plasma of patients with inflammatory bowel disease. Dig Dis Sci 1995;40:1883–1889. 14. Dahlback B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously recognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993;90: 1004–1008. 15. Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombosis Study. Lancet 1993;342:1503–1506. 16. Beauchamp NJ, Daly ME, Hampton KK, Cooper PC, Preston E, Peake IR. High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis. Br J Haematol 1994;88:219– 222. 17. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994;330:517–522. 18. Ridker PM, Hennekens CH, Lindpaintner K, Stampfer MJ, Eisenberg PR, Miletich JP. Mutation in the gene for coagulation factor V and the risk of myocardial infarction, stroke and venous thrombosis in apparently healthy men. N Engl J Med 1995;332:912–917. 19. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64–67. 20. Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH. Activated protein C resistance caused by Arg506Gln mutation in factor Va (letter). Lancet 1994;343:1361. 21. Voorberg J, Roelse J, Koopman R, Buller H, Berends F, ten Cate JW, Mertens K, van Mourik JA. Association of idiopathic venous thromboembolism with a single point-mutation at Arg506 of factor V. Lancet 1994;343:1535–1537. 22. Vandenbroucke JP, Koster T, Briet E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oralcontraceptive users who are carriers of the factor V Leiden mutation. Lancet 1994;1443–1457. 23. Heligren M, Svensson PI, Dahlback B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Obstet Gynecol 1995;173: 210–213. 24. Weitz IC, Israel VK, Liebman HA. Tamoxifen-associated venous thrombosis and activated protein C resistance due to factor V Leiden. Cancer 1997;79:2024–2027. 25. Gandrille S, Greengard JS, Alhenc-Gelas M, Juhan-Bague I, Abgrali JF, Jude B, Griffin JH, Alach M, and the French Network on the behalf of INSERM. Incidence of activated protein C resistance caused by the ARG 506 GLN mutation in factor V in 113 unrelated symptomatic protein C deficient patients. Blood 1995;86:219– 224. 26. Koeleman PC, van Rumpt D, Hamulyak K, Reitsma PH, Bertina RM. Factor V Leiden: an additional risk factor for thrombosis in protein S deficient families. Thromb Haemost 1995;74:580– 583. 27. Liebman HA, Sutherland D, Bacon R, McGehee W. Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance. Br J Haematol 1996;95:550–553. 28. Le DT, Griffin JH, Greengard JS, Mujumbar V, Rapaport SI. Use of a generally applicable tissue factor–dependent factor V assay to detect activated protein C–resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant. Blood 1995;85:1704–1711.
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29. Liebman HA, Carfagno MK, Weitz IC, Berard P, Diiorio JM, Vosburgh E, Simms RW. Excessive fibrinolysis in amyloidosis associated with elevated plasma single-chain urokinase. Am J Clin Pathol 1992;98:534–541. 30. Targen SR, Murphy LK. Clarifying the causes of Crohn’s. Nat Med 1995;1:1241–1243. 31. Nassif A, Longo WE, Mazuski JE, Vernova AM, Kaminski DL. Role of cytokines and platelet-activating factor in inflammatory bowel disease. Implications for therapy. Dis Colon Rectum 1996;39: 217–223. 32. Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Coltran RS, Gimbrone MA. Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci U S A 1986;83:4533–4537. 33. Nawroth PR, Stern DM. Modulation of endothelial hemostatic properties by tumor necrosis factor. J Exp Med 1986;163:740– 745. 34. Joren PG, Hermans CR, Haber I, Kocks MM, Vermylen J, Parizel GA. Acquired protein C and S deficiency, inflammatory bowel disease and cerebral thrombosis. Blut 1990;61:307–310. 