Association of Changes in D-dimer and Other Coagulation Markers with Changes in Marder Score After Treatment of Acute Venous Thrombosis

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1 Journal of Thrombosis and Thrombolysis 14(1), 73 78, C 2003 Kluwer Academic Publishers, Manufactured in The Netherlands. Association of Changes in D-dimer and Other Coagulation Markers with Changes in Marder Score After Treatment of Acute Venous Thrombosis Abstract. Aim: Coagulation markers are sensitive tools to assess ongoing thrombus formation. An association between changes in these markers and changes in venographic Marder scores in patients with acute deep veinthrombosis treated with low-molecular-weight (LMWH) or unfractionated heparin (UFH) has not been reported. Methods: We investigated differences in coagulation parameters before and at the end of a twelve days the treatment of patients with an improvement versus no improvement of the venographic findings at the end of the treatment with LMWH (n = 48) and UFH (n = 41). Results: Patients with lower values in the Marder score had lower D-dimer levels at day 12 compared to entry treated with UFH and LMWH (p < 0.001). Not improved Marder scores paralleled unchanged D-dimer levels at end of both treatments. Higher values of factor- Xa inhibition and Heptest assay (p < 0.001) were measured at the end of treatment in LMWH- in contrast to UFH-patients. Thrombin inhibition was lower and unchanged at day 12 in patients treated with LMWH and UFH, respectively. Thrombin generation inhibition and release of tissue-factor pathway inhibitor remained unchanged in both groups. Conclusion: An improved Marder score is associated with a decrease of D-dimer during UFH and LMWH treatment of deep vein-thrombosis. Job Harenberg, 1 Kirsten Merx, 1 Ursula Hoffmann, 1 Alexander R. Tolle, 2 Menno V. Huisman 3 1 Forth Department of Medicine, University Hospital, Faculty of Clinical Medicine Mannheim of the Ruprecht-Karls, University of Heidelberg, Mannheim, Germany; 2 Novartis Pharma, Nuremberg, Germany; 3 Department of General Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands Key Words. venous thromboembolism, Marder score, D-dimer, factor Xa inhibition, low-molecular-weight heparin, heparin Introduction Large clinical trials have demonstrated the equal efficacy and safety of low-molecular-weight heparins (LMWH) compared to unfractionated heparin (UFH) for both the treatment of deep-vein thrombosis (DVT) [1,2] or pulmonary embolism [3,4]. Initially, clinical trials used repeated venography to confirm the efficacy of LMWH for treatment of acute DVT. A meta-analysis of studies always showed a trend for reduction of the thrombus size in patients with deep-vein thrombosis and treatment with LMWH compared to aptt-adjusted intravenous UFH and the meta-analysis indicated significant reduction of thrombus size [5]. A relation of the outcome of the venographic improvement of the thrombus size with coagulation markers was found for the activated partial thromboplastin time in patients treated with UFH [6]. In patients treated with LMWH a relation of the anti-factor Xa (axa) activity and the reduction of thrombus size was found during treatment with Dalteparin [7,8], Enoxaparin (9) and Certoparin [10,11]. Despite monitoring axa plasma levels, Marder Score reduction was comparable in the body weight-adjusted and axa activity-adjusted group of patients of a prospective trial as determined on the control phlebography performed after a 10-day period of treatment [12]. Thus, a relation between the doses of LMWH and the clinical outcome on thrombus regression is still debated even if it looks likely that laboratory monitoring is not required. We have focussed on the question whether anti- Xa activity or other coagulation parameters may be relevant in relation to the improvement of the Marder score after treatment of DVT with aptt adjusted intravenous UFH or fixed dose, body weight-independent LMWH Certoparin b.i.d. subcutaneously. We were interested specifically to whether differences can be found after treatment with LMWH compared to UFH. Address for correspondence: Job Harenberg, MD, IV. Dept. of Medicine, University Hospital Mannheim, Ruprecht- Karls University Heidelberg, Theodor-Kutzer-Ufer 1-3, D Mannheim, Germany. Tel.: +49/621/ , 2789; Fax: +49/621/ ; j-harenberg@t-online.de 73

