Functional roles of Shp1 and Shp2 in hemostasis and thrombosis. (A) Platelet spreading on fibrinogen. Basal and thrombin (0.1 U/mL)-activated platelets were plated on a fibrinogen-coated surface. (i) Representative differential interference contrast (DIC) and phalloidin-stained images of platelets; n = 3-5 mice/genotype. (ii) Surface area of individual platelets in DIC images was measured; n = 250-500 platelets/condition; n = 3 mice/genotype; mean ± SD; *P < .05; **P < .01; bar represents 5 μm. (B) Platelet adhesion to collagen under flow. Anticoagulated blood was flowed through collagen-coated capillary tubes at 1000 s−1. (i) Representative DIC images of adherent platelets; n = 3-5 mice/genotype; bar represents 10 μm. (ii) Percentage of surface coverage was measured. Data presented are means (± SEM) of 3 independent experiments. (C) Tail bleeding assay. Shp1/2 DKO (n = 13) mice bled excessively compared with litter-matched Shp1/2 WT mice (n = 20). Symbols represent individual mice; horizontal lines represent means. Mann-Whitney test was used to compare sample medians and determine statistical significance. (D) Laser injury-induced thrombus formation in vivo. Mice were injected with Dylight488-conjugated anti-GPIbβ antibody (X488). Arterioles in cremaster muscles of recipients were subsequently injured by laser and the accumulation of platelets (green) into the thrombi was assessed. (i) Representative composite brightfield and fluorescence images from X488-labeled platelets after laser injury of arteriole are shown. Bar represents 10 μm. (ii) Each curve represents the mean integrated thrombus fluorescence intensity (± SEM, gray; WT, top; KO, bottom) in arbitrary units (a.u.); n = 19-27 thrombi induced in 5-7 mice/genotype. See also supplemental Figure 2 video 1, Figure 2 video 2, Figure 2 video 3, and Figure 2 video 4.
