Platelet function is impaired in Tln1^3mutmice. (A) Western blots showing Talin1, RIAM, Kindlin-3, and Rap1 levels in WT and Tln1^3mut platelets. Actin served as loading control. (B) Surface levels of integrins α2, β1, αIIb, β3, and α5 assessed by flow cytometry (n = 4 mice). (C-D) Platelet integrin activation of control and Tln^3mut platelets analyzed by JON/A (C) and fibrinogen binding (D) upon stimulation with thrombin (n = 11 WT/12 Tln1^3mut), ADP (n = 11 WT/12 Tln1^3mut), U46619 (n = 5), a combination of ADP and U46619 (n = 5), or collagen-related peptide (CRP; n = 5). Activation index represents mean fluorescence intensity (MFI) values of JON/A-PE or fibrinogen–Alexa 647 normalized to integrin β3 levels. MFI values of WT platelets stimulated with 0.1 U of thrombin are set to 1; n represents the number of mice. (E) Representative platelet aggregation curves measured upon stimulation with different concentrations of collagen, U46619, thrombin, and CRP. Quantification of the data is provided in supplemental Figure 5. (F-G) Heparinized whole blood was perfused through micro flow chambers coated with fibrillar collagen to assess adhesion under flow. (F) Representative images of adherent CFSE-stained platelets after perfusion. Scale bar, 80 μm. (G) Quantification of platelet surface coverage normalized to platelet counts (n = 7 mice). (H) Quantification of platelet spreading area on fibrinogen 5, 10, and 30 minutes after activation with 0.01 U of thrombin (n = 5 experiments and mice). (I) Relative F-actin levels in resting vs thrombin-activated (0.01 U of thrombin) platelets quantified by phalloidin–Alexa Fluor 546 staining and subsequent fluorescence-activated cell sorter analysis (n = 4 mice). (J) Relative distribution of WT and Tln1^3mut platelets at different spreading stages 5, 10, and 30 minutes after plating on a fibrinogen-coated surface (n = 5 experiments and mice). Values are given as means ± 95% confidence intervals. *P < .05, **P < .01.