2 74 Harenberg et al. Patients and Methods Treatment regimens Patients with acute deep venous thrombosis, confirmed by ascending venography, were randomly assigned to therapy with LMWH received a fixed dose of 8,000 international anti-factor Xa units (axa IU) of certoparin (Mono-Embolex R ) subcutaneously b.i.d. or aptt-adjusted intravenous UFH. The extension of thrombosis was quantified by Marder Score. Values were 23.6 ± 8.3 and 24.2 ± 8.2 in the group of patients randomised to LMWH and UFH, respectively (2 p = 0.18, Wilcoxon test). None of the patients had symptomatic pulmonary embolism. The heparin treatment period lasted 12 days. Oral anticoagulation (vitamin K antagonists) was started between the 3rd and 7th day and was continued for up to 6 months. Treatment with LMWH or UFH was stopped as soon as an International Normalised Ratio (INR) of 2.0 or more was achieved for a minimum of 2 consecutive days [12]. Plasma samples Plasma samples were collected from patients at days 1 and 12 of treatment at four participating centres. The results of the coagulation markers were divided into the following groups: patients with improvement or no improvement of the Marder score in the second phlebography as compared to that at entry and treated with LMWH or UFH. Patients assigned to therapy with LMWH received a fixed dose of 8,000 international axa units (IU) of Certoparin (Novartis Pharma GmbH, Germany) subcutaneously b.i.d. Patients randomised to therapy with UFH received an initial bolus of IU, followed by continuous intravenous infusion at an initial rate of 20 IU/kg/h for 12 days (range: 7 to 15 days). The dose of UFH was subsequently adjusted to a target activated partial thromboplastin time (aptt) of two- to threefold the reference value according to a nomogram [6]. The primary outcome measure was defined as a relevant reduction of the Marder score of 30% or more on the second venography as compared to the one obtained at entry [12]. Coagulation parameters For the study, citrated plasma samples were drawn at day 1 and day 12 at 8 a.m. during infusion with UFH or before the subcutaneous administration of LMWH. All blood samples were taken at selected subgroups the participating centres in plastic tubes containing 3.8% sodium citrate (v/v, 1/10, citrate/blood), centrifuged immediately at 1,800 g for 10 min at room temperature, shock frozen and stored at 25 C within 2 hrs after withdrawal for 4 weeks. Thereafter samples were transferred frozen to the central laboratory and stored at 85 C until analysed. Blood samples were taken at day 1 and 12 of treatment at 8 a.m. and in patients treated with LMWH before the morning injection. D-dimer assay (normal range: <500 µg/l) was performed with commercially available reagents (Gold EIA, Agen Biomedical Limited, Acacia Ridge, Australia) on microtiter plates. The absorption was measured at 405 nm on the microtiter plate reader MR 7000 (Dynex, Denkendorf, Germany) and the concentration of D-dimer was calculated from the reference curve using the Biolinx 2.22 software program. Factor Xa inhibition was measured using the chromogenic substrate S 2222, exogenous antithrombin, and purified bovine factor Xa (all reagents from Kabi, Munich, Germany). Normal factor-xa inhibitory activity in plasma was below 0.01 heparin units/ml. Heptest clotting assay was performed as described earlier [13]. Factor Xa and Recalmix were from Haemachem (St. Louis, USA). Normal values ranged from 13 to 20 sec. Coagulation assays were performed in a KC10 Coagulometer. Thrombin inhibition was analysed by the chromogenic S2238 substrate assay in the presence of exogenous antithrombin and human thrombin (Chromogenix, Essen, Germany) on microtiter plates according to the manufacture instructions. Normal values were below 0.03 IU heparin/ml plasma. Activated partial thromboplastin time (aptt) and thrombin clotting time (TCT) were measured using commercially available reagents (Pathromtin, and thrombin reagent, both reagents from Behringwerke AG, Marburg, Germany, normal values <40 sec and <20 sec, respectively). Thrombin generation inhibition assay (TGIA) was determined according to a previously published method [14]. In brief, plasma was incubated with H-Gly-Pro-Arg-Pro-OH, kindly provided by Drs. Prasa and Stürzebecher, to inhibit polymerisation of fibrin. Thrombin formation was induced at 37 C by tissue thromboplastin reagent (Thromborel S, Aventis, Marburg, Germany) dissolved in 0.25 M CaCl 2 in the presence of the chromogenic substrate TH 5134 (H-ß-Ala-Gly-Arg-pNA (Pentapharm Ltd., Basel, Switzerland) adjusted with Tris-buffer, ph 7.4. The absorption was read at 405 nm after 15 min and the optical density (OD) of the sample minus the background (pooled plasma without thromboplastin reagent) was calculated. TFPI was measured using the two-step chromogenic assay on microtiter plates as described previously [15]. Plasma samples were heated for 10 min. at 60 C. Purified not activated factor X and purified not activated factor VII were a gift from Dr A. Heimburger (Behringwerke AG, Marburg, Germany). Thromboplastin was used from rabbit brain (Thromboplastin, Behringwerke AG, Marburg, Germany). Chromogenic substrate S2222 was used to measure activated factor Xa. The addition of

