Loss of SHARPIN from platelets results in compromised thrombus formation on collagen under shear flow conditions. A predetermined volume of whole blood (130-200 μL) obtained from SHARPIN^fl/fl GPIbα-Cre⁻ (A,D,E) and SHARPIN^fl/fl GPIbα-Cre⁺ (B,F,G) mice was aspirated through matrix-coated iBidi μSlide VI^0.1 flow channels at the indicated shear rates. Adherent platelets were perfusion fixed and stained for αΙΙb, and images were acquired by deconvolution microscopy using a ×20 objective. The average platelet surface coverage was determined with Image Pro software (Media Cybernetics). (A-C) Fibrinogen matrix. n.s., not significant. (A-B) Deconvolved images of SHARPIN^fl/fl GPIbα-Cre⁻ (A) and SHARPIN^fl/fl GPIbα-Cre⁺ attached platelets (B). (C) Average 2-dimensional area per field of view of fluorescent platelets attached to fibrinogen (n = 5). (D-H) Collagen matrix. *P < .05; **P< .01. (D-G) Images of attached platelets and thrombi. Arrows indicate platelet aggregates. (D,F) G = 350 s⁻¹. (E,G) G = 1700 s⁻¹. (H) Six optical sections of 0.2 μm each were acquired, and 3D rendered images of platelets and thrombi were prepared. The average 3D surface area per field of view of fluorescent platelets on collagen was calculated using Image-Pro software (n = 5).