Vlk deficiency in platelets does not affect platelet morphology or levels of platelet receptors and cargo proteins. (A) Analysis of genomic DNA from Vlk^+/+::Pf4-Cre, Vlk^f/f, Vlk^f/+::Pf4-Cre, and Vlk^f/f::Pf4-Cre mice. Targeted loxP allele (fl) is 350 bp; wildtype allele (+) is 241 bp; and Pf4-Cre allele is 450 bp. (B) Western blot analysis of phosphorylated and total VLK immunoprecipitates (VLK-IP) prepared from Vlk^f/f (CTL) and Vlk-cKO (cKO) washed platelet lysates. Lysates from K4 synoviocytes stably expressing green fluorescent protein (GFP) or wild-type mouse VLK (VLK^WT) were used as controls for VLK protein. Eluates were treated with PNGase-F. Anti–P-selectin and GAPDH were used as controls for platelet-specific and total protein levels in input samples, respectively. (C) Representative image of electron microscopy analysis of resting Vlk^f/f and Vlk-cKO platelets. Dense granules are indicated by black arrows. Representative images were obtained at 13 000×. Protein levels in platelets from Vlk^f/f (CTL) and Vlk-cKO (cKO) mice (n = 3 mice per genotype). Anti-GAPDH and actin antibodies were used to examine total protein levels of platelet receptors integrin (ITG) β₃, integrin α_2b, glycoprotein Ibα (GpIbα), and P-selectin (D) as well as platelet cargo fibrinogen (FGN), von Willebrand factor (vWF), and PF4 (E).