This study was approved by the Ethics Committee of the medical faculty of the University Hospital Magdeburg (application number 88/18) and the subjects provided written informed consent prior to the blood drawing procedure.

1. Heparin stock preparation and blood sampling

1. Calculate the amount of blood needed for the entire experiments.
   NOTE: Almost, 5 mL of heparinized blood is required for each vascular device in duplicates (loops). Likewise, 5 mL of blood is required for each baseline and static condition which is kept aside at room temperature.
2. Prepare a heparin stock solution at the concentration of 100 IU/mL by diluting the unfractionated heparin (e.g., Rotexmedia) in deionized water. Use 150 µL of this solution for the heparinization of 10 mL fresh blood.
3. Calculate the amount of blood as well as plasma that are needed for whole blood cell count, ELISA and FACS assays.
   NOTE: The measurements represented in this study are obtained from two loops running in parallel (one as duplicate) with a total blood volume of 10 mL.
4. Based on the required amount of blood, fill 10 mL syringes with 150 µL of heparin stock solution to prevent blood coagulation with a final heparin concentration of 1.5 IU/mL blood.
5. Draw blood using a butterfly (size: 21 G). Fill the syringes very gently to avoid hemolysis or cell activation due to excessive vacuum.
   NOTE: Permission from the local ethical committee should be obtained before the commencement of the experiments. Obtain informed consent from each human blood donor. Ensure that the blood donor is healthy and does not take any medication, especially no antiplatelet agents or non-steroidal anti-inflammatory drugs for at least 10 days prior to the experiments. Of note, same donor is preferred when comparing different types of vascular devices for the first time as presented in this manuscript. To further evaluate the inter-individual differences, the protocol can be repeated with different donors.
6. Collect blood in a glass beaker, avoid excessive agitation.

2. In vitro hemodynamic loop assembly

1. Fill the water bath until the water level reaches up to the center of the rotation unit. Set water temperature to 37 °C.
2. Loop assembly for testing different tubing materials (polyPVC, hepPVC, PVC and latex)
   1. Cut two 50 cm long pieces of each tube material (inner diameter 5 mm) with the tube cutter. Ensure that the cutting surface is flat, as this is particularly important for perfect closure for the loops with small diameters so that blood flows without any distortion on the fitting edge.
      NOTE: This entire protocol utilizes the tubes with an inner diameter of 5 mm.
   2. To ease the handling and to avoid post-sample processing delay after rotation, run only four loops (2 materials in duplicates) in parallel.
   3. To generate a loop shape, plug the open endings of the tubes into a short piece of silicon tube fitting the outer diameter of the investigative tube.
   4. Use the polycarbonate tension bands to ensure proper closing of the loops. When used for the first time, adjust the length of the bands to fit the outer diameter of the loop by cutting the tension band with scissors in required lengths and secure it with a 3 mm torque wrench.
   5. Carefully tighten the locking screw of the tension band connector under inspection of the tube endings. Adjust the closing force so that no gap remains between the tube endings. If the locking screw is totally tightened and the tension of the polycarbonate band seems too low to close the gap between the tube endings, open the locking the system and cut a few mm of the tension band. Repeat this, until accurate loop closure is achieved (Figure 1A).
   6. If the tubing material is very soft and tends to slip inside the tension bands, fix the loop with electrical tape to the tension band (Figure 1B,C).
   7. Prepare an additional loop of PVC for temperature control with the same dimensions as the test loops.
   8. Secure the loops in the loop cradle of the rotation unit outside the water bath. Thereafter, attach the loop cradle to the rotation unit inside the water bath (Figure 1E).
   9. Dismantle the tension band partly from the loops and unplug one end of each tube to open the loop.
   10. Gently fill each loop with 5 mL of blood with a 5 mL serological pipette. Mix blood gently twice in the glass beaker by slowly pipetting up and down before loading into the loops.
   11. Take the disposable thermometer and place it inside the temperature control loop. If the thermometer is too large, cut it with scissors to fit smaller tubes. Fill the loop with 5 mL of deionized water at room temperature.
   12. Close the loops and ensure whether the loops, the tension bands and the rack are properly fitted.
   13. Set rotation speed to 30 rounds per minute (rpm) and rotate for 3 h.
3. Loop assembly for testing the impact of improper loop closure (gap)
   1. Prepare four loops (polyPVC) as described in 2.2.
   2. Close two of the loops properly with the tension bands and avoid any gap between the tube endings.
   3. For the other two loops plug the open endings into the bigger tube as described in 2.2.3, but leave a gap of 1-2 mm in between the loop endings. Do not use the tension bands for these loops (Figure 1D).
   4. Prepare one temperature control loop as described in 2.2.7 and 2.2.11.
   5. Fill all loops with blood as described in 2.2.10.
   6. Set the rotation speed to 30 rpm and rotate for 3 h.
4. Loop assembly for stent testing
   1. Prepare four loops as described in 2.2 and following.
      NOTE: To assess the blood biocompatibility of stents, the tubing material itself should be tested to be biocompatible in order to prevent masking of cell activation. If no data exists, the tube material itself can be tested as described in 2.2. Furthermore, the diameter range within the stent can be applied (see manufacturer’s instructions) and should fit the inner diameter of the tube material.
   2. Open two of the loops and take the tube out of the tension band system.
   3. Insert the stent into the middle of the tube as per the manufacturer’s instructions.
   4. Use the other two loops without stent as control. Fill all loops with blood as described in 2.2.10.
   5. Set rotation speed to 30 rpm and rotate for 3 h.
5. Temperature control
   1. At any time during duration and when rotation is stopped, the temperature of the blood inside the loops is indicated by the thermometer inside the temperature control loop. To read off the temperature, stop the rotation and immediately read off the temperature indicated by the thermometer.

