Translation of Human Tissue Factor Pathway Inhibitor-β mRNA Is Controlled by Alternative Splicing Within the 5′ Untranslated Region

Objective: Tissue factor pathway inhibitor (TFPI) blocks the initiation of coagulation by inhibiting TF-activated factor VII, activated factor X, and early prothrombinase. Humans produce two 3′ splice variants, TFPIα and TFPIβ, which are differentially expressed in endothelial cells and platelets and possess distinct structural features affecting their inhibitory function. TFPI also undergoes alternative splicing of exon 2 within its 5′ untranslated region. The role of exon 2 splicing in translational regulation of human TFPI isoform expression is investigated. Approach and Results: Exon 2 splicing occurs in TFPIα and TFPIβ transcripts. Human tissue mRNA analysis uncovered a wide variability of exon 2 expression. Polysome analysis revealed a repressive effect of exon 2 on TFPIβ translation but not on TFPIα. Luciferase reporter assays further exposed strong translational repression of TFPIβ (90%) but not TFPIα. Use of a Morpholino to remove exon 2 from TFPI mRNA increased cell surface expression of endogenous TFPIβ. Exon 2 also repressed luciferase production (80% to 90%) when paired with the β-actin 3′ untranslated region, suggesting that it is a general translational negative element whose effects are overcome by the TFPIα 3′ untranslated region. Conclusions: Exon 2 is a molecular switch that prevents translation of TFPIβ. This is the first demonstration of a 5′ untranslated region alternative splicing event that alters translation of isoforms produced via independent 3′ splicing events within the same gene. Therefore, it represents a previously unrecognized mechanism for translational control of protein expression. Differential expression of exon 2 denotes a mechanism to provide temporal and tissue regulation of TFPIβ-mediated anticoagulant activity.