# Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model
###### Abstract  
Coagulation factor V (FV) is a liver-derived protein encoded by the \(F5\) gene. Alternative splicing of \(F5\) exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPI\(\alpha\)) with high affinity, stabilising it in the circulation and potentially enhancing its anticoagulant activity. Accordingly, rare \(F5\) gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0-20 \(\mu\)M MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPI\(\alpha\) (activity) levels. coagulation; Factor V-short; \(F5\); alternative splicing; splicing modulation; morpholino; antisense oligonucleotides; bleeding +
Footnote †: journal: Applied  
10 September 2021  
13 October 2021  
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**Aticle**  
the recognition of these signals by the spliceosome [9]. Several studies support the ability of this technology to suppress unwanted splicing events (such as aberrant splicing induced by genetic mutations) in favour of alternative (correct or desirable) splicing events (see refs. [10; 11; 12; 13] for some coagulation-related examples). In fact, several antisense oligonucleotides have entered clinical trials or have already been approved as therapeutic agents for various diseases [14; 15]. Alternative splicing also contributes to the regulation of blood coagulation by generating variants of the same coagulation factor with different functional properties, as in the case of factor V (FV), encoded by the \(F5\) gene [16]. FV is a liver-derived plasma protein with an A1-A2-B-A3-C1-C2 domain-structure. Its main isoform, referred to as (full-length) FV, is kept inactive by a tight electrostatic interaction between a basic and an acidic region within the large B domain [17]. Step-wise proteolysis of this domain activates FV into the essential cofactor (FVA) of factor Xa (Fxa) in the conversion of prothrombin to thrombin, accelerating this reaction over a 1000-fold [18]. In the early stages of coagulation, the procoagulant activity of FV is regulated by the anticoagulant protein tissue factor pathway inhibitor \(\alpha\) (TFPI\(\alpha\)) [19]. In fact, the C-terminus of TFPI\(\alpha\) contains a basic region that can bind to the free acidic region of partially activated forms of FV, thereby preventing their incorporation in the prothrombinse complex [20], as well as their full activation to FVA [21]. In 2013, a minor splicing variant of FV, normally accounting for \(<\)5% of plasma FV, was discovered [22]. This so-called FV-short isoform arises from the removal of an optional intron within exon 13 (Figure 1), resulting in the in-frame deletion of 702 amino acids of the B domain, including the basic region. TFPI\(\alpha\) binds with high affinity to the unmatched acidic region of FV-short [22], thereby neutralising its constitutive prothrombinse activity [23].