3. Blood sample processing

1. After rotation, let the loops stand in the rack for 2 min in an upward position to let the blood accumulate at the bottom of the loops, avoiding any spilling while opening the loops.
2. Inspect the blood left for static conditions: If this blood is coagulated, an improper heparinization is ultimately suspected. In this case, repeat the experiment preferably also with another blood donor.
3. Carefully take the loops out of the rack. Open the connectors and let the blood flow into a 10 mL glass beaker.
4. Pool the blood from the tubes that are run in duplicates.
5. Draw fresh blood for baseline analysis from the same donor as described in 1.5 and 1.6.
6. Collection of sodium citrate blood
   1. For the collection of sodium citrate blood, fill four 1.5 mL tubes, each with 111 µL sodium citrate solution collected from sodium citrate tubes, to generate a final concentration of 3.2% of sodium citrate.
   2. Fill each tube with 1 mL of blood from the glass beakers as described in 1.6 and 3.3.
   3. Keep the blood at room temperature and use the this blood for FACS analyses as described in 12.
      NOTE: To change the length of the loops for different experimental setups, properly plan aliquots based on the amount of measurements to be made. It may be necessary to establish all required measurements with fresh blood prior to the real experiments to ensure the volume of blood in the loops is enough for all measurements.
7. Collection of ethylenediaminetetraacetic acid (EDTA) blood and plasma samples.
   1. From each glass beaker with blood (see 1.6 and 3.3) transfer 4.5 ml of blood into one 5 ml EDTA tube. Fill two EDTA tubes from each 10 ml glass beaker with blood.
   2. Gently mix the blood and keep it on ice.
   3. Transfer 2 mL of blood into a 2 mL locking centrifugation tube and use this blood for blood cell count and free hemoglobin measurement as described in 5. And 6.
   4. Centrifuge the remaining blood at 3,500 x g for 20 min.
   5. Carefully collect the plasma in the supernatant in 500 µL aliquots and immediately freeze at -80 °C.

4. Scanning electron microscopy and µCT images

1. After blood sample processing, rinse the emptied loops prepared as described in 2.3 with 10 mL of phosphate-buffered saline (PBS) each.
2. Carefully cut off a 1 cm long sample from each end of each tube with a scalpel.
3. Incubate sample overnight at 4 °C in a 2% glutaraldehyde solution.
   CAUTION: Inhalation of aldehyde vapors can cause nasal symptoms such as a runny nose ore persistent stuffiness and airway irritation, and contact with skin causes dermatitis. Aldehydes should be handled in a fume-hood while wearing gloves, a protective gown and safety goggles.
4. Rinse the sample (3 times) with PBS.
5. Prepare a 1% solution of osmium tetroxide (OsO₄) in deionized water and incubate sample for 15 min at room temperature.
   CAUTION: OsO₄ is a strong oxidizing agent, it can be reduced by exposure to light. To avoid the reduction during preparation, store OsO₄ in a brown glass bottle. OsO₄ is extremely volatile, its fumes are toxic to eyes, nose and throat. Always work under a fume-hood and use gloves and protecting clothing, ensuring that no part of the body is exposed to OsO₄. Contact your institution’s guidelines regarding handling of waste disposal and storage. In general, OsO₄ is storable for several months, but it needs special glass bottles with Teflon liner and a desiccator, because OsO₄ can discolor internal surfaces and contents of the refrigerator in the presence of leaking fumes.
6. Take out samples, transfer them in a new 15 mL centrifuge tubes and rinse samples 3 times with PBS.
7. Prepare a series of ethanol with varying concentrations (25%, 50%, 75%, 95%, 100%)
8. Dehydrate samples in ethanol: incubate for 20 min each in 25%, 50%, 75% and 90% and 30 min in 100%.
9. Take samples out of 100% ethanol and let it air dry overnight at room temperature.
10. Examine samples in the scanning electron microscope at an acceleration voltage of 5 kV and with the X-ray µCT scanner.

