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Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene
T C Hsu1, S K Shore, T Seshsmma
1Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Abstract:
Platelet factor XI is associated with the platelet plasma membrane and has an apparent Mr (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of approximately 1.9 kilobases was detected in megakaryocytic cells, and one of approximately 2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala91-Arg144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.
Insights
Platelet factor XI, a protein found on platelet membranes, is synthesized through alternative splicing of the factor XI gene. This process results in a unique form of factor XI specific to platelets and megakaryocytes.
Area of Science:
- Hematology
- Molecular Biology
- Genetics
Background:
- Platelet factor XI is associated with platelet membranes, but its synthesis site and nature are unknown.
- Its molecular weight differs from plasma factor XI, suggesting a distinct form.
Purpose of the Study:
- To investigate the synthesis and nature of platelet factor XI.
- To determine if platelet factor XI is derived from the same gene as plasma factor XI.
Main Methods:
- Reverse transcriptase polymerase chain reaction (RT-PCR) to amplify factor XI exons from platelet mRNA.
- In situ amplification and hybridization to detect factor XI mRNA in platelets and other blood cells.
- Northern hybridization to analyze factor XI mRNA transcripts in megakaryocytic and liver cells.
- Cloning and sequencing of factor XI cDNA from a megakaryocyte library.
Main Results:
- RT-PCR amplified 12 of 13 exons coding for mature plasma factor XI from platelet mRNA, with identical sequences.
- In situ hybridization confirmed the presence of factor XI mRNA (exon III) in platelets but not exon V, and neither in other blood cells.
- Northern hybridization detected a ~1.9 kb factor XI mRNA transcript in megakaryocytic cells and a ~2.1 kb transcript in liver cells.
- Sequencing of cloned megakaryocyte cDNA revealed the absence of exon V, with splicing of exon IV to VI, creating a variant protein lacking amino acids Ala91-Arg144.
Conclusions:
- Platelet factor XI is an alternative splicing product of the factor XI gene.
- This alternative splicing generates a platelet-specific isoform of factor XI.
- Platelet factor XI is localized to platelets and megakaryocytes and is absent in other blood cells.