Defective platelet activation and hemostasis in DKO mice is restored in TKO mice. (A) Anti-P-selectin-FITC, anti-triggering receptor expressed on myeloid cells-like transcript 1 (TLT-1)-FITC and fibrinogen-488 binding to washed platelets (2 × 107/mL) following stimulation with or without PAR4 peptide (AYPGKF) (100 μM or 500 μM, 20 minutes, room temperature) was measured by flow cytometry. The fold increase of the MFI relative to the corresponding unstimulated platelets was calculated, n = 5-7 mice/genotype. (B) Representative phalloidin-stained images of resting (basal) and thrombin-stimulated (0.1 U/mL, 5 minutes) platelets spread on fibrinogen-coated cover-slips (100 μg/mL, 45 minutes, 37°C, scale bar: 5 μm). (C) Mean surface area of individual platelets quantified by KNIME software, n = 6 mice/genotype (200 to 450 platelets/condition). (D) Hemostatic response was measured in saline tail bleeding assay by an excision of a 3-mm portion of the tail tip followed by immersion of the tail in 0.9% isotonic saline at 37°C. Plotted is the time to complete arrest of bleeding. Experiments were conducted in a double-blinded manner, n = 16 mice/genotype. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with Sidak test; mean ± SD.