Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/1/jth14391.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/2/jth14391-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148369/3/jth14391_am.pd

Genetic duplication of tissue factor reveals subfunctionalization in venous and arterial hemostasis.

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2-4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds

Complete loss of TF activity results in early lethality due to hemorrhage.

(A) Fish from a double heterozygous (Aa/Bb) f3 incross were genotyped at 8 weeks of age and found to have normal Mendelian ratios (n = 135, AA/BB = 10, AA/Bb = 17, AA/bb = 7, Aa/BB = 15, Aa/Bb = 36, Aa/bb = 20, aa/BB = 10, aa/Bb = 13, aa/bb = 9). Survival curves show that by 9 weeks, there was statistically significant loss of aa/bb offspring. By 400 days, there was a relatively even reduction of the remaining genotypes which is common in wild-type fish, and confirms that a single copy of f3a or f3b is sufficient for survival. (B) A repeat cohort was left undisturbed until genotyping at 6 months of age. The statistically significant loss of aa/bb confirms the early lethality of TF loss independent of genotyping stress. (C) A group of 7 double homozygous mutants were identified at 1 month of age and observed over the following 3 months. 6/7 were lost by 2 months with the single survivor passing at 112 days. (D) Early lethality was due to gross hemorrhage. Examples include the pericardial space (left) and head (right) (red arrows).</p

Synthesized recombinant TF gBlocks.

(DOCX)</p

Loss of TFb protects against spontaneous thrombosis in <i>at3</i>^<i>-/-</i> larvae but does not improve long term survival.

(A) Fibrin deposits were assessed at 5 dpf. Deficiency of TFa did not alter thrombosis scores while loss of TFb led to less deposition (B) When followed for >1 year, f3 genotype did not influence at3-/- survival; however, all aa/bb offspring were lost by 120 days, consistent with TF dependent lethality. Statistical significance by binomial proportion test: *** A* represents AA or Aa, B* represents BB or Bb.</p

Loss of TF contributes to risk of cardiac tamponade.

(A) Stress was chemically induced in 3 dpf larvae by exposure for 16 hours using 25 μM epinephrine and 0.01% hydrocortisone. Larvae were scored for the presence of erythrocytes in the pericardial space (noted by red arrows) by an observer blinded to genotype. Examples of control (left) and tamponade-positive fish (right) are shown. (B) TF-, prothrombin (f2)- and fibrinogen (fga)-deficient fish all showed significantly elevated rates of tamponade relative to controls. Furthermore, TF- and prothrombin-deficient larvae had comparable rates of tamponade and are both significantly increased relative to fga mutants. Statistical significance by binomial proportion test: * < 0.5, ** < 0.01, *** < 0.001. +/* represents +/+ or +/-.</p

TFa and TFb have evolved partial subfunctionalization according to <i>in vivo</i> vascular endothelial laser injury model and <i>in vitro</i> recombinant protein clotting assay.

(A) Laser-mediated venous endothelial injury at 3 dpf shows that the time to occlusion (TTO) was not altered by loss of TFb (AA/bb, Aa/bb) but was delayed in the complete absence of TFa (aa/BB, aa/Bb). Thrombus formation was absent after complete TF loss (aa/bb). (B) Laser-mediated arterial endothelial injury at 5 dpf shows that the ability to form occlusive thrombi was significantly reduced in fish lacking TFb (AA/bb, Aa/bb, aa/bb, indicated by grey bars) compared to BB and Bb groups (green and purple bars; respectively). (C) Endothelial injury at 2 dpf confirms the dependence on TFb in the arterial system prior to the presence of thrombocytes. Occlusive thrombi were nearly absent without TFb (Aa/bb, aa/bb). (D) Relipidated recombinant TFa or TFb proteins (173 nM) were mixed with citrated trout plasma, and recalcified. TFa-liposomes induced clot formation during a manual tilt test significantly faster than TFb-liposomes or empty liposome controls. The reaction was observed until 300 seconds, and then again at 15 minutes to assess for delayed clot formation (the latter observation is indicated by an open or closed circle at 300 seconds). (E) The assay was repeated with relipidated TF diluted in 0.5x HBS (20 mM HEPES pH 7.5, 50 mM NaCl) and plotted on log-log axes demonstrating 10 to 100-fold higher procoagulant activity of TFa over TFb. Statistical significance by Mann-Whitney U testing: ** < 0.01, *** < 0.001. All phenotypic data were collected by an observer blinded to genotype. Open squares in A and B indicate individual larvae that did not occlude within 120 seconds.</p

Oligonucleotides used in methods.

(DOCX)</p

Complete data set of laser injury on offspring from <i>f3 Aa/Bb;f9b</i>^<i>+/-</i> incrosses.

(DOCX)</p

Larval cardiac tamponade at 4 dpf.

(MP4)</p