Figure 1
Platelet morphology and biochemical, genetic, and functional analyses of ITGA2B R995W mutation. (A; left) DNA sequence analysis of ITGA2B. The entire coding regions of the patients' ITGA2B were amplified from genomic DNA by the polymerase chain reaction, and amplified DNA fragments were subjected to direct cycle sequence analysis. A C to T transition at nucleotide 3077, changing Arg995 to Trp (R995W), was detected. Nucleotide numbering for ITGA2B cDNA is according to Poncz et al.18 The arrow shows the position of the substitution. (Right) Allele-specific restriction analysis. DNA fragments amplified using primers 2Bg305/303 (supplemental Table 1) were digested with BspACI (SibEnzyme), electrophoresed on 2% agarose gels, and stained with ethidium bromide. The 3077C > T substitution abolishes a recognition site for BspACI, generating a new 231-bp band (arrowhead). The mutation was not found in 108 healthy controls or in the SNP database (www.ncbi.nlm.nih.gov/SNP). MW indicates HaeIII digest of ΦX 174 DNA; C, control; and P1, patient 1. (B) Immunoblot analysis of platelets. Triton X-100-soluble platelet lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4% to 12% gradient acrylamide slab gels (Invitrogen) and electroblotted onto polyvinylidine difluoride membranes. The blots were incubated with anti-β1 tubulin antibody NB2301,19 anti-GPIbα antibody PL524 (Takara), and anti-αIIb antibody SZ22 (Beckman-Coulter) and reacted with horseradish peroxidase-conjugated secondary antibody. The bound antibodies were visualized using an enhanced chemiluminescent substrate. C indicates control; and P1, patient 1. (C) Platelet morphology. Peripheral blood smears were stained with May-Grünwald-Giemsa for a normal control and patient 1 (original magnification, × 1000). The patient showed giant platelets with morphologically normal leukocytes. The number in each panel shows the mean platelet size (n = 200). Images were obtained using a BX50 microscope with a 100×/1.35 numeric aperture oil objective (Olympus). Images of the slides were acquired using a DP70 digital camera and DP manager software Version 1.2.1.107 (Olympus). (D) Activation state of platelet αIIbβ3. Washed platelets from patient 3 were resuspended in Tyrode buffer (137mM NaCl, 2.7mM KCl, 1.0mM MgCl2, 3.3mM NaH2PO4, 3.8mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 0.1% glucose, 0.1% bovine serum albumin, pH 7.4) and incubated with fluorescein isothiocyanate-conjugated PAC-1 or 125 μg/mL fluorescein isothiocyanate-labeled fibrinogen in the presence or absence of 10μM FK633 (αIIbβ3-specific peptidomimetic antagonist: black lines) or 10μM adenosine diphosphate (blue lines), and analyzed by flow cytometry. Numbers indicate the mean fluorescence intensity. Results are representative of 2 independent experiments. (E) Quantitation of the αIIbβ3 activation state. The activation state of αIIbβ3 was quantified as an activation index on transiently transfected 293T cells. The activation index was higher for αIIb-W995 than for β3-H723 but was weaker than for an activating mutant β3-N562. Activation index = (a − b)/(c − b), in which a is the mean fluorescence intensity of PAC-1 binding with buffer, b is the mean fluorescence intensity in the presence of FK633, and c is the mean fluorescence intensity in the presence of PT25–2 (anti-αIIbβ3 antibody, which induces the active conformation of αIIbβ3). Data are mean plus or minus SE (n = 3). (F) FAK phosphorylation. Washed platelets from patient 3 (left) or transiently transfected 293T cells (right) were incubated in suspension or seeded onto 100-μg/mL fibrinogen-coated plastic dishes for 1 hour. Cells were washed with phosphate-buffered saline and lysed with 1% Triton X-100 and 1mM sodium vanadate. FAK was immunoprecipitated from the lysates with anti-FAK antibody FAK(C903; Santa Cruz Biotechnology) and protein G-Sepharose, and phosphotyrosine was detected with the antiphosphotyrosine antibody 4G10 (Millipore). Note that 300-μg and 150-μg lysates from suspension and adhered platelets, respectively, and 200-μg lysates from suspension and adhered transfected 293T cells were used for immunoprecipitation analysis. To monitor the loading of gel lanes, the membrane was stripped and reprobed with the anti-FAK antibody FAK(A17; Santa Cruz Biotechnology). Results are representative of 2 and 3 independent experiments