Reduction of coagulability state one year after bariatric surgery

Author’s Accepted Manuscript Reduction of coagulability state one year after bariatric surgery Jérémie Thereaux, Fanny Mingant, Charles Roche, Hubert Galinat, Francis Couturaud, Karine Lacut www.elsevier.com/locate/buildenv

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S1550-7289(16)30729-8 http://dx.doi.org/10.1016/j.soard.2016.09.030 SOARD2784

To appear in: Surgery for Obesity and Related Diseases Received date: 1 June 2016 Revised date: 19 September 2016 Accepted date: 20 September 2016 Cite this article as: Jérémie Thereaux, Fanny Mingant, Charles Roche, Hubert Galinat, Francis Couturaud and Karine Lacut, Reduction of coagulability state one year after bariatric surgery, Surgery for Obesity and Related Diseases, http://dx.doi.org/10.1016/j.soard.2016.09.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REDUCTION OF COAGULABILITY STATE ONE YEAR AFTER BARIATRIC SURGERY

Jérémie Thereaux, M.D., Fanny Mingant, M.D., Charles Roche, M.D., Hubert Galinat, M.D., Francis Couturaud, M.D., Ph.D., Karine Lacut, M.D., Ph.D.

From the Department of General, Digestive and Metabolic Surgery (J.T., C.R.), the Laboratory of Hemostasis (F.M., H.G.), and the Department of Internal Medicine (F.C, K.L.), La Cavale Blanche University Hospital, Boulevard Tanguy Prigent, 29200 Brest. From the University of Bretagne Occidentale (UBO), EA 3878 (GETBO) (J.T., F.M., H.G., F.C., K.L.), 22 avenue Camille Desmoulins CS 93837 - 29238 Brest. From INSERM, CIC1412 (FC, KL), La Cavale Blanche University Hospital, Boulevard Tanguy Prigent, 29200 Brest. Address reprint requests to Dr. Jérémie Thereaux at the Department of General, Digestive and Metabolic Surgery), La Cavale Blanche University Hospital, Boulevard Tanguy Prigent, 29200 Brest, France, or at [email protected] (+33298347216)

Acknowledgment section Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE form for Disclosure of Potential Conflicts of Interest. Funding/Support: None Previous Presentation: None

1

ABSTRACT

Background: Obese patients are in a hypercoagulable state relative to normal-weight patients. Low grade inflammation may be a key factor for this condition. Objectives: Our study aimed to compare the coagulability state of morbidly obese patients before and one year after bariatric surgery (BS) using the Thrombin Generation (TG) test, a validated method to assess coagulation in vitro. Settings: University hospital. Methods: All patients undergoing BS between September 1, 2014 and April 30, 2015 were eligible for this prospective study (N = 42). Two distinct reagents were used for TG initiation based on the tissue factor concentration (Reagents LOW and HIGH). The main outcomes were endogenous thrombin potential (ETP) and peak height of TG. The rate of follow-up after one year was 97%. Results: One year after surgery, %Weight Loss was 32.5 ± 8.4%; CRP decreased from 9.0 (3.7-12.9) to 1.1 (0.3-2.8) mg/ml (P < 0.001) and fibrinogen from 4.2 ± 0.8 to 3.5 ± 0.8 g/L (P < 0.001). The ETP (%) decreased from (108.0 (95.0-117.0) to 78.0 (71.0-98.0) (P < 0.001) (LOW reagent) and from 113.0 (103.0-134.0) to 96.0 (86.0-107.0) (P < 0.001) (HIGH reagent). Peak height (%) decreased from (117.0 (92.0-139.0) to 82.0 (70.0-111.0) (P = 0.003) (LOW reagent) and from 106.0 (96.0-118.0) to 97.0 (87.8-105.2) (P = 0.003) (HIGH reagent). Conclusion: Our study shows a significant reduction in TG potential one year after BS in morbidly obese patients. Reduction of low grade inflammation may be one of the underlying mechanisms.