35. Aadland E, Odegaard OR, Roseth A, Try K. Free protein S deficiency in patients with Crohn’s disease. Scand J Gastroenterol 1994;29:333–335. 36. Wyshock E, Caldwell M, Crowley JP. Deep venous thrombosis. Inflammatory bowel disease and protein S deficiency. Am J Clin Pathol 1988;90:633–635. 37. Heijboer H, Brandjes DPM, Buller HR, Sturk A, ten Cate JW. Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. N Engl J Med 1990;321: 1512–1516. 38. Den Heijer M, Koster T, Blom HJ, Bos GMJ, Briet E, Reitsma PH, Vandenbroucke JP, Rosendaal FR. Hyperhomocysteinemia as a risk factor for deep venous thrombosis. N Engl J Med 1996;334: 759–762. 39. D’Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997;90:1–11. 40. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 38-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698–3703. 41. Martinelli I, Sacchi E, Landi G, Taioli E, Duca F, Mannucci PM. High risk of cerebral-vein thrombosis in carriers of a prothrombin-gene mutation and in users of oral contraceptives. N Engl J Med 1998;338:1793–1797. 42. Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995;346:1133–1134. 43. Simioni P, Prandoni P, Lensing AWA, Scudeller A, Sardella C, Prins MH, Villalta S, Dazzi F, Girolami A. The risk of recurrent venous thromboembolism in patients with an Arg506-Gln mutation in the gene for factor V (factor V Leiden). N Engl J Med 1997;336:399– 403. 44. Gaffney PR, O’Leary JJ, Doyle CT, Gaffney A, Hogan J, Smew F, Annis P. Response to heparin in patients with ulcerative colitis (letter). Lancet 1991;337:239–240.
Received February 24, 1998. Accepted June 30, 1998. Address requests for reprints to: Howard A. Liebman, M.D., Department of Medicine and Pathology, University of Southern California School of Medicine, Kenneth Norris Jr. Cancer Hospital, Topping Tower, Third Floor, 1441 Eastlake Avenue, Los Angeles, California 90033. e-mail: [email protected]; fax: (213) 7640060. Supported in part by grant 987 F1-2 from the American Heart Association Greater Los Angeles Chapter (to H.A.L.).
The Factor V Leiden Mutation Increases the Risk of Venous Thrombosis in Patients With Inflammatory Bowel Disease HOWARD A. LIEBMAN,* NADER KASHANI,* DOUGLAS SUTHERLAND,* WILLIAM MCGEHEE,* AND LORI KAM‡ Divisions of *Hematology and ‡Gastroenterology, Department of Medicine, University of Southern California School of Medicine, Los Angeles, California
Background & Aims: Thromboembolic disease is a significant cause of morbidity and mortality in patients with inflammatory bowel disease (IBD). The aim of this study was to determine the incidence and possible association of the factor V Leiden mutation with the development of thrombosis in patients with IBD. Methods: This retrospective study included 11 patients with IBD and arterial or venous thrombosis and 51 patients with IBD and no history of thrombosis who were matched for age, sex, ethnic/racial origin, and type of IBD (controls). The presence of the factor V Leiden mutation was determined by coagulation assay and confirmed by a polymerase chain reaction method. Results: Four of 11 IBD patients (36%) with thrombosis and 2 of 51 IBD controls (4%) were heterozygotes for the factor V Leiden mutation (relative risk, 14.00; 95% confidence interval, 1.55–169.25; P 5 0.009, Fisher exact test). All thrombotic events in the patients with activated protein C resistance were venous with a calculated prevalence of 50% (4 of 8 patients) and a relative risk of venous thrombosis in IBD patients with factor V Leiden of 23 (95% confidence interval, 2–294; P 5 0.005). Conclusions: In patients with IBD, inheritance of the factor V Leiden mutation results in a significant increased risk of venous thrombosis.
