Unaltered inside-out activation of integrins in RIAM-null platelets. (A) Western blot analysis reveals the absence of RIAM protein (Epitomics, EP2806; 110 kDa) in RIAM-null platelets. (B) Unaltered platelet number and (C) size in RIAM-deficient mice as assessed by flow cytometry (FACSCalibur; BD Biosciences) and with a standard blood cell analyzer (Sysmex). (D) Integrins are normally recruited to the surface of RIAM-null platelets upon stimulation with thrombin (0.01 U mL−1). Unaltered activation of platelet (E) αIIbβ3-integrin (JON/A-PE) translates into normal (F) fibrinogen-binding and (G) aggregation responses of RIAM-null platelets as determined by (D-F) flow cytometry or (G) turbidometric aggregometry (APACT). For aggregation studies with thrombin, collagen-related peptide (CRP), collagen, and U46619 washed platelets (1.5 × 105 platelets per μL) were used; aggregation studies with ADP were performed in platelet-rich plasma (1.5 × 105 platelets per μL). U46, U46619, a stable thromboxane A2 analog; Rhd, rhodocytin. Activation of platelet (H) β1-integrin (9EG7-FITC), as well as (I) adhesion and (J) thrombus formation of RIAM-null platelets under flow (1000 s−1) on collagen I (100 μg mL−1) was indistinguishable from wild-type controls. Images were acquired with a Zeiss Axiovert 200 inverted microscope (40×/0.6 oil objective). Scale bars in I and J represent 25 µm. (B-F,H-J) Values are mean ± standard deviation. Curves in G represent light transmission over time, with platelet-rich plasma set as 0% and platelet-poor plasma as 100% aggregation. The presented results are representative of ≥3 independent experiments with at least n = 3 vs 3 individuals per group.