Key Word: Bariatric surgery, thrombin generation, coagulability

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Introduction Severe and morbid obesity are of increasing concern worldwide. During the last four decades, the worldwide prevalence of obesity has increased from 3.2 to 10.8% in men and from 6.4 to 14.9% in women.1 In 2014, the prevalence of the most serious grade of obesity, morbid obesity, was estimated to be approximately 0.64% in men and 1.6% in women. High body mass index (BMI) is an important factor leading to reduced life expectancy, hence, for each increase in BMI of 5 kg/m2 , the adjusted hazard ratio is 1.15 for coronary heart disease and 1.04 for stroke.2 Venous thromboembolism disease is also of concern, with an estimated odds ratio of approximately 1.5-2 in obese patients.3 The main mechanism by which obesity may increase the risk of venous thromboembolism disease is the much higher frequency of hypercoagulability among obese patients than found in normal weight or overweight patients.4-9 For over a decade, bariatric surgery (BS) has been shown to be the most effective treatment for morbid obesity because of sustainable weight loss10 and reduction in mortality and cardio-vascular events.11,12 However, there are no studies assessing the effect of BS on coagulability in obese patients. The aim of our study was to compare pre- and postoperative coagulability of grade II-III obese patients undergoing BS after one year, using the Thrombin Generation Assay (TGA), a widespread validated test of global clotting function.13-15

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Patients and Methods

SELECTION OF PATIENTS From September 1, 2014 to April 30, 2015, 42 consecutive patients scheduled for BS were invited to participate in our prospective observational study. The study was approved by the local ethics committee. All patients were informed in detail about the study protocol and provided informed written consent for their participation in the study. Data were collected and stored in an electronic database registered with the French national data protection agency (“Commission Nationale Informatique et Libertés”). This study has been registered in a publicly

accessible

database

(http://www.researchregistry.com)

(Id

number

researchregistry1085) in accordance with the World Medical Association's Declaration of Helsinki. The indication for BS was in accordance with French guidelines for BS, which are very similar to those of the US National Institutes of Health. Briefly, BS is indicated for adult patients with a BMI ≥ 40kg/m2 or a BMI between 35.0 and 39.9 kg/m2 with severe coexisting conditions. Each patient was evaluated and followed for at least six months before surgery and the indication for BS was endorsed by a multidisciplinary team. All patients with history of inflammatory or hematological diseases, lifelong anticoagulant therapy, or chronic suppuration were excluded. Detailed clinical characteristics were collected. Hypertension was defined as blood pressure above 140 mmHg (systolic) and/or 90 mmHg (diastolic) or the use of antihypertensive medication. Diabetes was defined as fasting glycaemia above 7 mmol/l (1.26 mg/dl) on at least two different occasions or the use of antidiabetic medication. Dyslipidemia 4

was defined as the presence of at least one of the following criteria: a total cholesterol concentration above 5.7 mmol/l (220 mg/dl), a serum HDL-c (High Density Lipoproteincholesterol) concentration below 1.0 mmol/l (38.7mg/dl), a triglyceride concentration above 1.7 mmol/l (15.0 mg/dl), or the use of lipid-lowering medication. All patients had a nocturnal ventilatory polygraphy and were considered to have obstructive sleep apnea syndrome if the apnea–hypopnea index was > 10 events/h, or if they were already being treated by nocturnal continuous positive-airway pressure therapy. Maximal BMI was defined as the maximal BMI obtained before surgery. Baseline excess weight (kg) was calculated, according to the theoretical weight for a BMI of 25 kg/m², as [weight at baseline–theoretical weight]. The % of excess weight loss (%EWL) was assessed as 100*[weight loss/baseline excess weight] Weight loss (in kg) was defined as [weight at baseline - weight at follow-up]. The percentage of initial total weight loss (%TWL) was calculated as: 100*[weight loss/weight at baseline].

