Anti cd61

The 3D‐printed silk bioink was housed in a customized device manufactured using 3D Stereolithography (LSA) printing technology (FormLab). The model was created using Fusion 360 (AutoDesk), and PreForm software was used for slicing (FormLab). The printing was done using a long‐term non‐toxic biocompatible resin (FormLab). A glass window at the bottom ensured possibilities for live‐cell imaging and high‐resolution microscopy. For immunofluorescence imaging samples were fixed in 4% formaldehyde for 20 min, at room temperature. Samples were probed with anti‐CD34 (1:100), anti‐CD61 (1:100), anti‐CD41 (1:100) (Beckman Coulter), CD42b (1:100) (Invitrogen), or anti‐β1‐tubulin (1:200) (Abcam), overnight at 4 °C. Alexa Fluor secondary antibody (1:500) (Invitrogen) have been incubated for 2 h at room temperature. Nuclei were stained with Hoechst 33 258 (Sigma–Aldrich). Samples were imaged by an SP8 confocal laser scanning microscope (Leica). For live imaging, samples were stained with FITC conjugated anti‐CD61 or anti‐CD41 antibodies, or Cytopainter Cell Plasma Membrane Staining Kit and NBD Cholesterol Staining Dye Kit (Abcam). Nuclei were stained with BioTracker NIR694 Nuclear Dye (Sigma‐Aldrich). Isotype controls were used as a negative control to exclude non‐specific background signals. The acquisition parameters were set on the negative controls.

Platelet count and parameters were determined using an automated hematology analyzer (Sysmex, Kobe, Japan). Platelet reactivity was determined in citrated whole blood (3.2% sodium citrate, Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometry based assay as previously described between 1 and 3 h after blood collection^{52 (link)}. Platelets were ex vivo stimulated with ADP (1.2 and 125 μM; Sigma-Aldrich, Zwijndrecht, The Netherlands) and CRP-XL (a kind gift from Prof. Farndale, Cambridge, UK) for 20 min at room temperature. Platelets were stained using anti-CD61 (Beckman Coulter, Brea. CA, USA), anti-P-selectin (Biolegend, San Diego, CA, USA) and anti-fibrinogen (DAKO, Santa Clara, CA) antibodies and fixated in 0.2% paraformaldehyde. Platelets were identified based on Size (FSC), granularity (SSC) and their expression of CD61. Degranulation was determined as the membrane expression of α-granule protein P-selectin and platelet aggregation was quantified as the amount of fibrinogen binding to the activated integrin αIIbβ3. Platelet reactivity was measured on a FC500 flow cytometer (Beckman Coulter, Brea, USA). Data were extracted using Kaluza 2.1 (Beckman Coulter), normalized against quality controls to ensure measurement stability and are expressed as median fluorescence intensity (MFI). A gating strategy is provided in Supplemental Fig. S1.