Supplemental Material s16

SUPPLEMENTAL MATERIAL

Chronic antiplatelet therapy is not associated with alterations in the presentation, outcome or host response biomarkers during sepsis: a propensity-

matched analysis

Maryse A. Wiewel, Sacha F. de Stoppelaar, Lonneke A. van Vught, Jos F. Frencken, Arie J.

Hoogendijk,

Peter M.C. Klein Klouwenberg, Janneke Horn, Marc J. Bonten, Aeilko H. Zwinderman, Olaf L.

Cremer,

Marcus J. Schultz, and Tom van der Poll, on behalf of the MARS Consortium. METHODS

Biomarker measurements

Tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), IL-6, IL-8, IL-10, IL-

13, soluble intercellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sE-selectin) were measured using FlexSet cytometric bead arrays (BD Bioscience, San Jose, CA) using

FACS Calibur (Becton Dickenson, Franklin Lakes, NJ, USA). Angiopoietin-1, angiopoietin-2, protein C, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), antithrombin, matrix metalloproteinase (MMP)-8, tissue inhibitor of metalloproteinase (TIMP)-1 (R&D systems, Abingdon, UK), and D-dimer (Procartaplex, eBioscience, San Diego, CA) were measured by Luminex multiplex assay using BioPlex 200 (BioRad, Hercules, CA).

Propensity score matching and Cox proportional hazards regression

To account for differential likelihood of receiving antiplatelet therapy we constructed a propensity score [1], using logistic regression, including variables associated with use of antiplatelet therapy based on significant differences in baseline characteristics in the unmatched cohort and variables that we considered of relevance to our outcomes.

This score included age, gender, race (white), weight, cerebrovascular disease, chronic cardiovascular insufficiency, chronic renal insufficiency, congestive heart failure, chronic obstructive pulmonary disease (COPD), diabetes mellitus, hematologic malignancy, hypertension, immune deficiency, metastatic malignancy, history of myocardial infarction, peripheral vascular disease, angiotensin-converting enzyme (ACE)-inhibitors/ angiotensin receptor blockers, anticoagulants, beta-blockers, calcium channel blockers, diuretics, insulin, non-steroidal anti-inflammatory drugs, oral anti-diabetic drugs, statins and site of infection. Because one aim of our study was to determine the effect of chronic antiplatelet therapy on clinical presentation in the ICU, measures for disease severity, were not included in the propensity score model. Subjects were 1:1 matched by the estimated

2 propensity score using nearest neighbor matching with a caliper of 0.2SD of the logit of the propensity score. Standardized differences were determined to ascertain balance between the propensity-matched groups [2]. In order to adjust for potential differences in disease severity, Cox proportional hazards regression was used to estimate the effect of antiplatelet therapy on mortality rate including the APACHE IV acute physiology score

(APS) in the model. Proportional hazards assumptions were verified through examination of Schoenfeld residuals.

3 REFERENCES

1. Rubin DB (1997) Estimating causal effects from large data sets using propensity scores. Ann Intern Med 127: 757-763 2. Austin PC (2009) Balance diagnostics for comparing the distribution of baseline covariates between treatment groups in propensity-score matched samples. Stat Med 28: 3083-3107

4 SUPPLEMENTAL TABLES

Supplemental table 1. Sites of infection and causative pathogens

Unmatched cohort Propensity-matched cohort Antiplatelet No antiplatelet p Antiplatelet No antiplatelet p N = 267 N = 705 N = 150 N = 150 Site of infection Pulmonary (%) 119 (44.6) 318 (45.1) 0.88 70 (46.7) 70 (46.7) 1 Abdominal (%) 47 (17.6) 137 (19.4) 0.52 21 (14) 24 (16) 0.64 Urinary tract (%) 33 (12.4) 68 (9.6) 0.25 19 (12.7) 18 (12) 0.87 Other (%)a 42 (15.7) 97 (13.8) 0.49 23 (15.3) 26 (17.3) 0.62 Co-infection (%) 26 (9.7) 85 (12.1) 0.39 17 (11.3) 12 (8) 0.32 Causative pathogens Gram-positive bacteria 70 (46.7) 73 (48.7) (%) 131 (49.1) 333 (47.2) 0.95 0.82 Gram-negative bacteria 81 (54) 87 (58) (%) 163 (61) 388 (55) 0.44 0.63 Yeast/fungi (%) 27 (10.1) 74 (10.5) 0.73 16 (10.7) 12 (8) 0.56 Other (%) 31 (11.6) 97 (13.8) 0.30 19 (12.7) 22 (14.7) 0.73 Unknown (%) 47 (17.6) 116 (16.5) 0.92 29 (19.3) 21 (14) 0.29 a Site of infection: "other" includes cardiovascular infection, mediastinitis and skin infection.

