PUBLICATION

Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders

Authors

Fish, R.J., Freire, C., Di Sanza, C., Neerman-Arbez, M.

ID

ZDB-PUB-210115-1

Date

2021

Source

International Journal of Molecular Sciences 22(2): (Journal)

Registered Authors

di Sanza, Corinne, Fish, Richard, Neerman-Arbez, Marguerite

Keywords

fibrin, fibrinogen, thrombocytes, thrombosis, zebrafish

MeSH Terms

Plasmids/genetics
Biomarkers*
Disease Models, Animal
Gene Editing
Animals
Sequence Deletion
Blood Coagulation
Fibrinogen/chemistry
Fibrinogen/genetics
Fibrinogen/metabolism*
Zebrafish
Base Sequence
RNA, Guide, Kinetoplastida
Exons
Platelet Activation
Venous Thrombosis/blood*
Venous Thrombosis/diagnosis
Venous Thrombosis/etiology*
Gene Expression
Blood Platelets/metabolism*

(all 20)

PubMed

33440782 Full text @ Int. J. Mol. Sci.

Abstract

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga^-/- larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga^+/- larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding Aα^Δ19-56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα^+/Δ19-56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga^-/- mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα^+/Δ19-56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

Genes / Markers

Figures

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Expression

Gene	Antibody	Fish	Conditions	Stage	Anatomy	Assay	Figure
cp	Ab2-cp	fga^ug704/+ (TU)	standard conditions	Adult	blood plasma	WB	Figure 4
cp	Ab2-cp	fga^ug704/ug704 (TU)	standard conditions	Adult	blood plasma	WB	Figure 4
cp	Ab2-cp	TU	standard conditions	Adult	blood plasma	WB	Figure 4
EGFP		fga^ug701/+; la2Tg	light damage: posterior caudal vein	Day 5	thrombocyte	IFL	Figure 2
EGFP		fga^ug701/ug701; la2Tg	light damage: posterior caudal vein	Day 5	thrombocyte	IFL	Figure 2
EGFP		fga^ug704/+; la2Tg (TU)	light damage: posterior caudal vein	Day 5	thrombocyte	IFL	Figure 6
EGFP		fga^ug704/ug704; la2Tg (TU)	light damage: posterior caudal vein	Day 5	thrombocyte	IFL	Figure 6
EGFP		la2Tg	light damage: posterior caudal vein	Day 5	thrombocyte	IFL	Figure 2
EGFP		la2Tg (TU)	light damage: posterior caudal vein	Day 5	thrombocyte	IFL	Figure 6
fga	Ab1-fga	fga^ug704/+ (TU)	standard conditions	Adult	blood plasma	WB	Figure 4

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
TU + MO1-fga	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein blood coagulation, fibrin clot formation decreased process quality, abnormal	Figure 7
TU + MO1-fga	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein blood coagulation, fibrin clot formation disrupted, abnormal	Figure 7
TU + MO1-fga	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein platelet aggregation decreased process quality, abnormal	Figure 7
TU + MO1-fga	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein platelet aggregation disrupted, abnormal	Figure 7
fga^ug701/+	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein blood coagulation, fibrin clot formation normal process quality, normal	Figure 1
fga^ug701/+	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein platelet aggregation normal process quality, normal	Figure 1
fga^ug701/+; la2Tg	light damage: posterior caudal vein	Day 5	posterior caudal vein blood coagulation, fibrin clot formation normal process quality, normal	Figure 2
fga^ug701/+; la2Tg	light damage: posterior caudal vein	Day 5	posterior caudal vein platelet aggregation normal process quality, normal	Figure 2
fga^ug701/ug701	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein blood coagulation, fibrin clot formation disrupted, abnormal	Figure 1
fga^ug701/ug701	light damage: posterior caudal vein	Protruding-mouth	posterior caudal vein platelet aggregation disrupted, abnormal	Figure 1

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
la2Tg	Tg(-6.0itga2b:EGFP)	Transgenic Insertion
ug701		Indel	fga
ug704		Complex	fga

Human Disease / Model

Human Disease	Fish	Conditions	Evidence
congenital afibrinogenemia			TAS
thrombosis			TAS

Sequence Targeting Reagents

Target	Reagent	Reagent Type
fga	CRISPR2-fga	CRISPR
fga	MO1-fga	MRPHLNO

Fish

Fish
fga^ug701/+
fga^ug701/+; la2Tg
fga^ug701/ug701
fga^ug701/ug701; la2Tg
fga^ug704/+; la2Tg (TU)
fga^ug704/+ (TU)
fga^ug704/ug704; la2Tg (TU)
fga^ug704/ug704 (TU)
la2Tg
la2Tg (TU)

Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-fga	polyclonal	fga	IgG	Rabbit
Ab2-cp	polyclonal	cp		Rabbit

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP

Mapping

Fish et al., 2021 ZDB-PUB-210115-1 PMID:33440782

Venous Thrombosis and Thrombocyte Activity in Zebrafish Models of Quantitative and Qualitative Fibrinogen Disorders

Fish et al., 2021

ZDB-PUB-210115-1
PMID:33440782