hromboembolism is a well-described complication of inflammatory bowel disease (IBD) with a reported incidence of 1%–6%.1–5 Although clinically significant thrombosis occurs in only a fraction of patients with IBD, it contributes significantly to patient morbidity and mortality. Autopsy studies report a much higher incidence of thrombosis, ranging from 7% to 39%, which is often responsible for patient mortality.1,6–8 Thromboembolic complications are more frequently associated with clinically active disease,5 but laboratory studies using sensitive markers of activation of the coagulation system have documented endothelial injury and increased thrombin generation in patients with both active and quiescent IBD.9–13 With this growing evidence that most patients with IBD have active thrombin generation, the question
T
emerges as to what other factors contribute to the development of clinically overt thrombosis in patients with IBD. In 1993, Dahlback et al.14 were the first to recognize a new mechanism for familial thrombophilia. This disorder is characterized by in vitro resistance to the anticoagulant effects of activated protein C (APC). Subsequent population studies have found APC resistance (APCR) to be the most prevalent cause of inherited thrombophilia with a reported prevalence of 2%–5% in populations of European descent.15–18 The molecular defect responsible for APCR in a majority of patients has been identified and has been shown to result from a mutation in the factor V molecule (R506Q, factor V Leiden), resulting in the loss of an APC cleavage site necessary for rapid inactivation of factor Va.19–21 Recent studies show that inheritance of the factor V Leiden mutation potentiates the thrombotic risk in subjects with other acquired22–24 or congenital25,26 prothrombotic states. We hypothesized that the factor V Leiden defect would be one inherited prothrombotic risk factor that could significantly increase the risk of thrombosis in patients with IBD. Therefore, we screened 11 patients with a history of IBD and thrombosis for the factor V Leiden mutation and compared the prevalence of this mutation in them with that of 51 patients with IBD and no history of thrombosis. We show that APCR caused by factor V Leiden is a major contributory factor to the development of venous thrombosis in patients with IBD and the combined risk of venous thrombosis in patients with IBD, and that this mutation is a significantly greater risk factor than factor V Leiden mutation alone in causing thrombosis. Abbreviations used in this paper: APC, activated protein C; APCR, APC resistance; LAC-USC, Los Angeles County–University of Southern California (Medical Center). r 1998 by the American Gastroenterological Association 0016-5085/98/$3.00
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Materials and Methods The study population included 11 patients with a history of IBD and thromboembolic complications and 51 IBD controls. A review of the records from Los Angeles County– University of Southern California (LAC-USC) Medical Center Anticoagulation Clinic, the IBD clinic, and hematology consultation records identified 14 patients with IBD and a history of either arterial or venous thrombosis. Only 11 of these patients were available to perform studies for APCR and factor V Leiden. A control population of 51 patients with IBD without a history of thrombosis were selected to be matched to the patients with thrombosis for age, sex, ethnic/racial origin, and type of IBD (ulcerative colitis or Crohn’s disease). The prevalence of the factor V Leiden mutation in the IBD patients with and without thrombosis was compared with that of a group of 81 patients with a history of idiopathic venous thrombosis and 142 racially matched control subjects without a history of venous or arterial thrombosis. Many of the patients with thrombosis, with and without a history of IBD, have been described previously.27 Patients were seen at LAC-USC Medical Center, the Kenneth Norris, Jr–University of Southern California Cancer Center, or at USC University Hospital. Patients with a history of IBD and deep venous thrombosis had documentation