Protein S and purpura fulminans: What’s new?

R. Prince1, S. Calzavarini 1, M. Yasuhiro2, J. H. Griffin 3, J. A. Fernández3, F. Saller 4, A. Angelillo-Scherrer1 (1Bern, Switzerland, 2Chiba, Japan, 3La Jolla, USA, 4Le Kremlin-Bicetre, France)

Bleeding disorders, coagulation and fibrinolytic factors
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: Complete protein S (PS) deficiency causes purpura fulminans (PF) characterized by disseminated intravascular coagulation (DIC) and skin necrosis. PF is thought to result from the imbalance between pro- and anticoagulants. Beyond its role in coagulation, PS exerts cellular functions through receptor tyrosine kinases Tyro3, Axl and Mer. The aim of this work is to mimic severe PS deficiency and monitor PF development

Methods: Pros1+/- and Pros1+/+ mice were treated with warfarin (0.8mg/day). E15 embryonic dorsal skin vasculature was explored by immunostaining: anti-VE-Cadherin, anti-Ter119, anti-CD31, anti-Lyve1 and F4/80 antibodies. Colony forming assays with fetal liver cells allowed us to study erythropoiesis.

Results: Warfarin caused 95% mortality in Pros1+/- and 5% in Pros1+/+ mice, but only a few Pros1+/- mice developed PF. Early PF lesions showed vascular engorgement, intradermal edema and rare thrombosis. In advanced lesions, we noticed massive red blood cell (RBC) extravasation, intra-epidermal hemorrhagic blisters and necrosis. RBC extravasation in mice treated by warfarin pointed to vascular wall damage during PF. Immunostainings of embryonic dorsal skin (VE-Cadherin, Ter119) confirmed RBC extravasation while anti-CD31 showed areas with underdeveloped and less dense vascular networks and less vessels branch points in Pros1-/- than in Pros1+/+ mice. Massively enlarged lymphatic vessels and increased macrophage infiltration were observed in Pros1-/- embryos (Lyve1, F4/80). Blood vessel and liver histology from Pros1-/- embryos revealed numerous circulating immature RBC compatible with increased erythropoiesis because of severe bleeding due to DIC and vascular lesions. In addition, many isolated erythroblast nuclei were found indicating altered phagocytosis. Pros1-/- embryos displayed higher cytokine levels than Pros1+/+ embryos. No difference was found between Pros1-/- and Pros1+/+ embryos for BFU-E colonies, whereas Pros1-/- displayed 50% less CFU-E colonies than Pros1+/+ mice, pointing to a blunted response to erythropoietin (EPO) in the context of inflammation promoted by PF. These results were confirmed by flow cytometry

Conclusion: PF developed under warfarin and in Pros1-/- embryos and was characterized by thrombosis occurring together with vascular damage and inflammatory processes disturbing nuclei phagocytosis, iron metabolism and response to EPO.