for platelets and transfected cells, respectively. (G) Abnormal cytoplasmic protrusions in αIIb-W995/β3-transfected CHO cells. Stably transfected CHO cells were seeded onto 100 μg/mL fibrinogen-coated glass coverslips and incubated for 2 hours at 37°C. Cells were fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Coverslips were then stained with anti-CD41a antibody HIP8 (BD Biosciences) followed by Alexa-488-labeled goat antimouse IgG (Invitrogen) and tetramethylrhodamine isothiocyanate-phalloidin (Sigma-Aldrich). Images were obtained using a confocal microscope with a Plan-Apochromat 63×/1.4 oil DIC objective lens LSM5Pascal (Carl Zeiss). Arrowheads indicate membrane ruffling (middle panel) and abnormal cytoplasmic protrusions with the bulbous tips (right panel) in αIIb-W995/β3-transfected CHO cells. Representative images from 3 independent experiments are shown. (H) Abnormal proplatelet formation in αIIb-W995/β3-transfected megakaryocytes. Mouse fetal liver-derived megakaryocytes infected with EGFP-αIIb and Kusabira-Orange-β3 retrovirus were examined in suspension cultures under an IX71 fluorescence microscope with an LCPlanFI 40×/0.60 objective lens (Olympus). (i) The percentage of megakaryocytes extending proplatelets was evaluated manually under a fluorescence microscope 1 to 4 days after infection. For each specimen, at least 100 megakaryocytes were evaluated. The number of proplatelet tips per megakaryocyte (ii) and the size of the proplatelet tips (iii) were measured on acquired images by the ImageScope software Version 10.2.2 (Aperio Technologies). At least 10 megakaryocytes were analyzed for each sample. An unpaired, 2-tailed t test was used to analyze data. A value of P less than .05 was considered statistically significant. Data are mean plus or minus SD. *P < .05. **P < .01. ***P < .0001. (iv) Representative megakaryocytes from 3 independent experiments are shown. Note that the number of proplatelet tips/bulbous structures (arrowheads) is decreased and the size of the tips increased in αIIb-W995/β3-transfected megakaryocytes than in wild-type αIIb/β3-transfected megakaryocytes. Scale bar represents 10 μm.

Platelet morphology and biochemical, genetic, and functional analyses of ITGA2B R995W mutation. (A; left) DNA sequence analysis of ITGA2B. The entire coding regions of the patients' ITGA2B were amplified from genomic DNA by the polymerase chain reaction, and amplified DNA fragments were subjected to direct cycle sequence analysis. A C to T transition at nucleotide 3077, changing Arg995 to Trp (R995W), was detected. Nucleotide numbering for ITGA2B cDNA is according to Poncz et al.18  The arrow shows the position of the substitution. (Right) Allele-specific restriction analysis. DNA fragments amplified using primers 2Bg305/303 (supplemental Table 1) were digested with BspACI (SibEnzyme), electrophoresed on 2% agarose gels, and stained with ethidium bromide. The 3077C > T substitution abolishes a recognition site for BspACI, generating a new 231-bp band (arrowhead). The mutation was not found in 108 healthy controls or in the SNP database (www.ncbi.nlm.nih.gov/SNP). MW indicates HaeIII digest of ΦX 174 DNA; C, control; and P1, patient 1. (B) Immunoblot analysis of platelets. Triton X-100-soluble platelet lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4% to 12% gradient acrylamide slab gels (Invitrogen) and electroblotted onto polyvinylidine difluoride membranes. The blots were incubated with anti-β1 tubulin antibody NB2301,19  anti-GPIbα antibody PL524 (Takara), and anti-αIIb antibody SZ22 (Beckman-Coulter) and reacted with horseradish peroxidase-conjugated secondary antibody. The bound antibodies were visualized using an enhanced chemiluminescent substrate. C indicates control; and P1, patient 1. (C) Platelet morphology. Peripheral blood smears were stained with May-Grünwald-Giemsa for a normal control and patient 1 (original magnification, × 1000). The patient showed giant platelets with morphologically normal leukocytes. The number in each panel shows the mean platelet size (n = 200). Images were obtained using a BX50 microscope with a 100×/1.35 numeric aperture oil objective (Olympus). Images of the slides were acquired using a DP70 digital camera and DP manager software Version 1.2.1.107 (Olympus). (D) Activation state of platelet αIIbβ3. Washed platelets from patient 