SURGERY We use a standardized surgical technique for laparoscopic Roux-en-Y gastric bypass (LRYGB) and sleeve gastrectomy (SG). The type of procedure was chosen during a multidisciplinary meeting. Briefly, for SG, mobilization of the greater curve begins six cm proximal to the pylorus, and continues to the angle of His with importance given to the total exposure of the left crural pillar. Gastric resection involves using a 36 French bougie. The staple line is reinforced with a 2-0 absorbable running suture. For LRYGB, the stomach is first divided beneath the first vessel of the lesser gastric curve. The alimentary limb is created by dividing the jejunum 30-40 cm downstream from Treitz’s angle. The alimentary limb (Roux limb) length varies from 120 cm (BMI < 50 kg/m²) to 150 cm (BMI ≥ 50 kg/m²) depending on the preoperative BMI.

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FOLLOW-UP AND LABORATORY DETERMINATIONS The protocol included evaluations for all patients one month before surgery and six and 12 months after surgery. No patients underwent another BS during follow-up. Early postoperative complications were defined according to the Clavien-Dindo classification.16 Fasting blood samples were collected at each time point of the follow-up, between 09:00 and 11:00 AM, by a standard procedure from the cubital vein (butterfly device). All measurements were performed using fresh, unfrozen plasma, except for von Willebrand factor antigen, factor VIII measurements and the thrombin generation assay (TGA) where aliquots derived from centrifuged citrated plasma (at ambient temperature) were immediately frozen and stored (-80°C) until use.

Biochemical Parameters Fasting blood glucose (normal value (n.v.) < 5 .8 mmol/l), hemoglobin A1c (HBA1c; n.v. < 6%), total cholesterol (n.v. < 5.7 mmol/l), high-density lipoproteins (HDL) cholesterol (n.v. < 1 mmol/l), triglycerides (n.v. < 1.70 mmol/l), Serum glutamate-pyruvate transaminase (SGPT; n.v. < 49 UI/l)), and Serum glutamate-oxaloacetate transaminase (SGOT; n.v. < 34 UI/l) measurements were performed on an automated analyzer (Advia 1800 Siemens®). C Reactive Protein (CRP; n.v. < 5 mg/l) was analyzed using an immunoturbidimetry assay (Advia 1800 Siemens®) and fasting insulin (n.v. < 18 mUI/l) by ELISA (CIS Bio International®). BS is known to potentially induce micronutrient deficiency.17 Vitamin K (n.v. 150-900 ng/l), which is necessary for the production of some clotting factors, was tested preoperatively and after one year.

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Coagulation Parameters Platelet counts (n.v. 150-400 x 109/l) were measured on a Sysmex XE 5000 Analyzer. Prothrombin time (PT) (n.v. 70-100%), the activated partial thromboplastin time (aPTT) ratio (n.v. 0.85-1.2), fibrinogen (n.v. < 4.3 g/l), factor VIII (n.v. < 290%), von Willebrand factor antigen (n.v. < 200%), D-Dimer (n.v. < 0.42 µg/ml), and fibrin monomer (n.v. < 6 µg/ml) were also measured (Stago®, STA-R Evolution analyser).

Thrombin Generation Parameters Frozen platelet poor plasma (PPP) aliquots were thawed by incubation for 5 min in a water bath at 37°C before performing TGA. TGA was performed using the CAT method (Diagnostica Stago®). 80 µl of PPP was pipetted into the well of a microtiter plate together with 20 µl of PPP Reagent (Stago®). For this study, each TGA was performed using two distinct PPP Reagents based on the tissue factor (TF) concentration: PPP Reagent LOW (Stago®) (TF (1 pM) and phospholipids (4 µM)) and PPP Reagent HIGH (Stago®) (TF (20 pM) and phospholipids (4 µM)). PPP Reagent LOW is used for routine screening.13 PPP Reagent HIGH (named because of the high TF concentration) is used to extinguish the intrinsic pathway (mediated by phospholipids) and increase the extrinsic pathway (mediated by TF). The reaction was initiated by the addition of 20 µl of a mixture composed of the fluorogenic thrombin substrate and CaCl2 (FluCa kit; Stago®). A thrombin calibrator (Stago ®) was used to compensate for any quenching by the patient’s PPP. Fluorescence was read using a Fluoroscan Ascent® fluorometer (Thermo Labsystems, Finland) and the data recorded and treated using specific software (Thrombinoscope TM, Thrombinoscope BV, The Netherlands). Velocity, lag time (starting point of TG), peak height (maximum thrombin concentration), time to peak (point to reach the peak thrombin height), and endogenous

