1. Rap1-GTP–interacting adaptor molecule (RIAM) is dispensable for platelet integrin activation and function in mice
by Simon Stritt, Karen Wolf, Viola Lorenz, Timo Vögtle, Shuchi Gupta, Michael R. Bösl, and Bernhard Nieswandt
Blood
Volume 125(2):
January 8, 2015
©2015 by American Society of Hematology

2. Unaltered inside-out activation of integrins in RIAM-null platelets.
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.
Simon Stritt et al. Blood 2015;125:
©2015 by American Society of Hematology

3. RIAM deficiency does not affect platelet outside-in signaling and in vivo thrombus formation.
RIAM deficiency does not affect platelet outside-in signaling and in vivo thrombus formation. RIAM-null platelets display a normal (A) spreading and (B) reorganization of the actin (red, Phalloidin Atto647N) and tubulin (green, α-tubulin-Alexa F488) cytoskeleton on a fibrinogen-coated (100 μg mL−1) surface. Values in A represent mean. Images in B were acquired with a TCS SP5 confocal microscope (100×/1.4 oil STED objective; Leica Microsystems). Scale bars represent 3 µm. (C-D) Platelet clot retraction of RIAM-null platelets was indistinguishable from wild-type controls. Values in D are mean ± standard deviation. In vivo, RIAM deficiency neither interfered with (E) normal hemostasis as assessed by a tail bleeding time assay nor with (F-G) arterial thrombus formation upon FeCl3-induced damage of the endothelium. Each symbol in E represents 1 individual. Each symbol in F represents 1 mesenteric arteriole. Horizontal lines in E and F represent mean. Arterioles in G were visualized with an Axiovert 200 inverted microscope and a 10×/0.25 objective (Zeiss). The presented results are representative of ≥3 independent experiments with at least n = 3 vs 3 individuals per group.
Simon Stritt et al. Blood 2015;125:
©2015 by American Society of Hematology