Figure 2.
Figure 2. Compound heterozygous CalDAG-GEFI mutation causes CalDAG-GEFI deficiency in patient platelets and delayed integrin αIIbβ3 activation kinetics. (A-B) Sequencing results of CalDAG-GEFI showing the heterozygous 1178A>T mutation (K309X) in patient (top) and father (bottom) (A) and heterozygous 1331_1333 deletion (L360del) in patient (top) and mother (middle) (B). Genomic DNA isolated from peripheral blood mononuclear cells was used as a template for polymerase chain reaction and amplified DNA fragments were sequenced. Reverse transcription polymerase chain reaction analysis of platelet mRNA also confirmed these mutations (data not shown). (C) Western blot analysis of CalDAG-GEFI in platelets from control, patient, her mother, and her father. (D) Western blot for CalDAG-GEFI in 293T cells transfected empty vector, wild-type CalDAG-GEFI, or mutant CalDAG-GEFI (K309X and L360del) vector. (E) Platelet integrin αIIbβ3 activation kinetics determined by initial velocity assay. Washed control and patient platelets were mixed with 100 μM PAR1-AP at time “zero.” At each indicated time point, 20 μL FITC-PAC-1 was added to the mixture. After 30 seconds of incubation with FITC-PAC-1, bound FITC-PAC-1 was immediately assessed by flow cytometry (n = 3: mean ± SD; Student t test, *P < .05, **P < .01). (F) The percentage of FITC-PAC-1 binding to patient platelets relative to control platelets. PAC-1 binding to patient platelets relative to control platelets at each time point was indicated as percent control MFI (n = 3: mean ± SD; Student t test, *P < .05, **P < .01). (G) Shear-induced thrombus formation. Whole blood from control or patient was perfused over collagen-coated surface at a wall shear rate of 1250 seconds−1. Thrombus volumes were measured by MetaMorph and VoxBlast software every minute (n = 3: mean ± SD). Representative thrombus images after 10 minutes of perfusion were shown (top and side view). (H) Statistical analysis of thrombus area and height. Surface coverage by adhered platelets (left) and thrombus height (right) after 10 minutes of perfusion were analyzed (n = 3). (I) Platelet integrin αIIbβ3 activation kinetics in patient with OSP-1 determined by initial velocity assay. In platelets of OSP-1, rapid initial αIIbβ3 activation at 30 seconds was induced the same as control platelets. However, αIIbβ3 activation was transient and almost no αIIbβ3 activation velocity at 300 seconds. (J) β2 integrin activation in neutrophils. Whole blood was stimulated with 200 nM fMLP or 100 ng/mL PMA, and induced β2 integrin activation was determined by binding of monoclonal antibody 24, which recognizes the activated form of β2 integrin (n = 3: mean ± SD; Student t test). WT, wild type.

Compound heterozygous CalDAG-GEFI mutation causes CalDAG-GEFI deficiency in patient platelets and delayed integrin αIIbβ3 activation kinetics. (A-B) Sequencing results of CalDAG-GEFI showing the heterozygous 1178A>T mutation (K309X) in patient (top) and father (bottom) (A) and heterozygous 1331_1333 deletion (L360del) in patient (top) and mother (middle) (B). Genomic DNA isolated from peripheral blood mononuclear cells was used as a template for polymerase chain reaction and amplified DNA fragments were sequenced. Reverse transcription polymerase chain reaction analysis of platelet mRNA also confirmed these mutations (data not shown). (C) Western blot analysis of CalDAG-GEFI in platelets from control, patient, her mother, and her father. (D) Western blot for CalDAG-GEFI in 293T cells transfected empty vector, wild-type CalDAG-GEFI, or mutant CalDAG-GEFI (K309X and L360del) vector. (E) Platelet integrin αIIbβ3 activation kinetics determined by initial velocity assay. Washed control and patient platelets were mixed with 100 μM PAR1-AP at time “zero.” At each indicated time point, 20 μL FITC-PAC-1 was added to the mixture. After 30 seconds of incubation with FITC-PAC-1, bound FITC-PAC-1 was immediately assessed by flow cytometry (n = 3: mean ± SD; Student t test, *P < .05, **P < .01). (F) The percentage of FITC-PAC-1 binding to patient platelets relative to control platelets. PAC-1 binding to patient platelets relative to control platelets at each time point was indicated as percent control MFI (n = 3: mean ± SD; Student t test, *P < .05, **P < .01). (G) Shear-induced thrombus formation. Whole blood from control or patient was perfused over collagen-coated surface at a wall shear rate of 1250 seconds−1. Thrombus volumes were measured by MetaMorph and VoxBlast software every minute (n = 3: mean ± SD). Representative thrombus images after 10 minutes of perfusion were shown (top and side view). (H) Statistical analysis of thrombus area and height. Surface coverage by adhered platelets (left) and thrombus height (right) after 10 minutes of perfusion were analyzed (n = 3). (I) Platelet integrin αIIbβ3 activation kinetics in patient with OSP-1 determined by initial velocity assay. In platelets of OSP-1, rapid initial αIIbβ3 activation at 30 seconds was induced the same as control platelets. However, αIIbβ3 activation was transient and almost no αIIbβ3 activation velocity at 300 seconds. (J) β2 integrin activation in neutrophils. Whole blood was stimulated with 200 nM fMLP or 100 ng/mL PMA, and induced β2 integrin activation was determined by binding of monoclonal antibody 24, which recognizes the activated form of β2 integrin (n = 3: mean ± SD; Student t test). WT, wild type.

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