The etiology, diagnosis and treatment of venous thromboembolism Kraaijenhagen, R.A.

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1 UvA-DARE (Digital Academic Repository) The etiology, diagnosis and treatment of venous thromboembolism Kraaijenhagen, R.A. Link to publication Citation for published version (APA): Kraaijenhagen, R. A. (2000). The etiology, diagnosis and treatment of venous thromboembolism. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 27 Mar 2019

2 Chapter II High plasma concentration of factor VIIIc is a major risk factor for venous thromboembolism. Roderik A. Kraaijenhagen 1, Pieternella S. in 't Anker 1, Marianne M.W. Koopman 1, Pieter H. Reitsma 2, Martin H. Prins 3, Abraham van den Ende 4 and Harry R. Biiller 1 Department of Vascular Medicine 1, Laboratory of Experimental Internal Medicine 2, Department of Clinical Epidemiology and Bio statistics 3, Laboratory of Vascular Medicine 4 Academic Medical Center, Amsterdam, The Netherlands. Thrombosis and Haemostasis, 83, In press. -9-

3 CHAPTER II Abstract Background Established risk factors, including deficiencies of protein C, protein S or antithrombin and the factor V Leiden and prothrombin mutation, are present in about one third of unselected patients with venous thromboembolism. In addition to these inherited thrombophilic defects, elevated plasma levels of factor VIIIc have been suggested to be important in the pathogenesis of (recurrent) venous thromboembolism. The objective of this study was to assess the relevance of factor VIIIc plasma concentration in consecutive patients with venous thromboembolism. Methods We studied the prevalence of elevated plasma levels of factor VIIIc in 65 patients with a proven single episode and in 60 matched patients with documented recurrent venous thromboembolism. The reference group consisted of 60 age- and sexmatched patients who were referred for suspected venous thromboembolism, which was refuted by objective testing and longterm clinical follow-up. To minimalize the influence of the acute phase, blood was obtained at least 6 months after the thromboembolic event and results were adjusted for fibrinogen and C-reactive protein. Factor VIIIc was re-determined several years after the first measurement in a subset of patients to evaluate the variability over time. To study a possible genetic cause, a family study was done. Results In the control, single and recurrent episode group, the prevalences of plasma levels of factor VIIIc above 175 IU/dl (90 th percentile of controls) were 10% (95% CI: 4 to 21%), 19% (95 % CI: 10 to 30%) and 33% (95% CI: 22 to 47%), respectively. For each 10 IU/dl increment of factor VIIIc, the risk for a single and recurrent episode of venous thrombosis increased by 10% (95% CI: 0.9 to 21%) and 24% (95% CI: 11 to 38%), respectively. Both low and high plasma levels of factor VIIIc were consistent over time (R = 0.80, p=0.01). A family study indicated a high concordance for elevated factor VIIIc plasma concentrations among first degree family members. Adjustment for fibrinogen, C-reactive protein and known thrombophilic risk factors did not change the observed association of elevated factor VIIIc with thromboembolism. Conclusion Elevated plasma levels of factor VIIIc are a significant, prevalent, independent and -10-

4 High plasma concentration offactor VIIIc is a major risk factor for venous thromboembolism. dose-dependent risk factor for venous thromboembolism. It also predisposes to recurrent venous thromboembolism. Introduction Venous thromboembolism is a common disorder, with an annual incidence in the general population of 2-3 per 1000 inhabitants. 1 The pathogenesis of venous thrombosis can still be explained by the triad of Virchow, postulated in 1856, i.e. changes in the vessel wall, blood stream or blood composition. Although the precise pathogenic mechanisms induced by changes in the blood stream and vessel wall have only been partly elucidated, much progress has been made in understanding alterations in blood composition, associated with a tendency to venous thromboembolism. Isolated deficiencies of proteins involved in the inhibition of coagulation have been described that result in a continuous modest hypercoagulable state, that may result in (recurrent) venous thromboembolism, either spontaneously or following minor trauma. In consecutive patients with documented venous thromboembolism, the prevalence of these protein abnormalities, including deficiencies of antithrombin, protein C, protein S, factor V Leiden or the prothrombin mutation, and hyperhomocysteinemia adds up to approximately 40%. 27 Recently, another thrombophilic factor has been described i.e. an increased plasma concentration of factor VIIIc. 8 The impact on the pathogenesis of venous thrombosis of this new risk factor has only in part been established. Studies have shown that an elevated level of factor VIII procoagulant activity (factor VIIIc) is likely an independent, relatively prevalent risk factor for venous thrombosis What remains unclear however, is whether there is a critical cut-off value of factor VIIIc plasma concentration or that there exists a dose-dependent relationship with the risk of thrombosis. Furthermore, the cause of an elevated factor VIIIc, a known acute phase protein, is not understood. Although some of the published studies analysed factor VIIIc several months after the occurrence of deep venous thrombosis, the potential for long-term or chronic acute phase reaction could not be ruled out. 4,8 " 10 Finally, it is unclear whether an increased factor VIIIc concentration is familial. The aim of the present study was to compare the prevalences of elevated plasma levels of factor VIIIc in consecutive patients with a single episode of venous thrombosis and/or pulmonary embolism, as opposed to a patient control group. In addition, we investigated whether this abnormality is associated with a higher risk for recurrent venous thromboembolism and whether a dose-dependent relationship can -11-