of venous thrombosis by ultrasonography. In patients with a diagnosis of pulmonary embolism, the diagnosis was based on appropriate clinical and laboratory findings combined with a high-probability ventilation-perfusion lung scan. Arterial thrombotic events, including stroke and myocardial infarction, were documented by generally acceptable diagnostic criteria including computerized tomography and angiography. Blood samples were collected in 3.2% sodium citrate, and platelet-poor plasma was prepared immediately by centrifugation at 10,000 rpm at 4°C for 10 minutes. Plasma samples were stored at 220°C until tested. All plasma samples were assayed within 1 week of collection. Plasma samples were assayed for the presence of factor V Leiden using the tissue factor– dependent factor V assay, as originally described by Le et al.28 and reported by our laboratory.27 This coagulation assay was demonstrated to have 100% sensitivity and specificity for the factor V Leiden defect in a blinded study compared with a polymerase chain reaction–based assay for the molecular defect of factor V Leiden.27 All patients in whom the factor V Leiden defect was detected were further studied using the polymerase chain reaction assay.27 Genomic DNA was isolated from 200 µL of the cellular pellet, which remained after removal of plasma for coagulation studies, using the QAI amp Blood Kit (Qiagen, Chatsworth, CA). In 8 IBD patients without thrombosis, only the molecular analysis for factor V Leiden was performed because fresh plasma was not available for coagulation studies. All patients with IBD and a history of thrombosis were also evaluated with functional assays for antithrombin III, protein C, protein S, plasminogen, the Exner test for the lupus anticoagulant, and anticardiolipin antibody assays using com-
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mercial kits (Protein C, Proclot Protein C; ACL Instrument Laboratories, Lexington, MA; and Staclot Protein S, Diagnostica Stago, Asieres-sur-Seine, France) and according to methods reported previously.29
Results The characteristics of the study populations are shown in Table 1. There were no significant differences in age or sex between the IBD patients with thrombosis and patients without thrombosis. The average age of the patients with IBD at the time of their thrombotic event was 39 years (range, 21–64 years), and the patients were studied an average of 2.5 months (range, 4 days to 36 months) after their thromboembolic event. Ulcerative colitis was the most frequent form of IBD in patients with thrombosis, a finding in agreement with most published studies of thrombosis and IBD.5 Despite our efforts to match the control IBD population with the patients with a history of thrombosis, there was a greater number of IBD patients of Latino origin in the control population (36% vs. 53%); however, this difference was not statistically different (P 5 0.48). This racial imbalance reflects the dominant ethnic background of the patients seen in the IBD clinic at LAC-USC Medical Center. Previous studies in our patient population have found a prevalence of factor V Leiden of 2.9% (2 of 69) in Latino subjects without a personal or family history of thrombosis (Table 1). We compared our IBD patients with and without thrombosis with 81 patients who had a history of idiopathic venous thrombosis and 142 control subjects without a history of IBD or thrombosis. The non-IBD thrombotic patients tended to be older than the IBD patients with thrombosis; this difference was statistically significant (P , 0.05). Table 1. Characteristics of the Study Population IBD Thrombosis (n 5 11)
Non-IBD Controls (n 5 51)
Age, yr (mean, range) 39 (21–64) 44 (12–73) Sex (M/F) 8/3 35/16 UC/CD 7/4 31/20 APCR1 (factor V Leiden) 4 2 Racial background (%) European, non-Latino 6 (55) 21 (41) Latinoa 4 (36) 27 (53) African American 1 (9) 2 (4) Asian 0 (0) 1 (2)
Thrombosis (n 5 81)
Controls (n 5 142)
54 (28–74) 51 (26–84) 47/34 82/60 — — 15
6
39 (48) 34 (42)
60 (42) 69 (49)
7 (9) 1 (1)
11 (8) 1 (1)
UC, ulcerative colitis; CD, Crohn’s disease. individuals with family origins from Central or South America.