7

thrombin potential (Area under the curve; (ETP)) were recorded (Figure 1). The values are reported as the percentage of normal values previously determined on a local pool of donor samples, according to laboratory recommendations (Stago®) and a recent review.13 TGA is a test of global clotting function.15 ETP and peak height are predictive parameters for thrombosis because the risk of thrombosis increases with increasing ETP and peak height.18-21 Hence, ETP and peak height were chosen as the main outcomes of this study.

STATISTICAL ANALYSIS Data management and statistical analysis was performed using SPSS software (version 21.0; SPSS, Chicago, IL). Categorical data are presented as counts and percentages. Normality was evaluated using the Shapiro-Wilk test. Continuous variables are presented as the mean and standard deviation or the median and interquartile range (IQR), as appropriate. Paired student’s t-tests or Wilcoxon rank tests were used for numerical variables, as appropriate. A p-value of less than 0.05 was considered to be statistically significant.

Results SELECTION OF PATIENTS AND BASELINE CHARACTERISTICS After initial exclusion, 40 patients were included in our prospective study. The preoperative TGA (clotting) failed for one patient, one patient was lost to follow-up after one year, and one patient needed anticoagulant therapy for the onset of arrhythmia during the postoperative year. These three patients were excluded from the final analyses (Figure 2). At baseline (N = 37), a majority of patients was female (73.0%), the mean age was 44.0 ± 10.4 years and the mean BMI was 45.2 ± 5.5 kg/m². The primary coexisting conditions were hypertension (35.1%) and obstructive sleep apnea requiring nocturnal continuous 8

positive airway pressure (27.0%). Overall, 17 (46.0%) patients underwent SG and 20 (54.0%) LRYGB.

PERIOPERATIVE EVENTS No deaths or venous thromboembolisms occurred during a 30-day postoperative period. Two patients experienced grade IIIb complications requiring surgery: one had a small bowel obstruction and the other, perforation of the remnant stomach.

OUTCOMES AFTER SIX AND 12 MONTHS Anthropometric Variations Patients experienced significant weight loss relative to baseline during follow-up. TWL was 26.3 ± 5.5% after six months and 32.5 ± 8.4% after 12 months. The BMI decreased by 11.7 ± 2.4 kg/m² after six months and 14.5±3.8 kg/m² after 12 months. At one year, %EWL was 77.1±23.0 (Table 1).

Biochemical Variations Table 1 shows the change of biochemical markers after one year. One year after BS, fasting glycaemia, HbA1c, triglycerides, and SGPT levels decreased significantly relative to baseline: fasting glycaemia decreased from 5.5 (5.0-5.9) to 4.9 (4.6-5.2) mmo/l (P < 0.001), HbA1c from 5.7 (5.3-5.9) to 5.3 (5.0-5.5) % (P < 0.001), triglycerides from 1.4 ± 0.7 to 1.0 ± 0.5 mmol/l (P < 0.001); and SGPT from 28 (22-43) to 19 (17-25) UI/l (P < 0.001). The median vitamin K (ng/l) serum level did not change after one year relative to baseline: 98 (71-153) vs. 104 (64-170) (P = 0.74).

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Changes in Hemostatic, Clotting Factor, and Inflammatory Marker Levels One year after BS, the level of inflammatory markers CRP and fibrinogen decreased significantly relative to baseline: CRP decreased from 9.0 (3.7-12.9) to 1.1 (0.3-2.8) mg/ml (P < 0.001), and fibrinogen from 4.2 ± 0.8 to 3.5 ± 0.8 g/l (P < 0.001) (Table 1). Among hemostatic and clotting factors, only PT decreased significantly relative to baseline: 94 (90100) vs. 90 (85-98) % (P = 0.005) (Table 1).

