Aberrant and Alternative Splicing of von Willebrand Factor
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von Willebrand disease (VWD) is the most commonly inherited bleeding disorder in humans resulting from quantitative deficiencies or qualitative defects of von Willebrand factor (VWF). VWD can be caused by a variety of mutations throughout the VWF gene, the majority of which are missense changes. Approximately 10% of pathologic VWF mutations are thought to disrupt the process of VWF splicing leading to VWD; however, this primarily acknowledges canonical splice site mutations, where the nucleotides are most integral and highly conserved to accomplish proper splicing. We hypothesized that pathologic splicing of VWF is likely an under-recognized mechanism of VWD, caused by variation outside of canonical splice sites. This thesis characterized three VWF mutations acting through pathologic splicing defects using plasma and patient-derived blood outgrowth endothelial cells (BOEC). In Family 1 it was found that the c.3538G>A mutation in exon 26 induces transcription of three in-frame aberrant splice forms (1) skipping exon 23, (2) skipping exon 26, (3) skipping exons 23 & 26 together, leading to Type 1 VWD. In Family 2, the canonical c.5842+1G>C mutation causes VWD through production of intracellularly retained in-frame VWF, skipping exons 33-34. The affected family also produces a transcript skipping exon 33 which was found in three normal BOEC lines at 13±0.2%. Family 3 has an intronic branch site c.6599-20A>T mutation which causes Type 1 VWD through nonsense mediated decay (NMD) of the premature termination codon (PTC) containing VWF transcript skipping exon 38. In-frame aberrant transcripts were found to be decreased under high shear stress, whereas PTC-inducing transcripts were increased. The aberrant splice forms identified in these families were further characterized in a heterologous cell expression model using human embryonic kidney (HEK)293T cells for VWF expression, secretion, functionality and intracellular trafficking. All mutants except for skipping exon 33 showed reduced expression and secretion and most colocalized with the endoplasmic reticulum. In-frame skipping of exons 33-34 was the only mutant that was able to multimerize and retain functional platelet, collagen, and Factor VIII (FVIII) binding without co-expression of wildtype (WT) VWF. Alternative splicing is known to occur in approximately 95% of mammalian multi-exon genes; however, no alternative splice variants have been described for VWF thus far. We hypothesized that given the large size of the VWF gene alternative splice variants likely exist. This thesis investigated the possibility of alternative VWF splicing in normal endothelial cells (EC)s using mRNA from BOEC, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) grown in unstimulated static conditions or after stimulation by estrogen, histamine, plasma, DDAVP, and 15 dynes/cm2 laminar flow. The expression profiles of these EC varied by cell line; however, shear stress appeared to consistently modulate the expression of both in-frame and PTC-inducing alternative splicing transcripts. The results presented in this thesis provide insight into VWF biology and biosynthesis, through the removal of skipped exons and VWF trafficking, and gleans insight into potential alternative VWF splice forms which may be regulated by shear stress and act to control VWF expression or serve other functions in vivo.