5 CHAPTER II be demonstrated. In a subset of patients the factor VIIIc determination was repeated several years after the first measurement to evaluate consistency of factor VIIIc levels over time. Furthermore, to evaluate a possible genetic cause of elevated factor VIIIc plasma levels a family study was done. Methods Patients and Study Design Patients and controls were consecutively identified from the records of patients referred to the thrombosis unit of the Academic Medical Center, Amsterdam, The Netherlands because of clinically suspected venous thromboembolism. This unit is a diagnostic service for patients seen in primary or secondary care. Deep venous thrombosis was diagnosed by either compression ultrasonography and/or contrast venography, whereas pulmonary embolism was confirmed by highprobability ventilation-perfusion lung scanning and/or pulmonary angiography Controls were symptomatic subjects referred to the unit, but who were shown not to have venous thromboembolism upon (serial) objective investigations and who had an uneventful 3 month clinical follow-up. Patients and controls with known thrombophilic abnormalities, such as antithrombin, protein C, protein S deficiency as well as those on full dose warfarin were excluded. Patients with more recently established thrombophilic abnormalities; i.e. hyperhomocysteinemia, factor V Leiden mutation and the prothrombin mutation were not excluded, but we adjusted the results of factor VIIIc levels for these thrombophilic abnormalities. Sixty consecutive patients were identified with documented recurrent venous thromboembolism. These patients were matched for age and sex with 65 patients who experienced a single episode of thrombosis and/or pulmonary embolism. A total of 60 age and sex matched subjects without venous thromboembolism referred in the same time period served as the reference group. Also, the time period between the two thrombotic events in the recurrent group was matched with the time elapsed since the thrombotic episode in the single event group and since referral in the reference group. After written informed consent a standardised medical history was obtained and blood samples were taken. The time which elapsed between the acute phase and the blood sampling was at least 6 months. In a randomly selected subset of 28 subjects a new blood sample was obtained after a median of 50 months (range months) to assess the consistency of high and low plasma levels of factor VIIIc. In all patients -12-

6 High plasma concentration offactor VIIIc is a major risk factor for venous thromboembolism. with a factor VIIIc level above 200 IU/dl and a history of venous thromboembolism, the number of first degree living relatives was ascertained. The largest family was selected to evaluate a potential inheritance of high factor VIIIc plasma levels. The plasma concentration level of factor VIIIc was determined in all first degree relatives of this family. The laboratory studies were done independently and without knowledge of the presence or absence of venous thromboembolism. The study protocol was approved by the Institutional Review Board. Laboratory Studies Blood for factor VIIIc determination was collected in tubes containing mmol/l trisodium citrate. Plasma was obtained by centrifugation for 15 minutes at 1520 g, followed by centrifugation for 5 minutes at g, both at room temperature. Factor VIIIc was measured by a one-stage clotting assay with factor-vlll-deficient plasma (JTC Baker, Deventer, The Netherlands) and automated activated partial thromboplastin time (APTT; Actin FS, Dade inc., Aguada, USA). All measurements were performed in duplicate. The final result was considered to be the mean of these measurements. Pooled normal plasma, from one hundred healthy volunteers, was used as a reference. The median value for factor VIIIc was 100 IU/dl (normal range: IU/dl). As upper limit for abnormal values the 90 th percentile in the patient control group was used for comparison. The factor VIIIc plasma concentration found in the study subjects was divided in five categories, i.e. < 100 IU/dl, IU/dl, IU/dl, IU/dl and >200 IU/dl. Blood for determination of fibrinogen was collected in tubes containing molair citrate (1:9). Fibrinogen (in g/1) was determinated by Clauss' method. 13 All measurements were performed in duplicate. The normal range was g/1. Blood for determination of C-Reactive Protein (CRP) was collected in tubes containing mmol/l trisodium citrate. CRP was determined by CRP antigen-antibody reaction (Beckman).Values above 5.0 (lg/1 are considered to be abnormal. The factor V Leiden mutation, the prothrombin mutation and fasting homocysteine plasma levels were measured by standardised assays. 5 > 14,15 Statistical analysis The values of factor VIIIc plasma concentration were categorised and the distribution for both patients and controls was determined. We calculated the unadjusted odds ratios with 95% confidence intervals (95% CI) for the different categories of factor VIIIc concentration compared to the median value of the normal distribution -13-