Changes in the Thrombin Generation Assay Results We evaluated TGA for all 37 patients included in the study with PPP reagent LOW at baseline, and one year. It was not possible to determine ETP and other parameters of TGA with PPP reagent HIGH in four patients at baseline and in one patient at one year because of extreme results (Such hypercoagulation state that fluorometer cannot provide data). These patients were excluded from the analyses with PPP Reagent HIGH. It was possible to determine these values in the four patients with extreme results at baseline during the followup one year after surgery. ETP and peak height decreased significantly with PPP reagent LOW, and PPP reagent HIGH, after one year (Table 2 and Figure 3). The lag time was shorter after at one year (116.0 (100.0-128.0) vs. 100.0 (95.0-124.5) %; p = 0.01) with the PPP reagent HIGH.

Discussion We found that the in vitro coagulabality state significantly decreased one year after BS, as assessed by ETP and peak height, in this prospective interventional comparative study of 37 morbidly obese patients. One of the underlying mechanisms may be the significant reduction of low grade inflammation associated with weight loss. 10

Longitudinal studies originally demonstrated increased hypercoagulability in obese patients by a higher risk of venous thromboembolism disease or cardio-vascular disease.3,22-24 Obesity is a risk factor for in vitro hypercoagulability.4-7 Campello et al. reported a 50% increase of ETP for grade 3 obese patients relative to control subjects of normal weight.7 Our study confirms previous results suggesting that patients with lower grade obesity have a lower ETP than grade 3 patients, as the patients in our study had a mean BMI of approximately 30 kg/m² one year after surgery.7 Hence, a lower coagulability state could explain, at least in part, the reduction in cardio-vascular events after BS.12,25 We observed reduced ETP and peak height, two parameters that have been widely demonstrated to be key outcomes of enhanced in vivo coagulability in TGA.18-20 Several studies have assessed risk factors for enhanced ETP in obese patients. Many clotting factors are elevated in obese patients. Obese patients have higher levels of vWF Ag, factor VIII, and factor VII than control subjects of normal weight.4,7,26 However, we did not observe a postoperative decrease in factor VIII and vWF Ag in our study. High grade inflammation, often present in obese patients,27 is one of the main factors responsible for enhanced coagulability.4,8,26,28,29 The significant decreases of CRP and fibrinogen one year after BS exhibited by the patients in our study could explain the reduction in enhanced TG in patients undergoing BS. The level of total cholesterol has also been found to be predictive of enhanced ETP in a previous study.4 We failed to demonstrate lower levels of total cholesterol after one year; this may be explained by the lack of sufficient power in our study since a reduction in total cholesterol has been widely found after BS in larger studies.25 Surprisingly, we found a lower lag time for PPP Reagent HIGH and a trend for PPP Reagent Low one year after BS. These results appear to be in contradiction with lower ETP and peak height as a short lag time could be associated with a hypercoagulable state.13 Thus, after weight loss, thrombin generation initiation seems to be shortened but corresponds with

11

lower peak height and area under the curve (ETP). We are unable to provide an explanation for these observations. Modification of the extrinsic pathway may merit further exploration as the PPP Reagent HIGH extinguishes the intrinsic pathway by using a high TF concentration. The main strength of this study is that we provided full TG outcomes, with two distinct reagents based on TF concentration, preoperatively and through one year after BS in consecutive patients undergoing LSG and LRYGB. We also measured vitamin K serum levels for all patients to evaluate the potential influence of this parameter on the coagulabality state. Our results suggest that changes in TG were independent of vitamin K serum levels.

Only

Ay et al. have assessed TG after BS in a cohort of patients undergoing gastric bypass and gastric banding.30 However, they excluded patients with lower weight loss and did not provide the rate of loss to follow-up. They also did not use a standardized commercial TF reagent and did not express their results as a percentage of a local pool of donor samples according to laboratory recommendations and a recent review.13 Finally, they did not provide vitamin K serum levels. Our study has some limitations. We provided data at one year after surgery because it has been found to be the time of maximum weight loss.25 However, long-term studies are necessary to confirm the reduction in TG as weight regain is common after BS.25 We did not assess principal inhibitors of the clotting system such as the TF pathway inhibitor, for which an increase after BS could lead to lower ETP and peak height. Last, we cannot provide data on fibrinolysis which is known to be attenuated in obese patients.29

Conclusion In conclusion, we observed a significant reduction in potential TG one year after BS in morbidly obese patients. A reduction of low grade inflammation could be a key factor to explain this outcome. Larger studies are needed to better explore the predictive factors of TG 12

reduction and to eventually correlate these with the reduction of arterial and venous thromboembolism diseases described after BS.

