**4. Platelet morphology**

The platelets-coated testing surfaces were fixed with freshly prepared 2.5% glutaraldehyde for 20 minutes. After washing with PBS, the samples were dehydrated in a graded-ethanol series (50, 70, 90, and 100% v/v) for 15 minutes each and allowed to dry at room tempera‐ ture. The platelet-attached surfaces were carbon sputter coated under vacuum to a thickness of 100–200 Å and examined at 10 kV using a Cambridge StereoScan 200 microscope (Cam‐ bridge Scientific Instruments, UK).

#### **4.1. Platelet aggregation**

The blood samples were collected in tubes containing PPACK (D-phenylalanyl-L-prolyl-Larginine chloromethyl ketone) as anticoagulant. Platelet aggregation was measured by means of light transmission aggregometry using Born's turbidimetric procedure and the PPACK-4 Platelet Aggregation Chromogenic Kinetic System (Helena Laboratories, USA). Briefly, 250 μL of PRP were incubated with specimen surfaces for 10 (baseline) and 60 mi‐ nutes. Thereafter,the PRP were placed in a cuvette containing a metal stir bar in the absence or in the presence (positive control) of the pro-aggregation agent, adenosine diphosphate (ADP) 20 μM. Upon the addition of ADP the platelets started to aggregate thus increasing light transmission through the sample. The degree of platelet aggregation was expressed as the maximum percentage change in light transmission from PPP used as baseline. The ob‐ tained values were expressed as mean of two measurements.

#### **4.2. Complement activation assay**

The test, based on Complement Reagents Kit (Siemens Healthcare Diagnostic, Germany) was performed on BCT Siemens coagulometer (Siemens, Germany). The test focused on the ability of the complement system to lyse a standard suspension of sheep erythrocytes, sensi‐ tized with a rabbit anti-serum against sheep erythrocytes. Briefly, 1 mL of fresh blood sam‐ ples previously incubated for 1 hour with different substrates were incubated with sensitized erythrocytes to investigate the complement activation. Diminished levels of indi‐ vidual components (e.g. due to prior activation by a foreign surface) result in a prolongation of the time taken for lyses. The time necessary for the lyses of a defined amount of erythro‐ cytes is used as basis for determining the complement activity [14,15]. The results were eval‐ uated using a reference curve prepared by a serial dilution of standard plasma with isotonic saline to give 100% of complement activity, 75% (75% of plasma + 25% saline) down to 10% of complement activity (10% of plasma + 90% saline).

#### **4.3. Statistical analysis**

Data were expressed as means ± standard deviation (SD). Where not differently stated, measurements were conducted at least in triplicate. Chi-square test or Student's t-test on un‐ paired data was used to assess the statistical significance of the difference between the re‐ sults obtained from the tested specimens (Kaleida-Graph, Synergy Software, USA). Statistical significance was assumed at a confidence level of 95% (p < 0.05).