7 CHAPTER II i.e. for factor VIIIc 100 IU/dl. In addition, we adjusted the factor VIIIc odds ratios for categorised fibrinogen, CRP and homocysteine levels as well as for the factor V Leiden and the prothrombin mutation using the Mantel Haenszel procedure. Pearson's correlation coefficient was calculated to describe the relationship between the first measurement of factor VIIIc plasma concentration and the re-determination several years later. Furthermore, a potential continuous relation was evaluated between factor VIIIc plasma concentration and venous thromboembolism using a logistic regression analysis. Supplementary, we adjusted also these data for categorised fibrinogen, CRP and homocysteine levels as well as for the factor V Leiden and the prothrombin mutation. Results A total of 185 patients were enrolled in the three age-and sex-matched study groups. Sixty patients had no venous thromboembolism, 65 patients experienced a single episode of thrombosis or pulmonary embolism, while the remaining 60 patients had recurrent venous thromboembolism. The clinical characteristics of the three study groups are described in Table 1. The mean age was approximately 55 years and the majority of patients had idiopathic deep vein thrombosis. The median time between the first and recurrent episode of venous thromboembolism was 2.3 years in the recurrent group and the duration of observation in the other two groups was comparable. The prevalence in each of the five factor VIIIc categories in the group of patients with a single and recurrent venous thromboembolism and in the controls are shown in Table 2. Concentrations above 175 IU/dl were observed in 10%, 19% and 33% in the control, single episode and recurrent episode groups, respectively. The odds ratios for each of the various categories of factor VIIIc, compared with the level of factor VIIIc below 100 IU/dl for the group of patients with a single or recurrent episode of venous thromboembolism are shown in Table 3. Compared to controls the odds ratio for venous thromboembolism for factor VIIIc concentrations above 200 IU/dl was as high as 11 (95 % CI: 2-71) for a single episode, and 45 (95% CI: 6-370) for a recurrent episode of venous thromboembolism. Adjustment for plasma levels of fibrinogen, CRP or fasting homocysteine concentrations as well as for the presence of factor V Leiden or the prothrombin mutation did not change these odds ratios (data not shown). For each 10 IU/dl increase of factor VIIIc, the risk for venous thromboembolism in all patients increased by approximately 17% (odds ratio 1.17; 95% CI ). The -14-

8 High plasma concentration offactor VIIIc is a major risk factor for venous thromboembolism. Table 1 Clinical characteristics of patients with a single episode of venous thromboembolism, a recurrent episode of venous thromboembolism and controls. Characteristics patients, n male sex (% of patients) mean age (+/- SD), y type of venous thromboembolisrr deep venous thrombosis, % pulmonary embolism, % Time elapsed since first symptomatic presentation, median (range), y no venous single episode recurrent episode thromboembolism of venous of venous thromboembolism thromboembolism (+/- 17) 2.7( ) (+/-16) (0.6-10) (+/-15) ( Table 2 Number of patients (prevalence) in the various factor VIIIc plasma concentration (in lu/dl) categories in controls and those with a single episode or a recurrent episode of venous thromboembolism. factor VIIIc (in lu/dl) control population, n (%) patients with a single episode of venous thromboembolism, n (%) patients with recurrent venous thromboembolism, n (%) <100 18(30.0) 5 (7.7) 2 (3.3) (46.7) 39 (60.0) 24 (40.0) (13.3) 9 (13.9) 14(23.3) (6.7) 6 (9.2) 10(16.7) >200 2 (3.3) 6 (9.2) 10(16.7) Table 3 Odds ratios (95 percent confidence interval (CI)) for four categories of an elevated factor VIIIc plasma concentration compared to a factor VIIIc plasma concentration below 100 IU/dl in patients with a single or recurrent episode of venous thromboembolism. factor VIIIc (in IU/dl) Odds ratio for patients with a single episode of venous thromboembolism (95% CI) Odds ratio for patients with recurrent venous thromboembolism (95% CI) (2-15) 6.4 (1-30) (1-16) 15.8 (3-86) (1-26) 22.5 (3-145) > (2-71) 45.0 (6-370) -15-

