PB1397 - Red blood cell membrane sulfatide expression: possible procoagulant role

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Chatphatai Moonla, MD, MSc

Postdoctoral Research Fellow
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States

Co-Author(s)

AI

Anton Ilich

Postdoctoral Research Associate
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
YP

Yuriy Prokopenko

Postdoctoral Research Associate
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
BE

Brandie M. Ehrmann

Mass Spectrometry Director
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
RS

Rachel Sielaty

Researcher
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
IP

Izabela Pawlinski

Researcher
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
PE

Patrick Ellsworth

Assistant Professor
Division of Hematology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
RP

Rafal Pawlinski

Professor
Division of Hematology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
DM

Dougald M. Monroe

Professor
Division of Hematology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
NK

Nigel S. Key

Professor
Division of Hematology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States

Background: Sulfatide (3-O-sulfogalactosylceramide) is a membrane glycolipid derived from sphingomyelinase-mediated hydrolysis of sphingomyelin via the intermediary molecule, ceramide. When expressed on the red blood cell (RBC) membrane, sulfatide has been reported to support adhesion of sickle cell disease (HbSS) RBCs to several endothelial ligands. Although reagent sulfatide is a known activator of factor XII (FXII), it is unknown whether sulfatide can activate the contact pathway when expressed on RBCs.

Aims: To identify membrane expression of sulfatide on HbSS compared to healthy control (HbAA) RBCs, and to investigate its possible role in FXII activation.

Methods: We first synthesized sulfatide microvesicles (SM4-MVs) (25:75 porcine sulfatide:phosphatidylcholine) as a model system of FXII activation and sulfatide inhibition. Washed HbSS or HbAA RBCs (0.6 million/µL in PBS + 1% BSA and glucose 70 mg/dL) were similarly tested for FXII activation. RBC membrane lipidomic analysis was performed by mass spectrometry.

Results: Generation of activated FXII (FXIIa) from zymogen FXII by SM4-MVs was suppressed in a dose-dependent manner by anti-sulfatide antibody and by arylsulfatase (Figure 1). Lipidomic analysis demonstrated a reduced sphingomyelin:ceramide ratio in HbSS RBCs (P=0.02), indicative of sphingomyelinase-mediated hydrolysis of sphingomyelin (Figure 2). This was accompanied by increased total sulfatide expression in HbSS compared to HbAA RBCs (P=0.07). Although both HbSS and HbAA RBCs led to FXII activation, FXIIa generation induced by HbSS or HbAA RBCs was not statistically different.

Conclusion(s): Both sickle cell disease (HbSS) and healthy control (HbAA) RBCs activate FXII in a buffer system. However, as yet, we have been unable to ascribe this activity to RBC sulfatide, which does appear to be increased in HbSS compared to HbAA RBCs by lipidomic analysis. In ongoing experiments, anti-sulfatide antibody will be tested for its ability to inhibit FXIIa generation by HbAA and HbSS RBCs.