3 Coagulation Markers After Treatment of DVT 75 polybrene excluded a direct effect of heparin on the TFPI-activity. The TFPI-activity levels were expressed in U/ml, in reference to a pooled plasma from 20 healthy individuals (age years) which was defined to contain 1.0 U/ml TFPI. Statistical methods The statistical evaluation of the laboratory values was performed on the same basis as the primary endpoint was calculated [12]. Statistical evaluation included calculation of mean values and standard error, and analysis comparing values of coagulation parameters at day 1 and day 12 of each treatment group and with an improvement or no improvement of the Marder score at the end of therapy compared to entry. The signed rank test according Wilcoxon was used for calculation of p-values. T-test (age, weight) and Fisher s exact test analysed the baseline characteristics of the patient groups. In addition, the data between the groups were compared using the Wilcoxon two-sample test. Results The baseline characteristics of the patient groups were equally distributed (Table 1). Patients were treated for 12.3 ± 2.1 days with LMWH and 12.0 ± 2.7 days with UFH, respectively (p = 0.49, Wilcoxon two-sample test). The dose of UFH was 30,885 ± 8,074 IU/day at day 1 and was adjusted by determinations of the activated partial thromboplastin time during the treatment period (2- to 3-fold prolongation of the aptt) leading to 33,974 ± 10,572 IU/day at day 12. The centres included 89 patients 48 and 41 randomised to LMWH and UFH, respectively. Of these 42 and 29 had an improvement and 6 and 12 no improvement of the Marder score at the end of therapy with LMWH and UFH, respectively (no significant difference between the groups). Table 1. Biographic Data of Patients with DVT Treated with LMWH or UFH in Whom Phlebographies and Plasma Samples Were Available at Days 1 and 12 LMWH UFH Characteristics (n = 48) (n = 41) p-values Age (years) mean ± SD 56.8 ± ± Weight (kg) mean ± SD 82.0 ± ± Male 32 (66.7%) 27 (65.9%) 1.00 Symptoms of DVT (d) 6.8 ± ± Previous VTE 11 (22.9%) 17 (41.5%) 0.07 Varicose veins 19 (39.6%) 9 (22.0%) 0.11 Surgery in past 4 weeks 6 (12.5%) 2 (4.9%) 0.28 Bedrest in past 4 weeks 14 (29.2%) 10 (24.4%) 0.64 Known cancer 4 (8.3%) 6 (14.6%) 0.50 Cardiac insufficiency 2 (4.9%) 0.21 Cerebral ischemia 2 (4.9%) 0.21 In patients randomised to LMWH and an improved Marder score at the end of treatment, D- dimer was lower (p < 0.001) and axa activity (p < 0.002) and Heptest assay (p < 0.031) were higher at day 12 compared to day 1. TFPI activity and TGIA were not different day 12 compared to day 1. Thrombin inhibition (S2238 assay, p < 0.002, and thrombin clotting time, p < 0.006) was significantly lower at the end of the treatment. Patients with no improvement of the Marder score showed higher antifactor Xa (p < 0.031) and lower anti-factor IIa inhibition (p < 0.031) at day 12 compared to day 1. The other parameters did not differ between the two time points of determination (Table 2). The values of the coagulation markers did not differ between the groups at day 1 and 12, supporting the relevance of the differences between days 1 and 12 in each treatment group. Patients allocated to UFH and an improved Marder score at day 12 showed significant lower levels of D-dimer (p < 0.001) and no changes of the other coagulation parameters at day 12 compared Table 2. Results of the Coagulation Values (Mean and Standard Error) at Days 1 and 12 of Patients Treated with LMWH and with an Improved or Not Improved Marder Score on the Second Venogram Compared to that at Entry. p-values of the Differences of the Coagulation Values Between Days 12 Versus Day 1 are Given Marder score Marder score improved worsened Day (n = 42) (n = 6) p-values D-Dimer ± ± (mg/l) ± ± p < S ± ± ± ± p Heptest ± ± ± ± p S ± ± ± ± p TCT (sec) ± ± ± ± p TGIA ± ± ± ± p TFPI ± ± ± ± p

4 76 Harenberg et al. Table 3. Results of the Coagulation Values (Mean and Standard Error) at Days 1 and 12 of Patients Treated with UFH and with an Improved or Not Improved Marder Score on the Second Venogram Compared to that at Entry. p-values of the Differences of the Coagulation Values Between Days 12 Versus Day 1 are shown Marder score Marder score improved worsened Day (n = 29) (n = 12) p-values D-Dimer ± ± (mg/l) ± ± p < S ± ± ± ± p Heptest ± ± ± ± p S ± ± ± ± p TCT (sec) ± ± ± ± p TGIA ± ± ± ± p TFPI ± ± ± ± p to day 1. No changes of the coagulation markers occurred also in patients with any improvement of the Marder score at the end of therapy (Table 3). It can be seen from the results, that all coagulation markers showed lower values in those patients, in whom the Marder score did not improve at the second phlebography. There was no correlation between the changes of the Marder scores and the changes of the coagulation markers (data not shown). Discussion Unfractionated intravenous heparin has provided an effective therapy of venous thromboembolism for more than half a century, but the need to monitor therapy and establish therapeutic levels is a fundamental problem. However, even with the best care using