9 CHAPTER II risk for a single episode increased by 10% (odds ratio 1.10; 95% CI ), while the risk for recurrence increased by 24% (odds ratio 1.24; 95% CI ). In a random sample of 28 subjects (13 patients with recurrent venous thromboembolism; 8 patients with single episode of venous thromboembolism and 7 controls) the determination of factor VIIIc concentration was repeated several years after the first determination. The correlation coefficient between the first and second measurement was 0.80 (p= 0.01) (Figure 1). A family with 8 first degree family members, of an index patient with a venous value of the initial determination of factor VIIIc (IU/dl) Correlation coefficient (R) = 0.80 (p=0.01) Figure 1 Correlation between the plasma concentrations of factor VIIIc at the initial and the second measurement 4 years later. thromboembolic event and a factor VIIIc plasma concentration level >200 IU/dl, was selected to evaluate the possible inheritance of high factor VIIIc plasma levels. In this family all first degree relatives and the husband of the index patient were investigated. The results and the association with venous thromboembolism are shown in X index patient venous thromboembolism patient with venous thromboembolism f VIII: 147 f VIII: 226 f VIM: 268 è f VIII: 191 f VIM: 225 f VIII: 236 f VIII: 155 T f VIII: 125 f VIM: 105 f VIM: 160 Figure 2 Genetic resemblance of factor VIIIc in a family with venous thromboembolic events in three generations. -16-

10 High plasma concentration of factor VIIIc is a major risk factor for venous thromboembolism. Figure 2. Three first degree family members of the index patient had a factor VIIIc concentration above 200 IU/dl of whom 2 had experienced a venous thromboembolic event. Two of the three children of the index-patient had factor VIIIc plasma levels below 150 IU/dl. These three children were 9, 8 and 3 years old, respectively. Discussion In this clinical study we evaluated the association of elevated factor VIIIc plasma levels with venous thromboembolism. Our findings indicate that an elevated factor VIIIc plasma concentration is a prevalent, independent and strong risk factor for venous thromboembolism. Increased factor VIIIc levels (above > 175 IU/dl) maybe found in approximately one quarter of unselected patients with symptomatic venous thromboembolism, which is in agreement with earlier observations/ 8-10 ) The present study reveals that factor VIIIc is in fact a dose-dependent risk factor. For each 10 IU/dl increment, the risk for a single episode of venous thromboembolism increases by 10% (95% CI 0.9 to 21%). Interestingly, for recurrent disease this figure is 24% (95% CI: 11 to 38%), which suggests that elevated factor VIIIc is an even stronger risk factor for recurrent disease (Table 3). Our findings also point out that the factor VIIIc concentration of a given individual remains reasonably constant over time (correlation coefficient: 0.8), which may explain why the prothrombotic state associated with an elevated factor VIIIc persists. It should be emphasised that we used a one-stage clotting assay, while in previous studies an antigen method was also evaluated. It, therefore, appears that both methods are adequate for the evaluation of factor VIII as a prothrombotic riskfactor What are the possible causes of an increased factor VIIIc concentration? It has been well documented that factor VIIIc is an acute phase protein. In this study, however, an acute phase response was ruled out since factor VIIIc levels were measured several months after the acute thrombotic event and furthermore correction for C-reactive protein and fibrinogen, well known acute phase proteins, did not alter the observed odds ratios. The consistency of factor VIIIc concentration over time further supports the notion that it is the intrinsic factor VIIIc level which is responsible for the found association with venous thromboembolism. Von Willebrand factor and ABO bloodgroup are other determinants of the factor VIIIc plasma concentration and although we did not study this, previous investigations have convincingly shown that correction for these variables does not change the relation between factor VIIIc and thrombosis. 810 Another possibility is that the high plasma concentration of factor VIIIc is (partially) determined by genetic factors. Our small family study shows a -17-