5 Supplemental table 2. Association of antiplatelet therapy with 60-day mortality using cox-proportional hazards regression in unmatched and propensity-matched cohort

Unmatched cohort Propensity-matched cohort Unmatched cohort adjusted for propensity HR 95% CI p HR 95% CI p HR 95% CI p 1.00 0.97- 0.98 1.12 0.75-1.66 0.58 1.11 0.82-1.51 0.49 Antiplatelet therapy 1.27 1.02 1.02- <0.00 1.02 1.01-1.02 <0.00 1.02 1.02-1.02 <0.000 APS 1.02 01 01 1 APS, acute physiology score; HR, hazard ratio; CI, confidence interval.

6 Supplemental table 3. Association of antiplatelet therapy with 90-day mortality using cox-proportional hazards regression in unmatched and propensity-matched cohort

Unmatched cohort Propensity-matched cohort Unmatched cohort adjusted for propensity HR 95% CI p HR 95% CI p HR 95% CI p 0.93 0.74- 0.55 0.95 0.66-1.37 0.78 1.03 0.77-1.39 0.84 Antiplatelet therapy 1.18 1.02 1.02- <0.00 1.01 1.01-1.02 <0.00 1.02 1.02-1.02 <0.000 APS 1.02 01 01 1 APS, acute physiology score; HR, hazard ratio; CI, confidence interval.

7 Supplemental table 4. Association of antiplatelet therapy with 30-day mortality using Cox-proportional hazards regression in a subgroup of shock patients (defined as the use of vasopressors for hypotension during 50% of the ICU day) of the unmatched and propensity-matched cohort Unmatched cohort Propensity-matched cohort Unmatched cohort adjusted for propensity HR 95% CI p HR 95% CI p HR 95% CI p 0.93 0.62- 0.71 1.04 0.53-2.06 0.90 1.22 0.74-2.02 0.44 Antiplatelet therapy 1.40 1.02 1.01- <0.00 1.02 1.01-1.03 0.004 1.02 1.01-1.02 <0.000 APS 1.02 01 1 APS, acute physiology score; HR, hazard ratio; CI, confidence interval.

8 Supplemental table 5. Association of antiplatelet therapy with 30-day mortality using Cox-proportional hazards regression in the complete unmatched and propensity-matched cohort including the interaction with shock during the first admission day Unmatched cohort Propensity-matched cohort Unmatched cohort adjusted for propensity HR 95% CI p HR 95% CI p HR 95% CI p 1.18 0.85- 0.33 1.47 0.82-2.62 0.19 1.35 0.91-1.99 0.14 Antiplatelet therapy 1.65 1,02 1.02- <0.00 1.01 1.01-1.02 0.000 1.02 1.02-1.03 <0.000 APS 1.03 01 6 1 1.08 0.80- 0.60 1.80 0.94-3.43 0.08 1.11 0.82-1.49 0.51 Shock (50% of day) 1.46 0.76 0.45- 0.31 0.68 0.29-1.60 0.38 0.80 0.47-1.32 0.42 Shock*Antiplatelets 1.29 APS, acute physiology score; HR, hazard ratio; CI, confidence interval.

9 Supplemental table 6. Association of antiplatelet therapy with 30-day mortality using Cox-proportional hazards regression in a subgroup of shock (defined as the use of vasopressors for hypotension during at least 6 hours) patients of the unmatched and propensity-matched cohort Unmatched cohort Propensity-matched cohort Unmatched cohort adjusted for propensity HR 95% CI p HR 95% CI p HR 95% CI p 1.06 0.75- 0.74 1.21 0.68-2.17 0.51 1.29 0.81-2.04 0.28 Antiplatelet therapy 1.50 1.02 1.01- <0.00 1.01 1.00-1.03 0.005 1.02 1.01-1.02 <0.000 APS 1.02 01 1 APS, acute physiology score; HR, hazard ratio; CI, confidence interval.

10 Supplemental table 7. Association of antiplatelet therapy with 30-day mortality using Cox-proportional hazards regression in the complete unmatched and propensity-matched cohort including the interaction with shock during the first admission day Unmatched cohort Propensity-matched cohort Unmatched cohort adjusted for propensity HR 95% CI p HR 95% CI p HR 95% CI p 1.08 0.74- 0.68 1.32 0.68-2.57 0.41 1.28 0.82-1.99 0.27 Antiplatelet therapy 1.59 1.02 1.02- <0.00 1.01 1.00-1.02 0.002 1.02 1.01-1.03 <0.000 APS 1.03 01 1 1.31 0.97- 0.08 2.03 1.05-3.91 0.04 1.34 0.996- 0.053 Shock (>6hours) 1.76 1.81 0.96 0.57- 0.87 0.91 0.38-2.14 0.82 0.96 0.57-1.63 0.89 Shock*Antiplatelets 1.61 APS, acute physiology score; HR, hazard ratio; CI, confidence interval.

