Gas6 Targeting Promotes Major Bleeding and Aggravates Inflammation in Protein S Deficient Mice

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<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<0.003).

More immature RBC were found in Pros1^-/- than in Pros1^+/+Gas6^+/+ circulating blood (17% vs 12%, p<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<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<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.