11 CHAPTER II high concordance for factor VIIIc plasma concentration in first degree adult family members and it suggests a relation with the occurrence of venous thromboembolism. Two of the three young children of the index patient had normal factor VIIIc plasma concentrations (below 150 IU/dl). However, it should be noted that all three children were younger than 10 years of age and it is well known that factor VIIIc increases in adulthood. 16 ' 17 To further address the genetic influence more extensive family studies are indicated. In addition, further analysis of the factor VIIIc gene is warranted although the results of initial studies, in which common polymorphisms in the factor VIII gene, including its promotor, were evaluated, are disappointing At least one issue about our study design requires comment. As our control subjects, we selected patients referred with possible venous thromboembolism, but who proved not to have the disease, instead of healthy volunteers. This was done in order to assess the attributable risk associated with the studied abnormalities in the population of interest. The possibility that our patient control group may include undetected venous thromboembolism is very unlikely, since both serial non-invasive testing with ultrasonography and normal ventilation perfusion lung scanning rules out the presence of venous thromboembolism with an almost 100% certainty. Also, longterm follow-up in these subjects was uneventful. Hence, we consider our control group appropiate. In conclusion, an elevated plasma level of factor VIIIc is a significant, prevalent, independent and dose-dependent risk factor for venous thromboembolism. An elevated factor VIIIc level also predisposes for recurrent venous thromboembolism. The precise pathogenetic mechanism and cause of an elevated factor VIIIc concentration remains to be elucidated. -18-

12 High plasma concentration offactor VIIIc is a major risk factor for venous thromboembolism. References 1. Nordstrom, M., Lindblad, B., Bergqvist, D., Kjellstrom, T. A prospective study of the incidence of deep-vein thrombosis within a defined urban population. J Intern Med 1992, 232: Heijboer, H., Brandjes, D.P.M., Büller, H.R., Sturk, A., ten Cate, J.W. Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. NEJM 1990, 323: Pabinger, I., Brucker, S., Kyrie, P.A., et al. Hereditary deficiency of antithrombin III, protein C and protein S: prevalence in patients with a history of venous thrombosis and criteria for rational patient screening. Blood Coagul Fibrinolysis 1992, 3: Koster, T., Rosendaal, F.R., Briet, E., et al. Protein C deficiency in a controlled series of unselected outpatients: an infrequent but clear risk factor for venous thrombosis (Leiden Thrombophilia Study). Blood 1995, 85: Poort, S.R., Rosendaal, F.R., Reitsma, P.H., Bertina, R.M. A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996, 88: Dahlback, B., Carlsson, M., Svensson, P.J. Familial thrombophilia due to a previously unrecognized 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: Bertina, R.M., Reitsma, P.H., Rosendaal, F.R., Vandenbroucke, J.P. Resistance to activated protein C and factor V Leiden as risk factors for venous thrombosis. Thromb Haemost 1995, 74: Koster, T., Blann, A.D., Briet, E., Vandenbroucke, J.P., Rosendaal, F.R. Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet 1995, 345: O'Donnell, J., Tuddenham, E.G., Manning, R., Kemball-Cook, G., Johnson, D. & Laffan, M. High prevalence of elevated factor VIII levels in patients referred for thrombophilia screening: role of increased synthesis and relationship to the acute phase reaction. Thromb Haemost 1997, 77: Kamphuizen PW, Eikelboom JC, Roosendaal FR, et al. High prevalence of factor VIII antigen increase the risk of deep-vein thrombosis but are not related to common factor VIII gene variations. Blood 1998, submitted. 11. Lensing, A.W., Prandoni, P., Brandjes, D.P.M., et al. Detection of deep-vein -19-

13 CHAPTER II thrombosis by real-time B-mode ultrasonography. NEJM 1989, 320: Anonymous. Value of the ventilation/perfusion scan in acute pulmonary embolism. Results of the prospective investigation of pulmonary embolism diagnosis (PIOPED). The PIOPED Investigators. JAMA 1990, 263: Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol (Basel) 1957, 17: Bertina, R.M., Koeleman, B.P., Koster, T., et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994, 369: den Heijer, M., Koster, T., Blom, H.J., et al. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. NEJM 1996, 334: Balleisen, L., Bailey, J., Epping, P.H., Schulte, H., van de Loo, J. Epidemiological study on factor VII, factor VIII and fibrinogen in an industrial population: I. Baseline data on the relation to age, gender, body-weight, smoking, alcohol, pillusing, and menopause. Thromb Haemost 1985, 54: Kamphuisen, P.W., Houwing-Duistermaat, J.J., van Houwelingen, H.C., et al. Familial clustering of factor VIII and von Willebrand factor levels. Thromb Haemost 1998, 79: Mansvelt, E.P., Laffan, M., McVey, J.H., Tuddenham, E.G. Analysis of the F8 gene in individuals with high plasma factor VIII:C levels and associated venous thrombosis. Thromb Haemost 1998, 80: