Vlk deficiency in platelets alters platelet aggregation and granule exocytosis. Evaluation of aggregation of washed platelets (2 × 10⁸ platelets/mL) from control mice (gray) and platelet-specific VLK knockout mice (blue) stimulated with 100 μM PAR4 peptide AYPGKF (n = 11 per group) (A), 4 mg/mL collagen (n = 4 per group) (B), or 0.4 U/mL thrombin (n = 4 per group) (C). Representative tracings are shown in left panel and cumulative data in right panel. (D) Expression of P-selectin in platelets from control (gray) and platelet-specific VLK knockout mice (blue) in response to AYPGKF. (E) Protein immunoblots of lysates (Lys.) and supernatants (Sup.) from Vlk^f/f (CTL) and Vlk-cKO (cKO) platelets resting or stimulated with 100 µM PAR4 peptide AYPGKF for 15 minutes. Anti-Tsp1 and anti-PF4 antibodies were used to detect secretion of α-granule cargo, and anti-GAPDH antibody was used to determine total protein levels in lysates. (F) Concurrent monitoring of aggregation (blue tracing) and ATP release (red tracing) after exposure to 50 μM AYPGKF in platelets from control (left panel) and platelet-specific VLK knockout mice (right panel). (G) Aggregation studies in platelets from control (left panel) and platelet-specific VLK knockout mice (right panel) were performed as described in panel E except that ADP 2.5 μM was added together with AYPGKF. (H) Representative image of protein immunoblots of lysates and supernatants from CTL and cKO platelets resting or stimulated with 100 µM AYPGKF and/or 2.5 µM ADP for 15 minutes. Anti-Tsp1 antibody was used to detect secretion of α-granule cargo, and anti-GAPDH antibody was used to determine total protein levels. (I) Cumulative percent levels of Tsp1 in supernatants from CTL and cKO platelets stimulated with 100 µM AYPGKF and 2.5 µM ADP; n = 3 independent experiments. Quantifications were normalized to GAPDH protein expression.