OC 73.4 - Patients with protein C deficiency have increased NETosis, possibly mediated by reduced levels of circulating activated protein C

Wednesday, June 28, 2023

11:00 – 11:15 ET

Room: 520

Session Available As: On-Demand

Co-Author(s)

Julia Oto

Postdoctoral Researcher
Hemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain
Valencia, Comunidad Valenciana, Spain

Presenting Author(s)

Raquel Herranz, MSc (she/her/hers)

PhD Candidate
Hemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain
Valencia, Comunidad Valenciana, Spain

Co-Author(s)

EP

Emma Plana

Postdoctoral Researcher
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain; Angiology and Vascular Surgery Service, La Fe University and Polytechnic Hospital, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
FC

Fernando Cana

Laboratory Technician
Hemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
MC

Maria Castaño

Researcher
Hemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
IA

Isabel Aleman-Cabrera

PhD Student
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
AB

Amando Blanquer

Medical doctor
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain; Thrombosis and Haemostasis Unit, Haematology Service, La Fe University and Polytechnic Hospital, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
AC

Ana Cid

Medical doctor
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain; Thrombosis and Haemostasis Unit, Haematology Service, La Fe University and Polytechnic Hospital, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
SH

Saturnino Haya

Medical doctor
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain; Thrombosis and Haemostasis Unit, Haematology Service, La Fe University and Polytechnic Hospital, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
FF

Fernando Ferrando

Medical doctor
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain; Thrombosis and Haemostasis Unit, Haematology Service, La Fe University and Polytechnic Hospital, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
SB

Santiago Bonanad

Head of Thrombosis and Hemostasis Unit
Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain; Thrombosis and Haemostasis Unit, Haematology Service, La Fe University and Polytechnic Hospital, Valencia, Spain
Valencia, Comunidad Valenciana, Spain
PM

Pilar Medina

PI
Hemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group, Medical Research Institute Hospital La Fe, Valencia, Spain
Valencia, Comunidad Valenciana, Spain

Background: Neutrophil extracellular traps (NETs) are highly prothrombotic networks that activate the extrinsic and intrinsic coagulation pathways. NETs also inhibit natural anticoagulants like activated protein C (APC), which also neutralizes their anti-inflammatory and cytoprotective functions. In turn, APC inhibits NETosis, which would reduce thrombotic risk and inflammation, although its relevance in vivo remains unexplored.

Aims: To analyze the possible regulation of NETosis exerted by APC in families with protein C deficiency (PCdef).

Methods: We obtained citrated plasma from a total of 84 individuals, 23 probands with PCdef and at least one thrombotic episode, 25 relatives with PCdef and 36 relatives without. We quantified circulating APC levels using by ELISA; PCag, PCamid and PCcoag (Stago) levels; and NETosis markers cell-free DNA (cfDNA; PicoGreen, Life Technologies), citrullinated histone 3 (citH3; Cell Death Detection ELISAPlus Roche, modified), DNA-histone complexes (Cell Death Detection ELISAPlus) and calprotectin (Human Calprotectin ELISA Kit, Hycult Biotech). We performed the statistical analysis with SPSS v21.

Results: PCdef patients have lower levels of circulating APC than their relatives without PCdef (0.87 vs. 0.92 ng/ml, P=0.002), as well as PCag (53.3 vs. 84.2 ng/mL, P< 0.001). In addition, patients with PCdef have increased NETosis: H3cit markers (0.27 vs. 0.15 U.A., P=0.009) and DNA-histone complexes (0.16 vs. 0.12 U.A., P=0.031). H3cit correlated with DNA-histone complexes (0.409, P< 0.0001) and PCag (-0.244, P< 0.045), and PCag correlated with APC (0.347, P< 0.011).

Conclusion(s): The reduction in APC levels in patients with PC deficiency seems to favor the increase in NETosis, as evidenced in previous in vitro studies. This combination of reduced anticoagulant activity and hypercoagulability induced by NETs could favor thrombotic episodes, which should be verified in large families with PC deficiency and members with and without thrombosis. ISCIII-FEDER (PI20/00075, PI20/01171, FI21/00171), GVA-CIACIF/2021/192, López Borrasca Award 2020 SETH and Grants to SETH Working Groups 2019 & 2022.

Learning Objectives:

Investigate how neutrophil extracellular traps (NETs) are able to activate coagulation.
Understand how activated protein C (APC) is able to inhibit NETosis.
Assess how patients with protein C deficiency are a reliable model to evaluate the regulatory effect of APC on NETosis.