Journal: Journal of Thrombosis and Haemostasis

Article Title: Clustering of glycoprotein VI ( GPVI ) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets

doi: 10.1111/jth.13613

Figure Lengend Snippet: Comparison of distributions of glycoprotein VI ( GPVI ) dimer clusters, glycoprotein I b ( GPI b) and integrin α 2 β 1 on adhered platelets, and the effect of inhibiting α 2 β 1 on cluster formation and adhesion. (A, B) GPI b (set of 15 images in panel A) and α 2 β 1 (set of 15 images in panel B): confocal images of adhered platelets prelabeled with Alexa Fluor 488‐conjugated 204‐11 Fab (anti‐ GPVI ‐dimer; 4 μg mL −1 , green) and Alexa Fluor 647‐conjugated anti‐human CD 42 clone 486805 (anti‐ GPI b, 5 μg mL −1 , red) or Alexa Fluor 647‐conjugated 16B4 (anti‐α 2 β 1 ; 5 μg mL −1 , red) and then allowed to adhere on collagen III (Col III )‐coated, III ‐30‐coated or Horm collagen (Horm)‐coated dishes. Transmitted light images are included to show the degree of spreading, and magnified images of the platelet(s), indicated by white arrows, are included. The distributions of GPI b were different from those of the GPVI dimers for all three collagenous substrates, and were not associated with the Horm fibers. Integrin α 2 β 1 bound to the Horm fibers, following a similar pattern as the GPVI dimer clusters, coinciding with GPVI dimer clusters at some points, but not being associated with the lammelipodia of the platelets spread on Col III or III ‐30. (C, set of 8 gray scale images, right‐side of the figure) Total internal reflection fluorescence microscopy ( TIRFM ) images (15‐min time point, upper row in each four‐image group) and differential interference contrast ( DIC ) images (lower row in each four‐image group). Platelets were prelabeled with Alexa Fluor 488‐conjugated 204‐11 Fab, treated with Gi9 (anti‐α 2 β 1 , 5 μg mL −1 ; + Gi9) or an equal volume of phosphate‐buffered saline ( PBS ) (No Gi9), and allowed to adhere on Col III or Horm under TIRFM monitoring for 30 min. Gi9 treatment decreased but did not prevent adhesion and GPVI dimer clustering on fibrous Horm, but little or no adhesion was seen on Col III , even at the 30‐min time point. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The antibodies used were: 4G10 (anti‐phosphotyrosine; Millipore Merck, Billerica, MA, USA); anti‐human CD42b/GPIb, clone 486805 (R&D Systems, Abingdon, UK); 16B4 (mouse anti‐human CD49b/integrin α 2 chain; Bio‐Rad, Hercules, CA, USA); Alexa Fluor 647‐conjugated AffiniPure F(ab′)2 fragment goat anti‐mouse IgG, Fcγ‐specific (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA); Gi9 (anti‐integrin α 2 ; Abcam, Cambridge, UK); and Alexa Fluor 647‐conjugated anti‐human CD62P (Bio‐Rad).

Techniques: Comparison, Fluorescence, Microscopy, Saline