Related Experiment Videos
Human platelets and megakaryocytes contain alternately spliced glycoprotein IIb mRNAs
P F Bray1, C S Leung, M A Shuman
1Cancer Research Institute, University of California, San Francisco 94143.
Abstract:
The earliest marker of the megakaryocyte lineage in human bone marrow is a heterodimeric complex of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). GPIIb-IIIa is a member of the integrin family and serves as a receptor for fibrinogen and other adhesive glycoproteins on the surface of activated blood platelets. Mature platelet GPIIb is processed from a single precursor peptide into a disulfide-linked GPIIb alpha and GPIIb beta chain. To study possible mechanisms which could determine ligand specificity of the GPIIb-IIIa complex we investigated whether there are multiple forms of GPIIb mRNA by screening an unamplified lambda gt10 human erythroleukemia cell cDNA library. Four GPIIb clones were isolated and sequenced. Two contained a 102-nucleotide in-frame deletion corresponding to 34 amino acids in the extracellular domain of GPIIb beta. Polymerase chain reaction amplification of human erythroleukemia cell cDNA confirmed the presence of two different transcripts. Normal genomic DNA sequence was shown to contain consensus splice-donor and splice-acceptor sites indicating a 102-base pair exon that is spliced in or out. Using primers specific for either transcript, both forms of GPIIb mRNA were identified by the polymerase chain reaction in normal human platelets and megakaryocytes, but not in reticulocytes or two white blood cell lines. These data provide evidence for two forms of GPIIb in platelets and megakaryocytes and suggest a mechanism for variations in receptor ligand affinity or localization.
Insights
Researchers discovered two forms of glycoprotein IIb (GPIIb) mRNA in human platelets and megakaryocytes. This finding suggests a mechanism for variations in GPIIb-IIIa receptor ligand affinity and localization.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- The glycoprotein IIb/IIIa (GPIIb-IIIa) complex is a key integrin receptor on activated platelets, crucial for hemostasis.
- GPIIb is processed into alpha and beta chains, and its ligand specificity is critical for platelet function.
Purpose of the Study:
- To investigate the existence of multiple glycoprotein IIb (GPIIb) mRNA forms.
- To explore potential mechanisms influencing GPIIb-IIIa receptor ligand specificity.
Main Methods:
- Screening of a human erythroleukemia cell cDNA library to isolate GPIIb clones.
- Sequencing of isolated GPIIb clones to identify variations.
- Polymerase chain reaction (PCR) amplification of cDNA to confirm transcript diversity.
- Analysis of genomic DNA for splice sites.
- RT-PCR using specific primers to detect GPIIb mRNA forms in various blood cell types.
Main Results:
- Four GPIIb clones were isolated, with two revealing a 102-nucleotide in-frame deletion in the GPIIb beta subunit.
- PCR confirmed the presence of two distinct GPIIb transcripts in human erythroleukemia cells.
- Genomic analysis identified splice donor and acceptor sites consistent with alternative splicing of a 102-base pair exon.
- Both GPIIb mRNA forms were detected in normal human platelets and megakaryocytes, but not in reticulocytes or white blood cell lines.
Conclusions:
- Evidence supports the existence of two GPIIb mRNA forms in platelets and megakaryocytes.
- Alternative splicing of the GPIIb gene provides a mechanism for generating protein variants.
- These variants may contribute to differences in GPIIb-IIIa receptor ligand affinity or cell surface localization.