Lack of GPIIbIIIa (αIIbβ3) activation on platelets in the presence of normal CalDAG-GEF1 levels in the LAD1v syndrome. (A) PAC-1 binding to activated αIIbβ3 integrins as detected by flow cytometry (left panel) shows strongly decreased binding to patient platelets after thrombin stimulation (shaded histogram) compared with control platelets (open histogram, solid black line). The binding of CD61 antibody to measure total αIIbβ3 expression, as measured in parallel samples, is shown in the right panel. In both panels, the binding of negative mouse Ig-FITC to the stimulated patient platelets (thin black line) is shown for comparison. The slight shift observed with PAC-1 in the patient platelets was not observed without thrombin stimulation (not shown). (B) A homozygous sequence variant of CALDAGGEF1 in intron 15 is present in the patient. (C) The intronic gene variation results in normal splicing of mRNA. Both reverse-transcribed PCR and sequence analysis of control and patient cDNA that contains exons 12 to 18 gave the expected wild-type products of 802 bp, as shown here. The arrow in the sequence traces indicates the exon 15–exon 16 boundary. (D) Normal CalDAG-GEF1 protein expression on Western blotting at the expected relative molecular mass of approximately 75 kDa is found. Results are representative for 5 of the patients from families 1 through 7 tested in total.