5. Blood cell count

1. Take 2 mL of the EDTA blood obtained as described in 3.7.3
2. Insert the tube into the automated hematology analyzer and follow the manufacturer’s instructions.

6. Measurement of free hemoglobin (fHb) in plasma

1. Thaw one plasma sample from each condition obtained as described in 3.7.5. Store on ice after thawing.
   NOTE: Always thaw frozen plasma samples in a water bath at 37 °C and immediately transfer to ice, containing some water, to lower the temperature. This is important to avoid activation of blood components during thawing.
2. Use the fHb reagent and follow the manufacturer’s instructions. Avoid contamination after opening and protect the reagent from direct light (sun, UV light).
3. Use a pipetting scheme (see manufacturer’s instructions) with 1:5 dilution.
4. Add 1000 µL of the hemoglobin reagent in a 1.6 mL semi-micro cuvette. Use this cuvette to determine the blank value.
5. Add 1000 µL of the hemoglobin reagent and 250 µL of the plasma sample in another semi-micro cuvette.
6. Mix the contents in both cuvettes by flushing the pipette thoroughly by repeatedly filling with reaction mixture, and incubate at least 3 min at room temperature.
7. Determine the extinction (E) of the sample against fHb reagent as blank reagent. Calculate the fHb-concentration (mmol/l) of the sample: E₅₄₀-E₆₈₀ x 0.452.

7. Measurement of FPA

1. Thaw a plasma sample from each condition obtained as described in 3.7.5 and store on ice after thawing.
2. Use the FPA Elisa kit and follow the manufacturer’s instructions.

8. Measurement of sC5b9

1. Thaw a plasma sample from each condition obtained as described in 3.7.5 and store on ice after thawing.
2. Use the sC5b-9 ELISA kit and follow the manufacturer’s instructions.

9. Measurement of PMN

1. Thaw a plasma sample from each condition obtained as described in 3.7.5 and store on ice after thawing.
2. Use the PMN-Elastase ELISA kit and follow the manufacturer’s instructions.

10. Measurement of TNF

1. Microplates must be coated one day before running the ELISA. For coating, add 100 µL of capture antibody solution to all wells, seal plate and incubate overnight at 2 °C-8 °C.
2. Thaw one plasma sample from each condition obtained as described in 3.7.5. Store on ice after thawing.
3. Use the TNF ELISA kit and follow the manufacturer’s instructions.

11. Measurement of IL-6

1. Microplates must be coated with capture antibodies one day prior to the experiment. For coating, add 100 µL of capture antibody solution to all wells of the microplate provided with the set, seal plate and incubate overnight at 4 °C
2. Thaw a plasma sample from each condition obtained as described in 3.7.5. and store on ice after thawing.
3. Use the IL-6 ELISA kit and follow the manufacturer’s instructions..