Acknowledgment section Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE form for Disclosure of Potential Conflicts of Interest. Funding/Support: None Previous Presentation: None

References 1. Ezzati M. Trends in adult body-mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19·2 million participants. Lancet 2016;387(10026):1377-1396. 2. Lu Y, Hajifathalian K, Ezzati M, Woodward M, Rimm EB, Danaei G. Metabolic mediators of the effects of body-mass index, overweight, and obesity on coronary heart disease and stroke: a pooled analysis of 97 prospective cohorts with 1.8 million participants. Lancet 2014;383(9921):970-983. 3. Severinsen MT, Kristensen SR, Johnsen SP, Dethlefsen C, Tjonneland A, Overvad K. Anthropometry, body fat, and venous thromboembolism: a Danish follow-up study. Circulation 2009;120(19):1850-1857. 4. Pruller F, Raggam RB, Posch V, et al. Trunk weighted obesity, cholesterol levels and low grade inflammation are main determinants for enhanced thrombin generation. Atherosclerosis 2012;220(1):215-218. 5. Beijers HJ, Ferreira I, Spronk HM, et al. Body composition as determinant of thrombin generation in plasma: the Hoorn study. Arterioscler Thromb Vasc Biol 2010;30(12):2639-2647. 6. Campello E, Spiezia L, Zabeo E, Maggiolo S, Vettor R, Simioni P. Hypercoagulability detected by whole blood thromboelastometry (ROTEM(R)) and impedance aggregometry (MULTIPLATE(R)) in obese patients. Thromb Res 2015;135(3):548553. 7. Campello E, Zabeo E, Radu CM, et al. Hypercoagulability in overweight and obese subjects who are asymptomatic for thrombotic events. Thromb Haemost 2015;113(1):85-96.

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Characteristics

Baseline (N = 37)

12 Months† (N = 37)

P-Value‡

Weight (kg) BMI (kg/m²) %TWL BMI variation (kg/m²) %EWL

123.9 ± 19.1 45.2 ± 5.5 NA NA

83.2 ± 18.1 30.3 ± 5.8 32.5 ± 8.4 -14.5 ± 3.8 77.1±23.0

< 0.001 < 0.001 NA NA

5.5 (5.0-5.9) 5.7 (5.3-5.9) 13.3 (8.7-19.1) 4.9 ± 0.8 1.1 ± 0.2 1.4 ± 0.7 27 (20-36) 28 (22-43) 9.0 (3.7-12.9) 98 (71-153)

4.9 (4.6-5.2) 5.3 (5.0-5.5) 4.9 (3.8-5.8) 4.7 ± 1.1 1.5 ± 0.3 1.0 ± 0.5 23 (21-26) 19 (17-25) 1.1 (0.3-2.8) 104 (64-170)

