L718P mutation in the membrane-proximal cytoplasmic tail of beta 3 promotes abnormal alpha IIb beta 3 clustering and lipid microdomain coalescence, and associates with a thrombasthenia-like phenotype

Abstract

Background: Support for the role of transmembrane and membrane-proximal domains of alpha IIb beta 3 integrin in the maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bi-directional transmembrane signaling.

Design and methods: We report the functional characterization of a mutant alpha IIb beta 3 integrin carrying the Leu718Pro mutation in the membrane-proximal region of the beta 3 cytoplasmic domain, identified in heterozygosis in a patient with a severe bleeding phenotype and defective platelet aggregation and adhesion.

Results: Transiently transfected cells expressed similar levels of normal and mutant alpha IIb beta 3, but surface expression of mutant alpha v beta 3 was reduced due to its retention in intracellular compartments. Cells stably expressing mutant alpha IIb beta 3 showed constitutive binding to soluble multivalent ligands as well as spontaneous fibrinogen-dependent aggregation, but their response to DTT was markedly reduced. Fibrinogen-adherent cells exhibited a peculiar spreading phenotype with long protrusions. Immunofluorescence analysis revealed the formation of alpha IIb beta 3 clusters underneath the entire cell body and the presence of atypical high-density patches of clustered alpha IIb beta 3 containing encircled areas devoid of integrin that showed decreased affinity for the fluorescent lipid analog DiIC(16) and were disrupted in cholesterol-depleted cells.

Conclusions: These findings are consistent with an important role of the membrane-proximal region of beta 3 in modulating alpha IIb beta 3 clustering and lateral redistribution of membrane lipids. Since the beta 3 mutant was associated with a thrombasthenic phenotype in a patient carrying one normal beta 3 allele, these results support a dominant role of clustering in regulating integrin alpha IIb beta 3 functions in vivo.

Figure 1.

Platelet expression of αIIbβ3 and binding of fibrinogen to stimulated platelets. (A) Flow cytometry analysis of surface αIIbβ3 and GPIbα. Washed platelets were incubated with monoclonal antibodies against β3 (H1AG11), αIIb (2BC1), αIIbβ3 (P2) and GPIbα (AK2) as described in the Design and Methods section. (B) Western analysis of platelet αIIb and β3 content. (C) Flow cytometry analysis of fibrinogen binding. Washed platelets were stimulated for 5 min with 100 μM ADP plus 1 mM epinephrine, 40 μM TRAP-6, or 10 nM PMA in the presence of FITC-fibrinogen. Numbers in the figure panels represent the fluorescence values calculated as a product of the percent of gated positive cells and the value of the mean channel of fluorescence intensity. (D) Adhesion and spreading of platelets on immobilized fibrinogen. Washed platelets were seeded on fibrinogen-coated coverslips for 30 min at 37ºC and, then, fixed, labeled with anti-αIIbβ3 monoclonal antibody 2BC1, and analyzed with an epifluorescence microscope. Bars: 15 μm. All data are representative of determinations with platelets from two different extractions of blood samples.

Figure 2.

Identification of a de novo Leu718Pro mutation in β3. (A) Direct sequencing of the sense strand of the 3’ overlapping RT-PCR product showing a heterozygous T to C substitution that changes Leu718 to Pro in the β3 subunit of the thrombasthenic patient. (B) Detection of the T2231C mutation and the IVS14+9C>T polymorphism in family members. The haplotype of the deceased father was deduced from those of the other members. (C) Localization of the Leu718Pro mutation (encircled) in the membrane-proximal region (underlined) of the β3 cytoplasmic tail.

Figure 3.

Binding of fibrinogen and PAC-1 to normal or mutant αIIbβ3 expressed in CHO cells. (A) CHO cells stably expressing wild type or mutant αIIbβ3-P718 were preincubated in the absence or presence of 1 mM RGDS or 25 mM DTT for 5 min at room temperature. Cells were then incubated with FITC-PAC-1 for 30 min, washed, and analyzed by flow cytometry. (B) CHO cells were first labeled with anti-αIIb monoclonal antibody and then incubated with FITC-Fg in the absence or presence of thrombin as described in the Design and Methods section. (C) Soluble fibrinogen-dependent aggregation of CHO cell transfectants. Non-transfected cells (CHO) or αIIbβ3 expressing cells were incubated in serum-free medium containing 1 mg/mL fibrinogen in the absence or presence of 15 mM DTT. Aggregates were examined with a phase contrast microscope using a x4 objective. All the results are representative of, at least, three separate experiments.

Figure 4.

Fibrinogen-mediated spreading of CHO cell transfectants. (A) Fluorescence microphotographs of CHO cell transfectants seeded for 16 h on fibrinogen or serum proteins. Cells were fixed, labeled with anti-β3 monoclonal antibody H1AG11 and examined in an epifluorescence microscope with a x40 objective. Arrowheads point to the extending processes in cells expressing mutant αIIbβ3-P718. Bars: 50 μm. (B) Arrows point to the swelling at the tip of the extending protrusion in CHO-αIIbβ3-P718 cells examined with a x63 objective. Bar: 25 μm. (C) Tyrosine phosphorylation of FAK in CHO cells. Non-transfected (CHO) or cells expressing wild-type (W) or mutant αIIbβ3-P718 were maintained in suspension or adhered to fibrinogen for 30 min. Cell lysates were analyzed by western blot. All the experiments were performed at least three times and similar results were obtained.

Figure 5.

Clustering of mutant αIIbβ3-718P stably expressed in CHO cells. (A) CHO cells stably expressing wild-type or mutant αIIbβ3-P718 were seeded on fibrinogen-coated coverslips for 4 h and then fixed and labeled with the anti-αIIb monoclonal antibody 2BC1, and examined by confocal microscopy. (B) CHO cell transfectants expressing αIIbβ3-P718 were seeded on fibrinogen-coated cover-slips for 4 h and then labeled with anti-αIIbβ3 monoclonal antibody P2, and anti-FAK[pY576] polyclonal antibody or phalloidin, and analyzed by confocal microscopy. (C) CHO cells expressing αIIbβ3-P718 were plated on fibrinogen-coated coverslips for the indicated periods of time, fixed and labeled with 2BC1, and analyzed by confocal microscopy. The images are single confocal sections near the adherent surface of the cells. Bars: 30 μm. Arrowheads point to functional adhesion complexes. Asterisks point to cell regions where the atypical integrin clusters locate. These experiments were repeatedly performed (n>4) with the same results.

Figure 6.

Clustering of mutant αIIbβ3-718P induces coalescence of lipid domains. (A) Effect of cholesterol depletion on the clustering pattern induced by β3-P718. CHO cells were treated for 15 min at 37ºC with MβCD followed by plating on fibrinogen-coated coverslips. Cells were then fixed, labeled with 2BC1 and analyzed by confocal microscopy. In the bottom panels, MβCD-induced disruption of αIIbβ3-P718 integrin clustering is shown under high magnification. (B) Distribution of the fluorescent lipid analog DiLC16 in the plasma membrane of CHO cells expressing normal or mutant αIIbβ3 integrin. CHO cells stably expressing wild type or mutant αIIbβ3 were seeded on fibrinogen-coated coverslips for 4 h and then labeled with the anti-αIIbβ3 monoclonal antibody 2BC1 and the fluorescent lipid-analog DiIC16 as described in the Design and Methods section, and analyzed by confocal microscopy. Bars: 30 μm. Representative pictures of more than four separate experiments are shown.