aLatino:
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Four of the 11 patients (36%) with a history of thromboembolic disease had APCR (Tables 1 and 2). All 4 were subsequently determined to be heterozygotes for factor V Leiden. Only 2 of the 51 (4%) patients with IBD and no history of thromboembolic disease had APCR and were heterozygotes for factor V Leiden. This difference was highly significant (P 5 0.009, Fisher exact test), despite the small number of patients studied. Therefore, the risk of thrombosis in patients with IBD who are carriers of the factor V Leiden mutation compared with noncarriers is increased 14-fold (relative risk, 14.00; 95% Cl, 1.55–169.25). The presence of the factor V Leiden mutation was only associated with venous thromboembolism. Therefore, 4 of the 8 patients (50%) with a history of venous thrombosis had the factor V Leiden mutation, representing a relative risk of venous thrombosis in IBD patients with factor V Leiden of 23 (95% CI, 2–294; P 5 0.005). In comparison, 15 of our 81 (19%) non-IBD patients with a history of thrombosis had APCR due to factor V Leiden (2 homozygotes and 13 heterozygotes). In the ethnic and racially matched controls without a history of IBD or thrombosis, 6 of the 142 subjects were heterozygotes for factor V Leiden. Therefore, in the non-IBD patients, the risk of thrombosis in carriers of the factor V Leiden mutation is increased 5-fold (relative risk, 5.15; 95% confidence interval, 1.77–16.84). These findings strongly support the hypothesis that IBD significantly amplifies the thrombotic risk associated with the factor V Leiden mutation. We found no other clinical manifestations that distinguished patients with APCR and thrombosis from the patients with thrombosis who were APCR negative. However, it is interesting that the only patient with clinically quiescent IBD at the time of his venous thrombosis had the factor V Leiden (Table 2). Table 2. Characteristics of Patients With IBD and Thrombosis
Age, yr (mean, range) Sex (M/F) UC/CD Activity (1) a Thrombotic event DVT PE MI Stroke Other arterial
APCR1 (n 5 4)
APCR2 (n 5 7)
41 (27–64) 3/1 2/2 3/4
38 (21–56) 5/2 5/2 7/7
3 1 — — —
2 2 1 1 1
UC, ulcerative colitis; CD, Crohn’s disease; DVT, includes both lower and upper extremity venous throbmosis; PE, all patients in this study with pulmonary embolus had documented lower extremity venous thrombosis. aIBD active at time of thrombotic event.
Two of the patients with thrombosis had deficiencies in specific coagulation inhibitors. One patient had a functional protein S of 43% (normal, 65%–140%), with normal levels of protein C, antithrombin III, and plasminogen and a normal APCR. Another patient with a normal APCR was diagnosed with sepsis, venous thrombosis, and massive pulmonary embolism. She had clinical and laboratory evidence of severe liver disease with significant prolongations in her prothrombin time (23.6 seconds, ratio of 2.03) and activated partial thromboplastin time (67 seconds, ratio of 2.22). Coagulation studies also found low levels of antithrombin III (28%) and protein C (46%), probably resulting from decreased hepatic synthesis and consumption.
Discussion Thromboembolic complications are a rare but a significant complication of IBD. The reported incidence of thrombosis in three large retrospective studies was approximately 1.3%–6.4%.1,4,5 However, the contribution of thromboembolic complications to patient morbidity and mortality is significant. In the study by Talbot et al.,5 23 of 92 patients (25%) with documented thromboembolic complications died during their acute episodes. Therefore, a better understanding of the mechanisms contributing to the development of thrombosis in IBD could greatly reduce patient morbidity. There are growing experimental data confirming a relationship between IBD and increased intravascular thrombin generation.9–13 Activation of coagulation appears greatest during active IBD; this is consistent with the reported increased incidence of thrombosis occurring with exacerbations of active IBD.5 However, several studies have also shown the presence of increased plasma markers of activated coagulation during periods of clinical inactivity of IBD,9,13 suggesting an ongoing procoagulant state. These findings may be explained by the chronic production of the inflammatory cytokines reported to occur in both active and quiescent IBD.30,31 Cytokines such as tumor necrosis factor a and interleukin 1 have been shown to induce procoagulant changes in endothelial cells, platelets, and monocytes.32,33 Despite evidence of increased thrombin generation in a majority of patients with IBD, no study adequately explains why clinically significant thrombosis only occurs in a minority of patients. Several investigators have attempted to correlate the development of clinical thrombotic events with deficiencies of hemostatic inhibitory proteins such as antithrombin III, protein C, or protein S.13,34,35 Although some patients with IBD and an inherited deficiency of these
