Platelet characterization and molecular genetic analysis of Pat01∗ using a novel RASGRP2 variant. (A) Aggregation of platelet-rich plasma (light transmission %) from Pat01∗ (blue curves) and healthy control (dotted red curve), induced by ADP (4-20 μM), epinephrine (8-40 μM), collagen H (2-10 μg/mL), TRAP6 (5-10 μM), or arachidonic acid (0.5 mg/mL). Platelet agglutination was induced using ristocetin (1.2 mg/mL). (B) Flow cytometric analysis of diluted platelet-rich plasma, showing normal expression of αIIbβ3 (CD41). (C) Flow cytometry indicating impaired ADP-induced fibrinogen binding to Pat1∗ platelets, compared with those of CCFs (20 measurements from 6 controls; mean ± standard deviation). (D) Verification of a homozygous RASGRP2 canonical splice site variant by direct sequencing. (E) Pedigree after segregation analysis (brown symbol indicates affected Pat III.4; dotted symbol indicates heterozygous carrier). (F) Direct sequencing of platelet-derived cDNA showing skip of exon 5. (G) Absence of CalDAG-GEFI protein (CDGI) in patient platelets compared with that in a control subject via western blotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.