DGKζ negatively regulates GPVI-mediated platelet activation but enhances thrombin (Thr)-mediated platelet activation. (A) Whole lysates of washed platelets and megakaryocytes (MKs) from WT and DGKζ-KO mice were analyzed by western blotting for DGKζ and actin. The blot shown on the left is representative of 3 to 4 independent experiments. Quantification of band densities (right) is reported as the mean ± SEM (n = 3-4). (B) Platelet counts (left) and mean platelet volumes (MPVs; right) observed in WT and DGKζ-KO mice. Values represent means ± SEM (n = 20). (Ci) Aggregation of washed platelets from WT and DGKζ-KO mice was measured by lumiaggregometry in response to high (top) and low (bottom) doses of CRP, collagen (Col), Thr, adenosine 5′-diphosphate (ADP), and U46619. Each aggregation tracing is representative of 3 to 5 independent experiments. (ii) Dose-response curve of maximal platelet aggregation after CRP, Col, and Thr stimulation. Values represent the mean ± SEM of maximal platelet aggregation observed in 3 to 4 independent experiments. (D) P-selectin exposure (left) and Jon/A binding as a reporter of activation of integrin αIIbβ3 (right) were measured by flow cytometry in washed platelets after 20 minutes of stimulation with CRP, Thr, ADP, and U46619 (U46) at the indicated concentrations. Values represent the mean ± SEM of mean fluorescence intensity (MFI) observed in 5 independent experiments. (E) Quantification of peak adenosine triphosphate (ATP) released from platelets stimulated with CRP (2.5 μg/mL), Col (5 μg/mL), and Thr (0.5 U/mL) relative to WT. ATP release was measured by lumiaggregometry. Values represent the mean ± SEM observed in 3 to 5 independent experiments. Statistical analysis was performed by the unpaired Student t test. *P < .05, **P <.01, ***P < .001, ****P < .0001 of DGKζ-KO as compared with WT. n.s., not significant.