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proteins may present with a predisposition to recurrent clotting during acute exacerbations of their IBD,36 recent studies have shown that activation of coagulation in IBD patients seems not to be related to deficiencies in these proteins.13 Deficiencies of these anticoagulant proteins account for approximately 5%–9% of venous thrombosis cases in the general population37; therefore, it is not surprising that they are not a major contributory factor to the risk of clinical thrombosis in IBD. However, other newly characterized acquired and inherited prothrombotic defects such as hyperhomocysteinemia38,39 and the prothrombin G20210A mutation40,41 occur with greater frequency and could prove to be important cofactors in the development of thrombosis in IBD. APCR caused by factor V Leiden is now recognized as the most prevalent inherited cause of thrombophilia, accounting for 11%–20% of acute venous thrombotic events.17,18 The reported prevalence in populations of European descent is 2%–5%,15–18,42 and the mutation appears to be absent in native African, native American, and Asian populations.42 Inheritance of the factor V Leiden mutation seems to significantly increase the risk of thrombosis in patients with other procoagulant defects such as protein C deficiency,25 oral contraceptive use,22,23 or pregnancy.23 We therefore hypothesized that inheritance of factor V Leiden mutation maybe a major contributory factor in the development of thrombosis in IBD. In this retrospective study, we found the prevalence of factor V Leiden to be approximately 36% of patients with IBD and thromboembolic events. All thrombotic events reported in the IBD patients with factor V Leiden were of a venous origin, a finding consistent with previous studies that failed to show an association between factor V Leiden and arterial thrombotic events.18 Therefore, the prevalence of factor V Leiden in the patients with IBD and venous thrombosis is 50%. This represented a 23-fold increased risk of venous thrombosis in IBD patients who inherited this mutation, significantly greater than the 5-fold increased risk of venous thrombosis observed in our non-IBD subjects. All the patients with APCR reported in this study were heterozygotic for the factor V Leiden mutation. Therefore, the relative risk calculations made for this cohort are for the heterozygotic state only. It could be assumed that the risk of thrombosis for the rare IBD patients who are homozygotic for factor V Leiden is significantly greater, and we predict that the clinical manifestations of these patients would be dominated by thromboembolic events. Unanswered questions posed by this study relate to the treatment of IBD patients with and without a history of
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thromboembolic complications who have APCR. In patients who have experienced thrombotic events, evidence showing that even quiescent IBD may be associated with ongoing thrombin activation suggests that these patients should remain on long-term oral anticoagulation treatment. This recommendation is further supported by a recent study showing a significant increase in recurrent thrombotic events in patients with APCR.43 The more difficult question relates to the management of the patients with IBD who have no history of clinical thrombosis and who are found to have APCR. Despite an increased risk of thrombosis, there is insufficient evidence to suggest routine prophylatic anticoagulation. However, short-term anticoagulation during acute exacerbations would be justified, and recent studies on the use of minidose heparin in refractory ulcerative colitis suggest that this approach could be a safe method of management.44 In summary, we have found that inheritence of APCR due to the factor V Leiden mutation is a major contributory factor in the development of venous thrombosis in patients with IBD. Screening for this coagulation defect appears justified in all IBD patients of appropriate ethnic backgrounds who have a history of thrombosis or a family history of venous thromboembolic events.
