Loss of Arl6ip5 Gene Function Suppresses Thrombosis in Mice

Background: The genetic contributors to thrombosis have only been partly described. Heterozygous tissue factor pathway inhibitor deficiency ( Tfpi^+/-) is a powerful negative genetic modifier of the homozygous FVL ( F5^L/L) phenotype, demonstrating a nearly complete perinatal lethal genetic interaction in F5^L/LTfpi^+/- mice. To identifymutations suppressing lethal thrombosis, we used this F5^L/LTfpi^+/- phenotype for a sensitized genome wide ENU mutagenesis screen. We screened over 12,000 mice and generated a total of 31 thrombosis suppressor variant lines. We were able to identify ENU-induced mutations in 10 of these lines, each potentially representing an independent pathway for reducing thrombosis severity. One of the mutations that suppressed thrombosis in F5^L/LTfpi^+/- mice was a dominant loss of function mutation (i.e. mutation in a single allele) in the ADP Ribosylation Factor Like GTPase 6 Interacting Protein-5 ( Arl6ip5) gene. Interestingly, no direct role(s) of Arl6ip5 gene or protein in hemostasis have been reported. Therefore, the aim of our studies was to investigate the hypothesis that Arl6ip5 deficiency suppresses thrombosis by reducing blood coagulation and platelet reactivity in mice.

Methods: We previously generated a mouse model of Arl6ip5 ( Arl6ip5 ^-) by genome editing, creating a 151 base pair deletion removing an exon 1 splice site. After backcrossing mice carrying the Arl6ip5 ^- allele to the wildtype C57BL/6J (B6) background, we performed Arl6ip5 ^+/-intercrosses to determine a potential difference in genotype frequency at weaning. To investigate whether the Arl6ip5 ^- allele was able to produce a stable mRNA transcript we performed RT-qPCR on whole liver, lung, heart, kidney, and brain samples derived from Arl6ip5 ^+/+ Arl6ip5 ^+/- and Arl6ip5 ^-/- mice. Furthermore, complete blood cell counts were analyzed using the Advia 2120 (N ≥ 10 for each group), and to investigate potential blood coagulation defects, Prothrombin Time (PT) (N ≥ 5 for each group) and activated Partial Thromboplastin Time (aPTT) (N ≥ 8 for each group) assays were performed on plasma from Arl6ip5 ^+/+ Arl6ip5 ^+/- and Arl6ip5 ^-/- mice with the operator blinded to the genotype. Similarly, we performed platelet function analyses on whole blood from Arl6ip5 ^+/+ Arl6ip5 ^+/- and Arl6ip5 ^-/- mice using the Roche Multiplate, with ADP and collagen as agonists (N ≥ 6 for each group).

Results:Arl6ip5^+/- x Arl6ip5^+/- crosses resulted in normal Mendelian proportions of progeny observed at weaning (N > 400). No Arl6ip5transcripts were present in any of the Arl6ip5 ^-/- tissue samples analyzed, whereas heterozygous mice had the expected 50% Arl6ip5 expression levels across all organs as compared to wildtype mice. A significant prolongation was observed in aPTT between Arl6ip5^+/+ and Arl6ip5^-/- animals (p<0.01). Although no differences in platelet counts between genotypes were observed, platelet aggregometry showed significant differences between wildtype Arl6ip5 ^+/+and Arl6ip5 ^-/-littermates for platelet aggregation, velocity, and area under the curve in response to ADP agonist (normalized to platelet counts, p<0.01). In addition, collagen-induced aggregometry also yielded significant differences in aggregation velocity.

Conclusions: Our results support the hypothesis that Arl6ip5 deficiency contributes to thrombosis suppression by increasing the aPTT and reducing platelet reactivity. Since the PT in Arl6ip5 ^-/- mice is normal, we speculate that Arl6ip5 ^-/- is either directly or indirectly affecting one or more of the levels or activity of coagulation factors VIII, IX, XI and XII. Significant differences in ADP- and collagen-induced platelet aggregation between Arl6ip5 ^+/+wildtype littermates and Arl6ip5 ^-/- suggests that the lack of Arl6ip5affects an aggregation process common to both pathways. Additional analyses are being performed to refine these mechanisms. Understanding the mechanism by which Arl6ip5 deficiency suppresses thrombosis could provide insights into human thrombosis regulation.