11 Supplemental table 8. Plasma biomarkers in unmatched cohort.

Day 0, 2, Admission (day 0) Day 2 Day 4 4 Antiplatelet No antiplatelet Antiplatelet No antiplatelet Antiplatelet No antiplatelet p a therapy therapy p therapy therapy p therapy therapy p N = 226 b N = 594 N = 186 N = 488 N = 124 N = 356 Coagulation 0.0 Platelets (x 109/l) 0.002 187 [125-275] 176 [96-264] 0.03 189 [127-270] 166 [87-255] 2 190 [101-280] 169 [70-271] 0.07 0.4 D-dimer (µg/ml) 0.20 10.7 [4.7-18.8] 9.2 [4-17.1] 0.10 10.3 [4.6-18.9] 9.2 [4.4-17.3] 8 12.6 [7.1-19.6] 9.8 [5.2-17.7] 0.07 110.3 [83.9- 116.2 [89.8- 123.6 [88.5- 0.5 136.1 [96.9- 134.1 [93.8- Protein C (ng/ml) 0.44 119.6 [93.6-151] 155.1] 0.16 157.8] 160.4] 4 180.3] 184.9] 0.75 745.7 [495.2- 740.6 [511.9- 685.8 [435.6- 703.8 [439.1- 0.9 797.7 [562.5- 870.7 [569.6- Antithrombin (ng/ml) 0.99 1107.3] 1066.2] 0.88 1037.6] 1068.5] 0 1304.9] 1385.6] 0.42 Endothelial cell activation 0.4 sE-Selectin (ng/ml) 0.72 10.6 [4.8-25.1] 10.4 [5-24.3] 0.95 9.6 [4.5-20.3] 9.2 [4.2-18.9] 4 8.3 [4.1-14.6] 7.9 [4-14.9] 0.76 175.3 [107.2- 198 [105.9- 195.7 [116.7- 213 [119.9- 0.3 216.1 [136- sICAM-1 (ng/ml) c 0.69 299.1] 316.8] 0.20 328] 350.7] 8 351.4] 212 [128.7-344.5] 0.58 0.1 Ang-1 (ng/ml) 0.69 2.1 [0.9-5.8] 2 [0.8-5.6] 0.74 1.8 [0.8-4.5] 1.6 [0.7-3.5] 4 1.4 [0.6-4] 1.5 [0.7-3.6] 0.99 0.9 Ang-2 (ng/ml) 0.59 7.5 [3.1-14.8] 7.3 [3.5-15] 0.51 8 [3.5-19.7] 8.3 [3.3-19.4] 8 5.7 [2.6-11.5] 6.2 [2.7-14.4] 0.49 0.3 Ang-2/Ang-1 ratio 0.53 2.9 [0.7-12.2] 2.9 [0.8-13.1] 0.72 4.3 [1.1-20.8] 5.2 [1.4-21.2] 9 3.7 [1-15.7] 3.9 [1-15.9] 0.82 Metalloproteinases 0.5 MMP-8 (ng/ml) 0.47 3.2 [1.1-11.3] 3.6 [1.1-11.7] 0.73 2.9 [0.9-7.6] 2.4 [0.8-8] 6 1.5 [0.5-4.3] 1.5 [0.6-4.7] 0.51 542.6 [295- 601.1 [267.7- 506.6 [293.5- 507.5 [245.5- 0.6 390.5 [234.1- TIMP-1 (ng/ml) 0.94 1031.5] 1243.7] 0.36 878.2] 915.4] 5 733.3] 363 [216.6-690.8] 0.58 Pro-inflammatory cytokines 0.7 TNF-α (pg/ml) 0.90 1 [0.7-1.7] 1.1 [0.7-1.7] 0.78 1 [0.7-1.7] 0.8 [0.7-1.5] 5 0.9 [0.7-1.7] 1.1 [0.7-1.7] 0.99 0.4 IL1-β (pg/ml) 0.39 2.4 [1.1-4.4] 2.6 [1.1-5.6] 0.11 2 [1-3.3] 2.4 [1-3.4] 2 1.9 [1-2.9] 2.3 [1-3.4] 0.63 156.3 [45.8- 204.1 [41.2- 59.5 [22.7- 73.1 [26.1- 0.1 IL-6 (pg/ml) 0.09 740.4] 1568.6] 0.30 156.6] 285.1] 0 38.2 [14.3-103.2] 37.7 [14.7-114.5] 0.54 110.6 [49.3- 140.6 [48.1- 71.2 [29.2- 0.4 IL-8 (pg/ml) 0.24 382.7] 563.5] 0.34 151.5] 79.4 [27.5-214] 0 54.9 [18.5-135.7] 60.9 [22.8-148.6] 0.39