References 1. Bargen JA, Barker NW. Extensive arterial and venous thrombosis complicating chronic ulcerative colitis. Arch Intern Med 1936;58: 17–31. 2. Ricketts WE, Palmer WL. Complications of chronic non-specific ulcerative colitis. Gastroenterology 1946;7:55–56. 3. Dennis C, Karlson KE. Surgical measures as supplements to the management of idiopathic ulcerative colitis: cancer, cirrhosis and arthritis as frequent complications. Surgery 1952;32:892–912. 4. Edwards FC, Truelove SC. The course and prognosis of ulcerative colitis. Part III. Complications. Gut 1964;5:1–15. 5. Talbot RW, Heppell J, Dozois RR, Beart RW. Vascular complications of inflammatory bowel disease. Mayo Clin Proc 1986;61: 140–145. 6. Warren S, Sommers SC. Pathogenesis of ulcerative colitis. Am J Pathol 1949;25:657–674. 7. Sloan WP Jr, Bargen JA, Gage RP. Life histories of patients with ulcerative colitis: a review of 2000 cases. Gastroenterology 1950;16:25–38. 8. Graef V, Baggenstoss AH, Sauer WG, Spittell JA Jr. Venous thrombosis occurring in non-specific ulcerative colitis. 1966;117: 377–382. 9. Edwards RL, Levine JB, Green R, Duffy M, Mathews E, Brande W, Rickles FR. Activation of blood coagulation in Crohn’s disease. Increased plasma fibrinopeptide A levels and enhanced generation of monocyte tissue factor activity. Gastroenterology 1987;92: 329–337. 10. Hudson M, Hutton RA, Wakefield AJ, Sawyer AM, Pounder RE. Evidence for activation of coagulation in Crohn’s disease. Blood Coag Fibrinol 1992;3:773–778. 12. Stevens TRJ, James JP, Simmonds NJ, McCarthy DA, Laurenson IF, Maddison PJ, Rampton DS. Circulating von Willebrand factor in inflammatory bowel disease. Gut 1992;33:502–506.
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13. Souto JC, Martinez E, Roca M, Mateo J, Pujol J, Gonzalez D, Fontcuberta J. Prothrombotic state and signs of endothelial lesion in plasma of patients with inflammatory bowel disease. Dig Dis Sci 1995;40:1883–1889. 14. Dahlback B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously recognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993;90: 1004–1008. 15. Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombosis Study. Lancet 1993;342:1503–1506. 16. Beauchamp NJ, Daly ME, Hampton KK, Cooper PC, Preston E, Peake IR. High prevalence of a mutation in the factor V gene within the U.K. population: relationship to activated protein C resistance and familial thrombosis. Br J Haematol 1994;88:219– 222. 17. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994;330:517–522. 18. Ridker PM, Hennekens CH, Lindpaintner K, Stampfer MJ, Eisenberg PR, Miletich JP. Mutation in the gene for coagulation factor V and the risk of myocardial infarction, stroke and venous thrombosis in apparently healthy men. N Engl J Med 1995;332:912–917. 19. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64–67. 20. Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH. Activated protein C resistance caused by Arg506Gln mutation in factor Va (letter). Lancet 1994;343:1361. 21. Voorberg J, Roelse J, Koopman R, Buller H, Berends F, ten Cate JW, Mertens K, van Mourik JA. Association of idiopathic venous thromboembolism with a single point-mutation at Arg506 of factor V. Lancet 1994;343:1535–1537. 22. Vandenbroucke JP, Koster T, Briet E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oralcontraceptive users who are carriers of the factor V Leiden mutation. Lancet 1994;1443–1457. 23. Heligren M, Svensson PI, Dahlback B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Obstet Gynecol 1995;173: 210–213. 24. Weitz IC, Israel VK, Liebman HA. Tamoxifen-associated venous thrombosis and activated protein C resistance due to factor V Leiden. Cancer 1997;79:2024–2027. 25. Gandrille S, Greengard JS, Alhenc-Gelas M, Juhan-Bague I, Abgrali JF, Jude B, Griffin JH, Alach M, and the French Network on the behalf of INSERM. Incidence of activated protein C resistance caused by the ARG 506 GLN mutation in factor V in 113 unrelated symptomatic protein C deficient patients. Blood 1995;86:219– 224. 26. Koeleman PC, van Rumpt D, Hamulyak K, Reitsma PH, Bertina RM. Factor V Leiden: an additional risk factor for thrombosis in protein S deficient families. Thromb Haemost 1995;74:580– 583. 27. Liebman HA, Sutherland D, Bacon R, McGehee W. Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance. Br J Haematol 1996;95:550–553. 28. Le DT, Griffin JH, Greengard JS, Mujumbar V, Rapaport SI. Use of a generally applicable tissue factor–dependent factor V assay to detect activated protein C–resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant. Blood 1995;85:1704–1711.