12. FACS analyses

1. Prepare 500 mL of FACS buffer by adding 10 mL of fetal calf serum and 2 mL of EDTA solution (0.5 M) to 488 mL of 1x PBS. The FACS buffer can be stored for 4 weeks at 4°C.
2. Use 100 μL of the sodium citrate blood (see 3.6.3) for each staining procedure (monocyte platelet aggregates (MPA), platelet activation (PA), leukocyte integrins (LI)).
3. Pipette 100 μL of blood from each sample into a 5 mL FACS tube. From each sample, prepare 3 tubes for antibody staining and label with MPA (monocyte platelet aggregates) panel, PA (platelet aggregates) panel and LI (leukocyte integrin) panel.
4. Mix the rest of the 4 samples into one 5 mL FACS tube. Use this mix for unstained and fluorescence minus one (FMO) controls.
5. Prepare 6 FACS tubes by pipetting 100 µL of the mixed sample blood into each tube. Label 3 tubes as unstained and 3 tubes as FMO-CD41, FMO-CD62P and FMO-CD162.
6. Add 100 µL of 4% paraformaldehyde solution and incubate for 15 min in the dark at room temperature.
   CAUTION: Paraformaldehyde is toxic to skin and eyes. It can cause serious pulmonary irritation when inhaled and can lead to lung damage after prolonged exposure. Furthermore, it is classified as a carcinogen and a reproductive toxin. Always wear gloves and safety glasses and work under the fume hood.
7. Add 1 mL of wash buffer to each tube and centrifuge at 287 x g for 5 min. Discard the supernatant and repeat wash step two times.
8. For red blood cell (RBC) lysis, add 1 mL of 1x buffer RBC lysis buffer (dilute 10x RBC lysis buffer in deionized water), mix by slowly pipetting up and down and incubate for 5 min at RT in the dark.
9. Prepare compensation beads for single stains. To 1 mL of FACS buffer add 4 drops of each positive and negative beads. Vortex thorough and add 100 µL of beads solution to one 5 mL FACS tube.
10. Prepare 4 tubes with beads and label as CD14-FITC, CD41-BV421, CD45-APC and CD62P-PE. Add 1 mL of FACS buffer to each tube and centrifuge at 287 x g for 5 min. Discard supernatant and keep on ice.
11. After RBC lysis, add 1 mL of FACS buffer to each tube and wash as described in 12.7. Discard supernatant.
12. Prepare antibody cocktails:
    1. MPA panel: Add 4 µL of CD45-APC, 4 µL of CD14-FITC and 4 µL of CD41-BV421 to 388 µL of FACS buffer.
    2. PA panel: Add 1.6 µL of CD41-BV421 and 12 µL of CD62P-PE to 386.4 µL of FACS buffer.
    3. LI panel: Add 4 µL of CD45-APC and 8 µL of CD162-BV421 to 388 µL of FACS buffer. Keep on ice.
13. Prepare single stains: Add 0.5 µL to 499.5 µL of FACS buffer each for CD14-FITC, CD41-BV421 and CD45-APC. For CD62P-PE add 0.3 µL to 499.7 µL of FACS buffer. Keep on ice.
14. Prepare FMO controls: (i) MPA panel (FMO-CD41): Add 1 µL CD14-FITC antibody and 1 µL of CD45-APC antibody to 98 µL FACS buffer. (ii) PA panel (FMO-CD62P): Add 0.4 µL of CD41-BV421 to 99.6 µL of FACS buffer. (iii) LI panel (FMO-CD162): Add 1 µL of CD45-APC to 99 µL of FACS buffer. Keep FMO controls on ice.
15. Vortex antibody cocktails and add 100 µL of each antibody cocktail as prepared in 12.12 to each of the respective labeled tubes (MPA, PA and LI panel) as described in 12.3 and mix gently by pipetting up and down. Keep on RT in the dark.
16. Vortex single stain antibody dilutions and add 100 µL of the single stain antibody dilutions as prepared in 12.13. to each of the respective labeled tubes with beads as described in 12.7 and mix gently by pipetting up and down. Keep on RT in the dark.
17. Vortex FMO antibody cocktails and add 100 µL of each FMO-1 antibody cocktail as prepared in 12.14. to each of the respective labeled tubes (FMO controls) as described in 12.5. and mix gently by pipetting up and down. Keep at RT in the dark.
18. Incubate all tubes with antibody staining for 30 min at RT in the dark.
19. Take the three tubes for unstained control (see 12.4) and resuspend pellet in 250 µL of FACS buffer. Keep on ice.
20. After incubation time, wash all tubes except unstained controls one time with FACS buffer as described in 12.7. Resuspend pellet in in 250 µL of FACS buffer and acquire data on the flow cytometer.
21. Use unstained cells for negative settings and compensation mouse beads for positive settings in FACS software. Further, use FMO to control the gating strategy, where FMO-CD41 is used in MPA panel, FMO-CD62P is used in PA panel and FMO-CD162 is used in LI panel.
22. Acquire almost 0.5 x 10⁶ – 0.25 x 10⁶ events per tube for FACS. Save data and analyze with an analysis software (version 9.9.6).