A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype by Catherine Ruiz, Chao-Yan Liu, Qi-Hong Sun, Marianne Sigaud-Fiks, Edith Fressinaud, Jean-Yves Muller, Paquita Nurden, Alan T. Nurden, Peter J. Newman, and Nathalie Valentin Blood Volume 98(8):2432-2441 October 15, 2001 ©2001 by American Society of Hematology

Expression of GPIIb-IIIa in the patient's platelets Expression of GPIIb-IIIa in the patient's platelets.(A) Cell-surface expression was assessed using monoclonal antibodies (10 μg/mL) directed against the GPIIb-IIIa complex (6E1), GPIIb (SZ22), and GPIIIa (AP3) subunits. Expression of GPIIb-IIIa in the patient's platelets.(A) Cell-surface expression was assessed using monoclonal antibodies (10 μg/mL) directed against the GPIIb-IIIa complex (6E1), GPIIb (SZ22), and GPIIIa (AP3) subunits. Bound IgG was assessed by flow cytometry using FITC-conjugated goat anti–mouse antibody. Results are given as the percentage (mean + range from 4 separate experiments) of the binding of the same antibodies to control platelets, assigned as 100%. N.M.'s platelets expressed about 20% GPIIb-IIIa compared with normal platelets. Surface expression of GPIb was similar on both N.M.'s and control platelets as shown by the binding of SZ1. (B) Total platelet GPIIb-IIIa was evaluated by immunoblotting. SDS-soluble platelet proteins (12 μg) from both control and N.M.'s platelets were subjected to electrophoresis on a 7% polyacrylamide gel under reducing conditions, transferred to PVDF membrane, and incubated with 20 μg/mL rabbit polyclonal antibodies specific for GPIIb and GPIIIa. Bound antibodies were detected using alkaline phosphatase–conjugated goat anti–rabbit IgG, followed by color development using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Spontaneous binding of PAC-1 and fibrinogen to resting platelets Spontaneous binding of PAC-1 and fibrinogen to resting platelets.Platelets were washed and resuspended (2 × 106) in Tyrode-HEPES buffer containing 10−7 M PGE1 and incubated without stimulation with 20 μg/mL FITC–PAC-1 (A) or 250 nM FITC-fibrinogen (B) for 2... Spontaneous binding of PAC-1 and fibrinogen to resting platelets.Platelets were washed and resuspended (2 × 106) in Tyrode-HEPES buffer containing 10−7 M PGE1 and incubated without stimulation with 20 μg/mL FITC–PAC-1 (A) or 250 nM FITC-fibrinogen (B) for 20 and 40 minutes, respectively, at room temperature (RT). Samples were diluted and fixed in 1% formaldehyde and subjected to flow cytometry. The mean fluorescence intensities (MFIs) of PAC-1 and fibrinogen binding in the presence (dark histogram) or in the absence of 2 mM RGDS peptide (clear histogram) obtained for the patient and control are indicated on the histograms. The fact that the patient's platelets expressed only 20% of the usual levels of GPIIb-IIIa reinforces the difference in ligand binding between N.M. and control platelets. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Evaluation of LIBS exposure on the patient's platelets Evaluation of LIBS exposure on the patient's platelets.To assess spontaneous LIBS exposure on platelets, PRP (2 × 106 platelets) with 10−7 M PGE1 was incubated with 10 μg/mL anti-LIBS antibodies 7D6, AP5, and D3 in the presence of 2 mM RGDS or RGES peptides... Evaluation of LIBS exposure on the patient's platelets.To assess spontaneous LIBS exposure on platelets, PRP (2 × 106 platelets) with 10−7 M PGE1 was incubated with 10 μg/mL anti-LIBS antibodies 7D6, AP5, and D3 in the presence of 2 mM RGDS or RGES peptides for 60 minutes at RT. After 2 washes, platelets were incubated with FITC-labeled goat anti–mouse F(ab′)2 for 30 minutes, then samples were washed once and bound antibodies were analyzed by flow cytometry. LIBS antibody binding was expressed as a LIBS index (LI) by standardizing the MFI for each LIBS monoclonal antibody (mAb) to that obtained using AP3, the binding of which was not affected by the patient's mutation. LI = LIBS mAb MFI/AP3 MFI for the patient and for the control separately. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Detection of platelet-bound fibrinogen in whole blood Detection of platelet-bound fibrinogen in whole blood.A volume (5 μL) of freshly drawn citrated blood was incubated for 20 minutes at RT with 6.3 μg rabbit FITC-conjugated polyclonal rabbit anti–human fibrinogen. Detection of platelet-bound fibrinogen in whole blood.A volume (5 μL) of freshly drawn citrated blood was incubated for 20 minutes at RT with 6.3 μg rabbit FITC-conjugated polyclonal rabbit anti–human fibrinogen. Saturating concentrations of anti-FcγRII blocking antibody, IV.3, were added to prevent platelet activation through the Fc receptors by immune complexes formed between soluble fibrinogen and antifibrinogen antibody. In parallel, 5 μL whole blood was incubated with 5 μg/mL FITC-conjugated anti-GPIIb antibody SZ22 to assess the GPIIb-IIIa expression at the cell surface. The samples were analyzed by flow cytometry and expressed as the MFI. These data are representative of 4 separate experiments. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Ultrastructural analysis of N. M. 's platelets Ultrastructural analysis of N.M.'s platelets.All studies were performed on platelets fixed in PRP and incubated with rabbit antifibrinogen antibody (dilution 1:50) for 1 hour at RT. Bound antibodies were detected using a species-specific anti-IgG antibody (... Ultrastructural analysis of N.M.'s platelets.All studies were performed on platelets fixed in PRP and incubated with rabbit antifibrinogen antibody (dilution 1:50) for 1 hour at RT. Bound antibodies were detected using a species-specific anti-IgG antibody (dilution 1:10) coupled to 10-nm gold particles overnight at 4°C before embedding. (A,D) Platelet sections are from a control donor. (B,C,E) Selected sections of platelets from patient N.M. (A) A nonstimulated control platelet is illustrated; few gold particles are present on the surface. (B) A platelet from N.M. shows a normal discoid shape and internal organization. Few gold particles are present at the platelet surface. (C) Another discoid platelet with larger vacuoles or a distended surface canalicular system is shown. The labeling for fibrinogen on the surface of this platelet was much more intense. (D) An example of TRAP6-activated control platelets (5 minutes). Even without continuous stirring, some aggregates have formed in the PRP, and labeling for fibrinogen is seen at the periphery of the aggregate. (E) TRAP6-activated platelets from the patient remained isolated or as small aggregates composed of 2 or 3 platelets; inside the platelet, large vacuoles are present and the granules have disappeared. Labeling at the platelet surface continues to be seen. Bars = 0.5 μm Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