3 were resuspended in Tyrode buffer (137mM NaCl, 2.7mM KCl, 1.0mM MgCl2, 3.3mM NaH2PO4, 3.8mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 0.1% glucose, 0.1% bovine serum albumin, pH 7.4) and incubated with fluorescein isothiocyanate-conjugated PAC-1 or 125 μg/mL fluorescein isothiocyanate-labeled fibrinogen in the presence or absence of 10μM FK633 (αIIbβ3-specific peptidomimetic antagonist: black lines) or 10μM adenosine diphosphate (blue lines), and analyzed by flow cytometry. Numbers indicate the mean fluorescence intensity. Results are representative of 2 independent experiments. (E) Quantitation of the αIIbβ3 activation state. The activation state of αIIbβ3 was quantified as an activation index on transiently transfected 293T cells. The activation index was higher for αIIb-W995 than for β3-H723 but was weaker than for an activating mutant β3-N562. Activation index = (a − b)/(c − b), in which a is the mean fluorescence intensity of PAC-1 binding with buffer, b is the mean fluorescence intensity in the presence of FK633, and c is the mean fluorescence intensity in the presence of PT25–2 (anti-αIIbβ3 antibody, which induces the active conformation of αIIbβ3). Data are mean plus or minus SE (n = 3). (F) FAK phosphorylation. Washed platelets from patient 3 (left) or transiently transfected 293T cells (right) were incubated in suspension or seeded onto 100-μg/mL fibrinogen-coated plastic dishes for 1 hour. Cells were washed with phosphate-buffered saline and lysed with 1% Triton X-100 and 1mM sodium vanadate. FAK was immunoprecipitated from the lysates with anti-FAK antibody FAK(C903; Santa Cruz Biotechnology) and protein G-Sepharose, and phosphotyrosine was detected with the antiphosphotyrosine antibody 4G10 (Millipore). Note that 300-μg and 150-μg lysates from suspension and adhered platelets, respectively, and 200-μg lysates from suspension and adhered transfected 293T cells were used for immunoprecipitation analysis. To monitor the loading of gel lanes, the membrane was stripped and reprobed with the anti-FAK antibody FAK(A17; Santa Cruz Biotechnology). Results are representative of 2 and 3 independent experiments for platelets and transfected cells, respectively. (G) Abnormal cytoplasmic protrusions in αIIb-W995/β3-transfected CHO cells. Stably transfected CHO cells were seeded onto 100 μg/mL fibrinogen-coated glass coverslips and incubated for 2 hours at 37°C. Cells were fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Coverslips were then stained with anti-CD41a antibody HIP8 (BD Biosciences) followed by Alexa-488-labeled goat antimouse IgG (Invitrogen) and tetramethylrhodamine isothiocyanate-phalloidin (Sigma-Aldrich). Images were obtained using a confocal microscope with a Plan-Apochromat 63×/1.4 oil DIC objective lens LSM5Pascal (Carl Zeiss). Arrowheads indicate membrane ruffling (middle panel) and abnormal cytoplasmic protrusions with the bulbous tips (right panel) in αIIb-W995/β3-transfected CHO cells. Representative images from 3 independent experiments are shown. (H) Abnormal proplatelet formation in αIIb-W995/β3-transfected megakaryocytes. Mouse fetal liver-derived megakaryocytes infected with EGFP-αIIb and Kusabira-Orange-β3 retrovirus were examined in suspension cultures under an IX71 fluorescence microscope with an LCPlanFI 40×/0.60 objective lens (Olympus). (i) The percentage of megakaryocytes extending proplatelets was evaluated manually under a fluorescence microscope 1 to 4 days after infection. For each specimen, at least 100 megakaryocytes were evaluated. The number of proplatelet tips per megakaryocyte (ii) and the size of the proplatelet tips (iii) were measured on acquired images by the ImageScope software Version 10.2.2 (Aperio Technologies). At least 10 megakaryocytes were analyzed for each sample. An unpaired, 2-tailed t test was used to analyze data. A value of P less than .05 was considered statistically significant. Data are mean plus or minus SD. *P < .05. **P < .01. ***P < .0001. (iv) Representative megakaryocytes from 3 independent experiments are shown. Note that the number of proplatelet tips/bulbous structures (arrowheads) is decreased and the size of the tips increased in αIIb-W995/β3-transfected megakaryocytes than in wild-type αIIb/β3-transfected megakaryocytes. Scale bar represents 10 μm.

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