Gpibα

Platelet lysates were prepared using 1X NP-40 lysis buffer added to washed platelets. GRIP1 (#AB5547, Millipore), 14-3-3 (#sc-629, Santa Cruz), GPIbα (#M040-0, Emfret Analytics), or GPIbβ (#M050-0, Emfret Analytics) antibodies were added to WT and GRIP1^-/- platelet lysates and incubated overnight at 4°C. Protein A/G beads (#sc-2003, Santa Cruz) were added for a minimum of 2 hours. Bead-antibody complexes were washed once with PBS and Laemmli running buffer was added. Tris-Glycine gels (4–15%, Bio-Rad) were used for Western blots with nitrocellulose membrane transfer. All primary antibodies (GRIP1 (#611318, BD), 14-3-3 (#sc-629, Santa Cruz), 14-3-3ζ (#sc-1019), GPIbα (#M040-0, Emfret Analytics), or GPIbβ (#M050-0, Emfret Analytics) were incubated overnight at 4°C. Infrared fluorescent secondary antibodies (anti-mouse Alexa Fluor 750, #A-21109, Thermo Fisher Scientific and anti-rabbit Alexa Fluor 680, #A-21037, Thermo Fisher Scientific) were added for detection on Licor (Licor).

The receptor levels on the surface of resting platelets were determined by labeling 10 μL of washed platelets at 2 × 10⁸ ml⁻¹ with 50 μL of Tyrodes containing 5 μl of the relevant FITC conjugated antibody (i.e. CD41, GPVI, CD49b, Clec2 or GPIbα; Emfret Analytics) and incubating in the dark for 30 min. Samples were diluted with 200 μl of modified Tyrodes buffer and analyzed using an Accuri C6 flow cytometer (BD Biosciences). Initial gating of platelets was performed using Forward Scatter/Side Scatter plots and then the fluorescence intensity (in FL1 channel) of 10,000 platelets in this gate was recorded for each sample. An isotype control antibody was used to subtract background and values presented as mean fluorescence intensity (MFI).
For P-selectin exposure and fibrinogen binding assays, platelets were stimulated with 0.1 U ml⁻¹ thrombin for 2 min at 37°C or with PBS (unstimulated controls) before staining with either anti-P-selectin-FITC or Alexa488-fibrinogen for 30 min and flow cytometry as described above.

For morphological bone analysis, femurs were fixed in 4% buffered formalin for at least 24 hours. Decalcification was performed in EDTA solution at room temperature for 2‐3 days. The decalcified bones were embedded in paraffin and cut in 3‐μm‐thick sections and stained with haematoxylin and eosin (H&E). Spleens were fixed in 4% formalin‐ and paraffin‐embedded. For histology, 3‐µm‐thick sections were cut and stained with H&E and Gomori stain. Immunohistochemistry was performed on an automated immunostainer (Ventana Medical Systems, Inc) according to the company's protocols for open procedures with slight modifications. All slides were stained with the antibody GPIbα (Emfret Analytics). Appropriate positive and negative controls were used to confirm the adequacy of the staining. MKs were counted at 20× HPF (magnification 200×).