a heparin protocol [6], some patients treated with intravenous heparin will receive subtherapy thus limiting heparin treatment seriously. Results of clinical trials have shown that the intensity of initial heparin treatment must be sufficient to prevent effectively recurrence of venous thromboembolism. Therapy with low-molecular-weight heparin, which does not require monitoring and dose finding, is the likely practical solution to these dilemmas [4]. Measurement of D-dimer, a specific fibrin degradation product, demonstrated a decrease during heparin therapy. Decreased D-dimer levels at day 5 were associated significantly with an improved venographic outcome after 6 months [17]. At initiation of heparin therapy, a sharp decrease in D-dimer levels occurs within 1 3 days, followed by a continuous and slow decline towards the normal range [18]. Here, we demonstrated that D-dimer levels decreased significantly and in the same order during the initial therapy of DVT after the subcutaneous administration of fixed dose LMWH and intravenous UFH. The decrease of D-dimer occurred only in patients with an improved Marder score at the end of therapy. D-dimer levels did not change significantly if the Marder score did not improve during therapy with either heparin. The discrepancies between increase of the anti-xa and Heptest activities in contrast to the decrease of the antithrombin activities at day 12 compared to day 1 after treatment with LMWH require explanation. These changes were not observed after treatment with UFH. The higher anti-xa/anti-iia ratio of LMWH may explain this phenomenon. Accordingly, the results show that LMWH maintained or enhanced its anti-xa activity and had lower anti- IIa activity in patients after a 12 days treatment. It may be assumed that less thrombin circulates in the patients due to an organisation of the thrombus. Less circulating thrombin may result in an enhanced availability of LMWH for inhibition of factor Xa. This mechanism does not account for UFH resulting in unchanged intensities of inhibition of factor Xa and thrombin-inhibition methods. Heptest assay and thrombin clotting time showed these differences only in patients randomised to LMWH and an improvement of the Marder score. The differences in the changes of the D-dimer levels and of the factor Xa and IIa inhibition may be explained by the above mentioned arguments. The results indicate that inhibition of factor Xa reduces D-dimer generation significantly, while thrombin is inhibited only to a small extent during LMWH treatment. In contrast, UFH inhibits both factor Xa and IIa and reduces by that the formation of D-dimer. The reduction of thrombus size during therapy of acute venous thrombosis with both heparins leads to a reduced formation of D-dimer and to an elevation of free UFH and LMWH due to a reduced binding to thrombin bound fibrin. Accordingly, UFH and LMWH bind to thrombin attached to fibrin in the same magnitude if the thrombus size does not decrease during therapy. Further studies have to

5 Coagulation Markers After Treatment of DVT 77 investigate the prognostic value of these findings in patients with acute venous thrombosis undergoing therapy with UFH and LMWH. TGAI and TFPI assays remained unchanged in all groups of patients at the end compared to the start of treatment. It has been shown that both parameters are influenced sensitively by LMWH and UFH after subcutaneous administration in healthy persons. In the present study, TGAI is similar after administration of s.c. LMWH and i.v. administration of UFH. TFPI is released in both treatment groups to a similar extend. It is of interest, that TFPI is not exhausted in patients with acute deep-vein thrombosis during therapy with continuous intravenous heparin in contrast to repeated intravenous bolus injections in healthy persons [19] or in patients with deep-vein thrombosis [20]. The absolute values of TFPI did not differ after subcutaneous administration of the same dose of LMWH in healthy persons [21] compared to patients of the present study. Coagulation parameters such as axa activities and D-dimer levels appear to be influenced to a large extent by Certoparin, given in a fixed and body weight-independent dose to patients with acute DVT. The prospective value of D-dimer and factor Xa inhibition in relation to the outcome of patients with acute venous thromboembolism treated with lowmolecular-weight heparin remains to be determined. Acknowledgment We gratefully acknowledge the technical assistance of Mrs. Ch. Giese and A. Hagedorn during the entire study. References 1. Levine M, Gent M, Hirsh J, et al. A comparison of lowmolecular-weight heparin administered primarily at home with unfractionated heparin administered in the hospital for proximal deep-vein thrombosis. N Engl J Med 1996;334: Koopman MMW, Prandoni P, Piovella F, et al. 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