< 0.001 < 0.001 < 0.001 0.21 < 0.001 < 0.001 0.03 < 0.001 < 0.001 0.74

Biochemical Markers Fasting glucose (mmol/l) HbA1c (%) Fasting Insulin (mUI/l) Total cholesterol level (mmol/l) HDL cholesterol level (mmol/l) Triglyceride level (mmol/l) SGOT level (UI/l) SGPT level (UI/l) C-Reactive Protein (mg/l) Vitamin K (ng/l) Coagulation Markers Platelet counts (x109) 274 ± 65 259 ± 65 0.07 PT (%) 94 (90-100) 90 (85-98) 0.005 aPTT ratio (%) 1.04 (0.96-1.1) 1.05 (1.0-1.1) 0.77 Fibrinogen (g/l) 4.2 ± 0.8 3.5 ± 0.8 < 0.001 VIII Factor (%) 140 (114-172) 140 (111-166) 0.81 vWF ag (%) 125 (101-159) 112 (95-154) 0.81 D-Dimer (µg/ml) 0.33 (0.26-0.44) 0.27 (0.25-0.32) 0.16 Fibrin Monomer (µg/ml) 2.9 (2.2-3.5) 3.2 (2.7-4.4) 0.21 *Plus-minus values are mean (SD) or median (IQR), as appropriate. Normal values are provided in the “Methods” chapter † 1 patients lost to follow-up ‡ Values were calculated with paired student-t tests or Wilcoxon signed-rank tests, as appropriate %TWL: % Total Weight Loss BMI: Body Mass Index (weight in kilograms divided by the square of the height in meters) HbA1c: Haemoglobin A1c HDL cholesterol: High Density Lipoprotein cholesterol SGOT: Serum glutamate-oxaloacetate transaminase SGPT: Serum glutamate-pyruvate transaminase PT: Prothrombin Time aPTT: activated partial thromboplastin time vWFag: von Willebrandt Factor antigen NA: Not Applicable

Table 1. Weight loss, changes in biochemical and coagulation markers at one year of follow-up (N = 37)*

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Thrombin Generation Characteristics

Baseline

(N =37)

12 Months

P-Value†

(N = 37)

Reagent Low ETP (%) Peak Height (%) Lag Time (%) Time to peak (%) Velocity (%)

Reagent High‡ ETP (%) Peak Height (%) Lag Time (%) Time to peak (%) Velocity (%)

108.0 (95.0-117.0) 117.0 (92.0-139.0) 126.0 (113.0-140.0) 105.0 (98.0-118.0) 124.0 (98.0-169.0) (N =32)

78.0 (71.0-98.0) 82.0 (70.0-111.0) 110.0 (102.0-130.0) 99.0 (88.0-111.0) 94.0 (75.0-145.0) (N = 32)

< 0.001 0.003 0.62 0.13 0.13

113.0 (103.0-134.0) 106.0 (96.0-118.0) 116.0 (100.0-128.0) 103.0 (97.0-114.0) 106.0 (95.0-126.0)

96.0 (86.0-107.0) 97.0 (87.8-105.2) 100.0 (90.5-124.5) 100.0 (93.0-107.8) 99.0 (84.5-118.2)

< 0.001 0.003 0.01 0.07 0.11

*Plus-minus values are median (IQR). † Values were calculated using Wilcoxon signed-rank tests ‡ 5 patients excluded (uninterpretable data because of extreme outcomes) Reagent LOW: PPP with 1 pmol TF and 4µmol phospholipids Reagent HIGH: PPP with 20 pmol TF and 4 µmol phospholipids ETP: Endogenous Thrombin Potential IQR: Interquartile Range PPP: Poor Platelet Plasma TF: Tissue Factor

Table 2. Thrombin generation test parameters variations at one year of follow-up (N = 37)*

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Figure 1: Model of thrombin generation curve

Figure 2: Flow Diagram TGA: Thrombin Generation Assay

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160 140 N

120

Values (%)

100 80 60

* *

*

*

*

**

* Preope rative 6 Months 12 Months

40 20 0 ETP ETP Peak Height Peak Height (Reagent (Reagent (Reagent (Reagent LOW) HIGH) LOW) HIGH) Thrombin Generation Assay according to PPP Reagent

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Figure 3. Thrombin generation assay based on PPP Reagent Reagent LOW: PPP with 1 pmol TF and 4 µmol phospholipids (N = 37) Reagent HIGH: PPP with 20 pmol TF and 4 µmol phospholipids (N =3 2) Values are expressed as the median (Interquartile Range). P-values (relative to baseline) were calculated using Wilcoxon signed-rank tests *< 0.05 **< 0.01 ***< 0.001 ETP: Endogenous Thrombin Potential TF: Tissue Factor NS: Non-significant PPP: Poor Platelet Plasma

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