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29. Liebman HA, Carfagno MK, Weitz IC, Berard P, Diiorio JM, Vosburgh E, Simms RW. Excessive fibrinolysis in amyloidosis associated with elevated plasma single-chain urokinase. Am J Clin Pathol 1992;98:534–541. 30. Targen SR, Murphy LK. Clarifying the causes of Crohn’s. Nat Med 1995;1:1241–1243. 31. Nassif A, Longo WE, Mazuski JE, Vernova AM, Kaminski DL. Role of cytokines and platelet-activating factor in inflammatory bowel disease. Implications for therapy. Dis Colon Rectum 1996;39: 217–223. 32. Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Coltran RS, Gimbrone MA. Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci U S A 1986;83:4533–4537. 33. Nawroth PR, Stern DM. Modulation of endothelial hemostatic properties by tumor necrosis factor. J Exp Med 1986;163:740– 745. 34. Joren PG, Hermans CR, Haber I, Kocks MM, Vermylen J, Parizel GA. Acquired protein C and S deficiency, inflammatory bowel disease and cerebral thrombosis. Blut 1990;61:307–310. 35. Aadland E, Odegaard OR, Roseth A, Try K. Free protein S deficiency in patients with Crohn’s disease. Scand J Gastroenterol 1994;29:333–335. 36. Wyshock E, Caldwell M, Crowley JP. Deep venous thrombosis. Inflammatory bowel disease and protein S deficiency. Am J Clin Pathol 1988;90:633–635. 37. Heijboer H, Brandjes DPM, Buller HR, Sturk A, ten Cate JW. Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. N Engl J Med 1990;321: 1512–1516. 38. Den Heijer M, Koster T, Blom HJ, Bos GMJ, Briet E, Reitsma PH, Vandenbroucke JP, Rosendaal FR. Hyperhomocysteinemia as a risk factor for deep venous thrombosis. N Engl J Med 1996;334: 759–762. 39. D’Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997;90:1–11. 40. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 38-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698–3703. 41. Martinelli I, Sacchi E, Landi G, Taioli E, Duca F, Mannucci PM. High risk of cerebral-vein thrombosis in carriers of a prothrombin-gene mutation and in users of oral contraceptives. N Engl J Med 1998;338:1793–1797. 42. Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995;346:1133–1134. 43. Simioni P, Prandoni P, Lensing AWA, Scudeller A, Sardella C, Prins MH, Villalta S, Dazzi F, Girolami A. The risk of recurrent venous thromboembolism in patients with an Arg506-Gln mutation in the gene for factor V (factor V Leiden). N Engl J Med 1997;336:399– 403. 44. Gaffney PR, O’Leary JJ, Doyle CT, Gaffney A, Hogan J, Smew F, Annis P. Response to heparin in patients with ulcerative colitis (letter). Lancet 1991;337:239–240.
Received February 24, 1998. Accepted June 30, 1998. Address requests for reprints to: Howard A. Liebman, M.D., Department of Medicine and Pathology, University of Southern California School of Medicine, Kenneth Norris Jr. Cancer Hospital, Topping Tower, Third Floor, 1441 Eastlake Avenue, Los Angeles, California 90033. e-mail: [email protected]; fax: (213) 7640060. Supported in part by grant 987 F1-2 from the American Heart Association Greater Los Angeles Chapter (to H.A.L.).