Gas6 Targeting Promotes Major Bleeding and Aggravates Inflammation in Protein S Deficient Mice
Authors: Prince Raja Eladnani , Calzavarini Sara , Quarroz Claudia , Reina Caro Maria Desiré , Schuepbach Manuel , Saller Francois , Angelillo-Scherrer Anne 
Published: November 2019

Background: Protein S (PS) and growth arrest-specific gene 6 (Gas6) are vitamin K-dependent proteins. PS is an anticoagulant whose pivotal role is illustrated by purpura fulminans (PF), a life-threatening condition characterized by purpuric skin lesions, disseminated intravascular coagulation and thrombosis. Unlike PS, Gas6 displays a procoagulant effect. Its complete deficiency has anti-hemostatic, pro-inflammatory and anti-erythropoietic effects. In tissues, PS and Gas6 have an impact on cellular functions by binding to and activating receptor tyrosine kinases of the TAM family. PS complete deficiency-induced PF is thought to result from the imbalance between procoagulant and anticoagulant forces. However, its pathophysiological mechanism has not yet been fully elucidated. Previous studies showed that Pros1-/-mice die in utero with a PF phenotype similar to the one observed in human. We therefore considered Pros1-/- mice as a suitable model of the human PF. The aim of this work was to investigate if Gas6 deficiency could rebalance hemostasis in PS deficient mice and rescue them from PF.
Methods & results: Pros1 +/- Gas6 -/- mice were intercrossed to obtain Pros1-/-Gas6-/- mice. Embryonic mortality was higher in Pros1+/-Gas6-/- than in Pros1+/- mattings (6% vs 3 % at E14, 21% vs 18 % at E16, 30% vs 20 % at E17, respectively). About 7.5% of dead embryos from Pros1+/-Gas6-/- mattings were found necrotic and macerated versus only 3.8% inPros1+/- mattings. Macroscopically, 74% of Pros1-/-Gas6-/- embryos displayed maximal bleeding scores (3) corresponding to intracranial and major body bleedings compared to 47% in Pros1-/- embryos (p&lt;0.02). Microscopically, all Pros1-/-Gas6-/- embryos had a bleeding score of 3, whereas 50% of Pros1-/- embryos displayed a milder bleeding score (2). Major blood vessels of E16 Pros1-/- and Pros1-/-Gas6-/- embryos contained a high number of non-phagocyted red blood cells (RBC) expelled nuclei (38% and 58%, respectively) while only few of them were found in Pros1+/+Gas6+/+ embryonic vessels (5%), indicating that altered phagocytosis due to the lack of PS was further aggravated by Gas6 deficiency (p&lt;0.003).
More immature RBC were found in Pros1-/- than in Pros1+/+Gas6+/+ circulating blood (17% vs 12%, p&lt;0.004), suggesting increased erythropoiesis in Pros1-/- embryos in response to bleeding. These data were confirmed using FACS analysis of CD71/Ter119 stained liver single cell suspensions. E14 Pros1-/- embryos had more immature RBC compared to Pros1+/+Gas6+/+ embryos (2.3% vs 1.4%, p&lt;0.08). However, Pros1-/-Gas6-/- embryos displayed less immature RBC in their circulating blood than Pros1-/- embryos. The number of erythroid burst-forming units was lower in Pros1-/-Gas6-/- fetal livers compared with Pros1-/- fetal livers (5±1.3 vs 8±2, p&lt;0.08) confirming reduced erythropoiesis in Pros1-/-Gas6-/- embryos. Perls staining showed increased iron stores in fetal liver and numerous intra-vascular hypochromic RBC in Pros1-/-Gas6-/- embryos pointing to a functional iron deficiency. We also investigated E15 embryonic dorsal skin by immunofluorescence. We found RBC extravasation (VE-cadherin and Ter119 stainings), vascular network outgrowth impairment with fewer vessel branches (CD31 staining), massively enlarged lymphatic vessels and increased macrophages infiltration (Lyve1 and F4/80 stainings) in Pros1-/-Gas6-/- and Pros1-/- indicating an ongoing inflammatory process.
Conclusion: Gas6 deficiency did not rebalance hemostasis in Pros1-/-embryos and prevent PF. Notably, Gas6 and PS combined deficiency leads to a more dramatic phenotype than PS deficiency alone with a higher mortality rate, more major bleedings, phagocytosis impairment, more inflammation and an erythropoietic defect compatible with anemia of inflammation.

Disclosures
No relevant conflicts of interest to declare.