The Cys560Arg mutation in N. M. 's GPIIIa The Cys560Arg mutation in N.M.'s GPIIIa.DNA sequence analysis was performed using N.M.'s genomic DNA. All exons, including intron-exon junctions, of both GPIIb and GPIIIa genes were amplified using PCR primer pairs hybridizing in the intron sequence flankin... The Cys560Arg mutation in N.M.'s GPIIIa.DNA sequence analysis was performed using N.M.'s genomic DNA. All exons, including intron-exon junctions, of both GPIIb and GPIIIa genes were amplified using PCR primer pairs hybridizing in the intron sequence flanking each exon. The resulting PCR fragments were purified and subjected to direct cycle sequencing. The nucleotide sequence of exon 10 of N.M.'s GPIIIa gene reveals a g1776T>C substitution (underlined codon), which results in the Cys560Arg mutation. The nucleotide substitution was also confirmed by sequence analysis performed by using reverse primer (not shown). Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Flow cytometric analysis of GPIIb-Arg560IIIa expressed on the transfected CHO cell surface.CHO cells were incubated with 40 μg/mL AP3 (lower), AP2 (upper), or IgG as a negative control for 60 minutes at 4°C; washed; and further incubated with 1:100 dilution... Flow cytometric analysis of GPIIb-Arg560IIIa expressed on the transfected CHO cell surface.CHO cells were incubated with 40 μg/mL AP3 (lower), AP2 (upper), or IgG as a negative control for 60 minutes at 4°C; washed; and further incubated with 1:100 dilution of FITC-conjugated anti-IgG for 60 minutes before flow cytometric analysis using a FACScan. MFIs are indicated on the histograms. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Flow cytometric analysis of LIBS exposure on surface-expressed GPIIb-Arg560IIIa.(A) Transfected CHO cells were incubated with 20 μg/mL LIBS antibodies for 45 minutes at RT, followed by FITC-conjugated goat anti–mouse IgG. Flow cytometric analysis of LIBS exposure on surface-expressed GPIIb-Arg560IIIa.(A) Transfected CHO cells were incubated with 20 μg/mL LIBS antibodies for 45 minutes at RT, followed by FITC-conjugated goat anti–mouse IgG. The binding of LIBS mAbs to GPIIb-IIIa was expressed as a LIBS index (LI) by normalizing the MFI of each LIBS antibody to that obtained using AP2, a complex-specific mAb the binding of which is unaffected by the Cys560Arg mutation (LI = LIBS mAb MFI/AP2 MFI). (B) Flow cytometric analysis of the binding of the activation-dependent fibrinogen-mimetic antibody PAC-1 (20 μg/mL) to GPIIb-Arg560IIIa (bold histogram) was performed in the presence of buffer (left), 2 mM RGDW (middle), or RGEW (right). Peptides were synthesized at the Peptide Core Laboratory of the Blood Research Institute (Milwaukee, WI). The MFIs are indicated on the histograms. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Expression of the internal pool of GPIIb-IIIa on the patient's platelets and subsequent aggregation after TRAP6 activation.(A) A total of 2 × 106 platelets in PRP were activated with 50 μM TRAP6. Expression of the internal pool of GPIIb-IIIa on the patient's platelets and subsequent aggregation after TRAP6 activation.(A) A total of 2 × 106 platelets in PRP were activated with 50 μM TRAP6. Platelets were then incubated with anti–GPIIb-IIIa antibody 6E1 (clear histogram) and isotype control (dark histogram) for 30 minutes at RT. After the addition of 1% formaldehyde in PBS, samples were analyzed by flow cytometry. The MFIs are indicated on the histograms. (B) Aggregation of N.M.'s platelets in PRP after secretion of the internal GPIIb-IIIa pool. Citrated PRP (2 × 108platelets/mL) was stimulated with 7 μM and 50 μM TRAP6 for the control and the patient, respectively. The values (percent of maximal secretion) for PF4 release, as assessed with an ELISA, are in parentheses. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology

Platelet adhesion and spreading on immobilized fibrinogen Platelet adhesion and spreading on immobilized fibrinogen.Adhesion and spreading of platelets were performed on dishes coated with 100 μg/mL fibrinogen or 5 mg/mL BSA as a control of adhesion. Platelet adhesion and spreading on immobilized fibrinogen.Adhesion and spreading of platelets were performed on dishes coated with 100 μg/mL fibrinogen or 5 mg/mL BSA as a control of adhesion. A total of 3 × 108 gel-filtered platelets were allowed to attach on a matrix for 90 minutes at 37°C. Catherine Ruiz et al. Blood 2001;98:2432-2441 ©2001 by American Society of Hematology