12 Anti-inflammatory cytokines d 0.0 IL-10 (pg/ml) 0.03 12 [4.2-43.6] 14.3 [4.7-57.2] 0.19 5.9 [2.7-14] 7.5 [3-22.2] 5 3.9 [1.9-11] 5.3 [2.3-14.2] 0.04 Renal injury 0.000 0.0 Creatinine (µmol/l) 0.0005 139 [84-224] 110 [71-177] 1 110 [74-183] 95 [61-166] 1 104 [66-168] 85 [58-143] 0.02 289.7 [169.1- 286.3 [143.7- 264.1 [148.4- 249.2 [115.6- 0.2 209.8 [129.5- 180.6 [106.2- NGAL (ng/ml) e 0.21 518.7] 525.8] 0.55 481.5] 476] 3 387.3] 329.2] 0.12 0.0 Cystatin C (µg/ml) 0.05 1.5 [1-2.4] 1.3 [0.9-2.3] 0.06 1.6 [1.1-2.5] 1.3 [0.9-2.3] 5 1.6 [1-2.4] 1.5 [1-2.3] 0.40 Plasma levels on day 0, 2 and 4 after intensive care unit admission. Results are presented as medians and interquartile ranges. Ang, angiopoietin; IL, interleukin; MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase-associated lipocalin; sE-selectin, soluble E-selectin; sICAM-1, soluble intercellular adhesion molecule-1; TIMP, tissue inhibitor of metalloproteinase; TNF-α, tumor necrosis factor-α. a Mixed-effects models comparing antiplatelet users with non-users over time. b Number of patients of whom plasma was available for measurement of biomarkers; platelet counts and creatinine levels were available for all patients. c Soluble intercellular adhesion molecule-1 also originates from leukocytes. d Levels of the anti-inflammatory cytokine IL-13 were below detection limit of the assay in the vast majority of patients and not different between groups (data not shown). e NGAL levels can also be elevated in sepsis as a consequence of leukocyte activation.

13 Supplemental table 9. Plasma biomarkers in healthy volunteers.

Healhy volunteers N = 27 Coagulation Platelets (x 109/l) a 150-400 D-dimer (µg/ml) 0.7 [0.3-1.6] Protein C (ng/ml) 148 [128.8-209] 2268 [1355- Antithrombin (ng/ml) 8307] Endothelial cell activation sE-Selectin (ng/ml) 4.4 [1.9-9.5] sICAM-1 (ng/ml) b 80.9 [19.9-105] Ang-1 (ng/ml) 10.6 [5.6-16.9] Ang-2 (ng/ml) 1.4 [0.6-2.1] Metalloproteinases MMP-8 (pg/ml) 68 [44.7-207.1] 110.7 [92.4- TIMP-1 (ng/ml) 147.6] Pro-inflammatory cytokines TNF-α (pg/ml) 0.7 [0.7-1.7] IL1-β (pg/ml) 2.8 [1.1-3.8] IL-6 (pg/ml) 0.9 [0.4-1.8] IL-8 (pg/ml) 4.2 [2.3-6.6] Anti-inflammatory cytokines c IL-10 (pg/ml) 0.7 [0.7-0.9] Renal injury Creatinine (µmol/l) a 75-110 NGAL (ng/ml) d 54.6 [41.3-68.6] Cystatin C (µg/ml) 0.8 [0.7-1.1] Results are presented as medians and interquartile ranges. Ang, angiopoietin; IL, interleukin; MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase-associated lipocalin; sE-selectin, soluble E-selectin; sICAM-1, soluble intercellular adhesion molecule-1; TIMP, tissue inhibitor of metalloproteinase; TNF-α, tumor necrosis factor-α. a Clinical laboratory reference ranges. b Soluble intercellular adhesion molecule-1 also originates from leukocytes. c Levels of the anti-inflammatory cytokine IL-13 were below detection limit of the assay in the vast majority of patients (data not shown). d NGAL levels can also be elevated in sepsis as a consequence of leukocyte activation.

14 SUPPLEMENTAL FIGURE

Supplemental figure 1: Distribution of propensity scores in matched and unmatched users and non-users.