Truncation of Glycoprotein (GP) IIIa (▵ 616-762) Prevents Complex Formation With GPIIb: Novel Mutation in Exon 11 of GPIIIa Associated With Thrombasthenia by Milagros Ferrer, Jianming Tao, Gema Iruı́n, Matilde Sánchez-Ayuso, José González-Rodrı́guez, Roberto Parrilla, and Consuelo González-Manchón Blood Volume 92(12):4712-4720 December 15, 1998 ©1998 by American Society of Hematology

Flow cytometric analysis of GPIIb-IIIa content in platelets from the proband, her parents, and her brother. Flow cytometric analysis of GPIIb-IIIa content in platelets from the proband, her parents, and her brother. The fluorescence analysis was performed in a Coulter cytometer, model Epics XL. Results are expressed as semilog plots of cell number versus fluorescence intensity. The upper panel shows the fluorescence signals of GPIIIa and GPIIb from control platelets. The negative control represents the fluorescent signal of platelets without antibodies. The middle panel shows the labeling of GPIIIa with the MoAb P95.2 in platelets from the proband, her parents, and her brother. The lower panel shows the results of labeling GPIIb with the MoAb M3 in platelets from the proband, her parents, and her brother. For the sake of clarity, the original plots have been redrawn. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology

SSCP analysis of exon 11 of GPIIIa amplified from genomic DNA SSCP analysis of exon 11 of GPIIIa amplified from genomic DNA. Genomic DNA fragment of 198 bp comprising exon 11 of GPIIIa and intronic flanking regions was amplified as described in Materials and Methods. SSCP analysis of exon 11 of GPIIIa amplified from genomic DNA. Genomic DNA fragment of 198 bp comprising exon 11 of GPIIIa and intronic flanking regions was amplified as described in Materials and Methods. The PCR products were digested with HinfI to yield fragments of 140 and 58 bp and electrophoresed in nondenaturing 16% acrylamide slab gels containing 8.7% glycerol. The arrow points to a distinct band shown by the patient, her parents, and her brother that is absent in the control (wt) DNA. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology

Identification of a G1846→T mutation in exon 11 of GPIIIa Identification of a G1846→T mutation in exon 11 of GPIIIa. Exon 11 of GPIIIa was amplified from genomic DNA as described in Materials and Methods. Identification of a G1846→T mutation in exon 11 of GPIIIa. Exon 11 of GPIIIa was amplified from genomic DNA as described in Materials and Methods. The amplification products were cloned in a T-vector and the primary nucleotide sequence of pooled DNA was determined in both directions. The figure shows a fragment of the sequencing ladder of the sense strand. The arrows point to a homozygous G1846→T transversion that changes Glu616→Stop. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology

Specific amplification of normal and [T1846]-GPIIIa alleles. Specific amplification of normal and [T1846]-GPIIIa alleles. A DNA fragment of 148 bp encompassing exon 11 and intronic flanking regions of GPIIIa was amplified from genomic DNA of a control, the proband, her parents, and her brother. Each DNA was amplified using a sense primer whose 3′ end was complementary to either the normal sequence (Wt) or to the mutant base (Mut). The amplification products were electrophoresed in a 3% agarose gel and the DNA bands were stained with ethidium bromide. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology

PCR-based determination of platelet mRNA-GPIIIa. PCR-based determination of platelet mRNA-GPIIIa. PCR-based quantitation of β-actin and GPIIIa mRNAs in platelets from the proband, heterozygous relatives, and normal individuals was performed using the instrumentation and the fluorogenic probes of the Perkin-Elmer Cetus LS-50B TaqMan System as described in Materials and Methods. R and Q denote the fluorescence of the reporter and the quencher dyes, respectively. Values were corrected for internal quenching and expressed as GPIIIa/β-actin fluorescence ratios. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology

Immunoprecipitation of biotin-labeled surface or total (surface and intracellular) proteins from CHO cells cotransfected with cDNAs encoding GPIIb and either normal or [T1846]GPIIIa. Immunoprecipitation of biotin-labeled surface or total (surface and intracellular) proteins from CHO cells cotransfected with cDNAs encoding GPIIb and either normal or [T1846]GPIIIa. CHO cells were transiently cotransfected with cDNAs encoding GPIIb and either normal or [T1846]GPIIIa in the expression plasmid pcDNA3. (A) Protein labeling was performed by exposure of intact cells to biotin-NHS, and the GPIIb-IIIa complexes were immunoprecipitated with either anti-GPIIIa (P37) or anti-GPIIb (M3) MoAbs as described in Materials and Methods. (B) The experimental design was similar to that described in (A), except that total cell lysates rather intact cells were exposed to biotin-NHS and the immunoprecipitation was also performed with a GPIIb-IIIa complex-specific antibody (CII). Mock cells were transfected with void pcDNA3 plasmid. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology

Pulse-chase analysis of the stability of normal or ▵GPIIIa-IIb complexes. Pulse-chase analysis of the stability of normal or ▵GPIIIa-IIb complexes. CHO cells were transiently cotransfected with cDNAs encoding GPIIb and either normal or [T1846]GPIIIa in the expression plasmid pcDNA3. Cells were pulse-labeled with [35S]-methionine for 30 minutes and then chased with medium containing unlabeled methionine. At the indicated times, the cells were lysed and labeled proteins were immunoprecipitated with the indicated antibodies. The immunoprecipitates were analyzed by electrophoresis as described in Materials and Methods. Milagros Ferrer et al. Blood 1998;92:4712-4720 ©1998 by American Society of Hematology